gene function and cellular pathways in higher vertebrates, including humans, have increasingly shown to be highly conserved thr isj 6: 21, 2009 issn 1824-307x review erratum to: immunorecognition and immunoreceptors in the cnidaria [6: 7-14, 2009] sr dunn center for marine studies, university of queensland, australia in the above article affiliation was reproduced incorrectly, and it should have appeared as below: sr dunn center for marine studies and arc centre for excellence for coral reef studies, the university of queensland, st lucia, brisbane, qld 4072, australia 21 isj094.pdf isj 2: 28-31, 2005 issn 1824-307x short communication effect of pyridoxine on the reproduction of the mulberry silkworm, bombyx mori l. (lepidoptera: bombycidae) si faruki department of zoology, rajshahi university, rajshahi, bangladesh accepted march 30, 2005 abstract the present investigation reports the effects of vitamin b6, also known as pyridoxine supplemented feed on the reproductive potential of bombyx mori l. all the concentrations of the vitamin significantly reduced the fecundity. egg-viability was also reduced at all the concentrations, but the differences were not significant in comparison to control. key words: vitamin; reproduction; bombyx mori l introduction in silk industry, fecundity and fertility of the bombyx mori l female are two major factors because these are directly correlated with silk-production determining the number of offspring which offers the production of considerable amount of raw silk. nutrition is an important growth regulating factor in silkworm. it has been reported that the vitamins of b-complex group and certain essential sugars, proteins, amino acids, minerals etc. are responsible for the proper growth and development of the silkworm, b. mori (horie and ito, 1963; horie et al. 1966; sengupta et al., 1972; khan and saha, 1997a; faruki, 1998). a number of researchers works on the effects of vitamin enriched food on the reproduction of b. mori females (khalequzzaman and mannan, 1982; faruki et al., 1992; khan and saha, 1996,; saha and khan, 1996, 1999). pyridoxine also known as vitamin b6, is part of the b group vitamins and is water soluble. it stimulates growth and is important in protein metabolism, and its deficiency in mammals results in a decrease in phosphorylases (anonymous, 1998). the present work, therefore, was attempted to determine the effects of pyridoxine on the fecundity and egg-viability of the two strains of b. mori. corresponding author: saiful islam faruki department of zoology, rajshahi university, rajshahi, 6205, bangladesh e-mail: faruki64@yahoo.com materials and methods to determine the effect of pyridoxine, the eggs of the two multivoltine strains of b. mori viz. nistari-m and urboshi-1 were collected from the germplasm bank of the bangladesh sericultura research and training institute, rajshahi. they were then disinfected with a 2% formalin solution and kept in the rearing room for hatching. the rearing room and other appliances used in the present experiment were disinfected with a 5% formalin solution. newly-hatched larvae were brushed to wooden rearing trays (40 x 29 x 7.5 cm) and were reared on finely-chopped fresh, tender mulberry (morus alba l.) leaves up to the second moult. for this study the source of pyridoxine was pyrol® (pyridoxine hydrochloride hcl, 25) supplied by jayson pharmaceuticals ltd., bangladesh the concentrations of pyridoxine used in the present experiment, viz. 10, 100, 500 and 1000µg/ml were prepared by adding the requisite amounts of the vitamin in distilled water. the leaves were treated by dipping in these solutions and dried by fanning. treated leaves were then supplied to the silkworm larvae from the first day of third instar till spinning. the larvae of a control batch was simultaneously reared on fresh mulberry leaves dipping in distilled water only and dried by fanning. from the fourth instar onwards entire mulberry leaves of both treated and untreated groups were supplied to the larvae. feeding was supplied four times a day at six hour intervals. three replications, each with 50 larvae, were made for each pyridoxine concentration. the rearing trays were kept in fine-netted cabinets. 29 table 1. effect of pyridoxine on the fecundity of b. mori females (n = 15) concentrations (µg/ml) eggs laid mean ± sd prc f-ratio silkworm strains 0 (control) 365.53 ± 35.62 � 10 292.33 ± 13.96 20.03 nistari m 100 300.47 ± 19.90 17.80 4.78* 500 316.80 ± 18.79 13.33 1000 335.00 ± 29.89 8.35 0 (control) 474.87 ± 49.43 � 10 361.00 ± 35.83 23.98 urboshi 1 100 401.47 ± 17.37 15.46 3.73* 500 417.40 ± 37.78 12.10 1000 429.87 ± 35.56 9.48 note: * significant at p = 0.05; prc = percent reproduction control; f = variance ratio table 2. effect of pyridoxine on the viability (%) of b. mori eggs silkworm strains concentrations (µg/ml) mean ± sd prc f-ratio 0 (control) 81.31 ± 5.12 � 10 72.73 ± 3.90 10.55 nistari m 100 75.65 ± 4.14 6.96 1.08 ns 500 77.50 ± 4.33 4.69 1000 79.39 ± 2.94 2.36 0 (control) 97.44 ± 1.20 � 10 90.98 ± 3.45 6.63 urboshi 1 100 93.97 ± 3.25 3.56 2.09 ns 500 93.37 ± 2.98 4.17 1000 93.16 ± 1.92 4.39 note : ns = not significant; prc = percent reproduction control; f = variance ratio 30 mature larvae were transferred to bamboo-made mountages for spinning cocoons. after spinning and pupation the cocoons were harvested and stored according to their sexes. the sex was determined by cutting cocoons with a sharp blade and observing the external genitalia. freshly-emerged moths of opposite sexes were allowed to mate and the females were retained for oviposition. for each concentration, 15 females were observed for their fecundity. the number of eggs laid by individual female was carefully counted and the eggs were incubated to observe their viability. the number of larvae hatched out from the known number of eggs was carefully counted and viability (%) was calculated. data on fecundity and egg-viability were subjected to analyses of variance. here, the variance ratio f was calculated from the ratio between treatment mean square and residual mean square and the value was compared with the tabulated value for significance. the percent reproduction control (prc) was calculated by the formula of rizvi et al. (1980) as: prc = v1 – v2 / v1 x 100, where, v1 = eggs laid/ hatched from untreated control, v2 = eggs laid/ hatched from treated groups. all the experiments were conducted at a mean room temperature of 26 ± 1oc. results and discussion the average egg-productivity in the female b. mori moths of both the strains resulting from various concentrations of the vitamin was significantly (p < 0.05) reduced at the concentration used (table 1). the eggviability was reduced at all the concentrations in comparison to control but statistically the result was not significant (table 2). in present experiment, it was found that lowest number of eggs was produced at the lowest concentration (10µg/ml) and at higher concentrations it was increased but lower than control in case of both the strains. the same trend was also true for egg-viability. the present results are in agreement with the findings of faruki et al. (1992) who observed that para-amino benzoic acid (paba) significantly reduced the fecundity and egg-viability of b. mori females. pai et al. (1988) recorded a reduced reproductive potentials in the females of b. mori resulting from paba treatment. similarly, banerjee and khan (1992) observed that vitamin b6 enhance the oviposition of the bacillus thuringiensis var. kurstaki-infected b. mori, but the rate was lower than the controls. khan and saha (1997b), saha and khan (1997a) and khan et al. (2002) reported that the reproductive ability of female b. mori drastically reduced when reared on feed enriched with vertebrate sex-hormones. saha and khan (1997b) and khan and saha (2003) reported that higher concentrations of vitamins reduced the fecundity and fertility of b. mori. the fecundity and fertility of the silkworm females were reduced when they were reared on the feed supplemented with different artificial nutrients (khalequzzaman and mannan, 1982). the present work is a preliminary study on the use of vitamin b6 for the determination of the reproductive ability of the most commercially important insect. therefore, future more comprehensive works are very much solicited in this line with highest concentrations on this species is required. acknowledgements the author would like to extend the sincere thanks to the chairman of the department of zoology (rajshahi university) for providing necessary laboratory facilities and to the director of the bangladesh sericulture research and training institute (rajshahi) for kindly supplying the experimental eggs of the silkworm. references anonymous. instruction manual of pyrol®. jayson pharmaceuticals ltd., bangladesh. 1998. banerjee sk, khan ar. effect of vitamin b6 on the fecundity of bacillus thuringiensis var. kurstaki-treated bombyx mori l. bangladesh j. zool. 20: 361-362, 1992. faruki si, khan ar, mannan a. fecundity and fertility of the silkworm, bombyx mori l. fed on mulberry leaves supplemented with para-amino benzoic acid. bangladesh j. zool. 20: 351-353, 1992. faruki si. nutritive effects of thianomin® enriched mulberry leaves on the silkworm, bombyx mori l. univ. j. zool. rajshahi univ. 17: 39-44, 1998. horie y, ito t. vitamin requirements of the silkworm. nature 197: 98-99, 1963. horie y, watanabe h, ito t. nutrition of the silkworm, bombyx mori – xiv. further studies on the requirements for b vitamins. bull. seric. exp. sta., tokyo 20: 393-409, 1966. khalequzzaman m, manan ma. effect of artificial diet on certain bilological aspects of the silkworm, bombyx mori l. (bombycidae: lepidoptera). univ. j. zool. rajshahi univ. 1: 71-76, 1982. khan an, khan ar, rahman s. effect of lynestrenol® on the growth and development of the mulberry silkworm, bombyx mori l. (lepidoptera: bombycidae). sericologia 42: 573-578, 2002. khan ar, saha bn. nutritive effects of fe-plus® (ferous fumarate + folic acid) on the silkworm, bombyx mori l. bangladesh j. zool. 24: 195-203, 1996. khan ar, saha bn. nutrition of the mulberry silkworm, bombyx mori on feed supplemented with calcium lactate. j. ecobiol. 9: 53-58, 1997a. khan ar, saha bn. effect of vertebrate sex-hormones, mestranol and norethindrone, on growth and development of bombyx mori l. (lepidoptera). bangladesh j. zool. 25: 103109, 1997b. khan ar, saha bn. nutritive effects of thiamine on the silkworm, bombyx mori l. bangladesh j. zool. 31: 169-176, 2003. pai ik, hedge sn, krishnamurthy nb. paba induced genotoxic effects in silkworm, bombyx mori. proc. intl. symp. nature of genetic variation in man. xii annual conf. emsi, hyderabad, india, pp 54, 1988. rizvi sjh, pandey sk, mukerji d, mathur sn. 1,3,7trimethylxanthane, a new chemosterilant for stored grain pest, callosobruchus chinensis l. z. angew. ent. 90: 378381, 1980. saha bn, khan ar. effect of dietary supplementation of vitamins and minerals on the growth and development of bombyx mori l. bangladesh j. zool. 24: 125-131, 1996. saha bn, khan ar. effect of vertebrate sex-hormones on bombyx mori l. sericologia 37: 19-25, 1997a. saha bn, khan ar. the nutritive effects of sinafort®-b on bombyx mori l. entomon 22: 29-34, 1997b. 31 saha bn, khan ar. the growth and development of the silkworm, bombyx mori l. on feed supplemented with nicotinic acid. bangladesh j. life sci. 11: 103-109, 1999. sengupta k, singh bd, mustafi jc. nutrition of the silkworm, bombyx mori l. i. studies on enrichment of mulberry leaf with various sugars, proteins, amino acids and vitamins for vigorous growth of worms and increased cocoon crop production. indian j. seric. 11: 11-19, 1972. isj 5: 50-xx, 2008 isj 5: 50-53, 2008 issn 1824-307x short communication cytotoxic activity by the mussel mytilus galloprovincialis and the venus clam chamelea gallina in the adriatic sea in 2007 d malagoli, l casarini, f fiori1, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy 1coop m.a.r.e. soc. coop. a.r.l., cattolica (rn), italy accepted may 13, 2008 abstract given the ecological and economic importance of bivalve molluscs, the evaluation of their welfare is one of the primary aims for both biologists and people working in shell fishing. after a three year-long period monitoring the cytotoxic activity exerted by the hemolymph from the mussel mytilus galloprovincialis, we have concluded that cytotoxicity represents a useful parameter to evaluate the status of the immune activity and therefore the health of mussels in a specific period of the year. during 2007, we compared the mussel cytoxicity with that of the venus clam chamelea gallina from contiguous areas of the northern adriatic sea. our observations indicate that the cytotoxicity of the hemolymph of the two species follows a similar course during the year, suggesting that cytotoxic activity is primarily determined by the life/reproductive cycles. key words: mytilus galloprovincialis; chamelea gallina; mussel; venus clam; cytotoxicity; immunity; mollusc health introduction in view of their feeding behavior and their localization along the coasts or in internal waters, the identification of biomarkers able to represent reliably the welfare of bivalve molluscs is one of the primary goals for both biologists and people interested in the economic/environmental importance of these species (baršienė et al., 2006; pellacani et al., 2006). bivalves are usually considered as bioindicators, and their immune system is frequently chosen as the main target of these studies (ballarin et al., 2003; canesi et al., 2007a, b; malagoli et al., 2007b, 2008; novas et al., 2007a, b). as a result, the understanding of the parameters on which mollusc health relies has improved and led to a better evaluation of the real impact of exceptional situations connected with environmental catastrophes (laffos et al., 2006). since 2005, we have been studying the seasonal variations in the cytotoxic activity of the hemolymph from mytilus galloprovincialis grown in mussel farms in the northern part of adriatic sea, in order to understand ___________________________________________________________________________ corresponding author: davide malagoli department of animal biology via campi 213/d 41100 modena, italy e-mail: davide.malagoli@unimore.it whether this activity could be a useful parameter to evaluate the health of a specific mussel population (malagoli et al., 2006, 2007a). from these studies, we have concluded that cytotoxicity in m. galloprovincialis hemolymph is subject to cyclic modifications during the year (malagoli et al., 2006, 2007). to verify whether our considerations could also be extended to other bivalves, we here present data concerning the fluctuations of cytotoxic activity in both m. galloprovincialis and the venus clam, chamelea gallina, during 2007. moreover, to assess the impact of shell fishing on the health of molluscs, we have compared results collected from clams in areas in which the fishing was closed with those obtained from animals where fishing was regularly undertaken. materials and methods animals specimens of the bivalve mollusc, mytilus galloprovincialis, (minimum length 60 mm, except for september) were obtained monthly from local fishermen in the cesenatico area (fc, italy). immediately after collection, 40 animals were utilized to obtain the hemolymph as described below. a similar procedure was applied for 50 chamelea gallina (minimum length 25 mm), but the clams were sampled only four times in the year 2007: january (winter), may (spring), september (summer) and november (autumn). the c. gallina specimens came from the riccione area (rn, italy), about 23 miles from mussel farms in cesenatico. for each single period, 2 lots of 120 clams/each were subjected to hemolymph withdrawal. of the 2 lots, one came from a restricted area in which any kind of fishing was forbidden from august 2006 and for the whole period of the project (i.e. 15 months), while the second lot was collected about 1 mile away where clam fishing was regularly undertaken. hemolymph preparation and cytotoxicity assay the detailed procedure for the hemolysis assay in mussels has already been described elsewhere (malagoli and ottaviani, 2005), but there were some differences in procedure for m. galloprovincialis and c. gallina. briefly, the mussel hemolymph was collected by exerting gentle aspiration with a sterile syringe inserted between the mussel valves and filtered into sterile tubes (0.2 μm filter porosity). for c. gallina, the valves were opened with a razor blade, and the hemolymph was aspired with a syringe and subjected to centrifugation (13,000xg for 5 min). the supernatant was then carefully recovered in order to leave the pellet containing circulating cells and debris undisturbed. filtration was not used with c. gallina, since the collected volumes per animal where lower than 500 μl and therefore not compatible with the size of the syringe filters. the filtered/recovered hemolymph from each animal was kept separately and never pooled. in order to evaluate the hemolytic activity of the hemolymph from the two species, the cytolysis of human a positive erythrocytes following incubation for 1 h at 25 °c (hubert et al., 1997; malagoli and ottaviani, 2005) and subsequent centrifugation at 3000xg for 5 min at 4 °c was quantified using a spectrophotometer (abs 541 nm). samples with an optical density (od) exceeding the fixed threshold od level of 0,5 where considered cytotoxic (malagoli and ottaviani, 2005). for each lot of molluscs, the percentage of cytotoxic animals was then calculated. the experiments were repeated twice and in duplicate for each animal. results and discussion the monthly evaluation of the hemolytic activity of m. galloprovincialis hemolymph confirmed that significant fluctuations in the percentage of cytotoxic animals occur during the year (fig. 1a). the comparison of these data with those already collected in 2005 and 2006 confirmed that higher values are seen during the summer (malagoli et al., 2007a). the major discrepancies observed between data collected in 2007 and results in 2005 and 2006 (malagoli et al., 2007a) concerns the findings for january (nearly 100 % of animals positive in 2007) and september (nearly 0 % of animals positive in 2007) (fig. 1a). while for the data collected in january we have no indication of exceptional situations, the countertendency observed in september is most probably related to the introduction of new mussels (length < 20 mm). in that fig. 1 course state of mytilus galloprovincialis and chamelea gallina cytotoxicity of contiguous areas throughout 2007. a) course state of mussel grown in mussel farms of cesenatico; b) course state of venus clams coming form fishing or no fishing areas of cattolica. each histogram represent for every month the result of one out of a set of four independent experiments. period, hemolymph had to be taken from animals of significantly different size and age compared to the specimens usually sampled during the rest of the year. this result suggests the hypothesis that molecules responsible for hemolytic activity in m. galloprovincialis are less abundant in young animals. to our knowledge, there are no data in the literature concerning the cytotoxic activity of the hemolymph of c. gallina. consequently, the data we present here cannot be compared with other information related to this model. however, as with the mussel, we have also observed in the clam a fluctuation in the number of cytotoxic animals during the year (fig. 1b). no differences were seen between clams from restricted and open fishing areas (fig. 1b), and there were no differences between the characteristics of the water in the two areas during the year. therefore, normal fishing activity does not appear to have an impact on the health of the population. as far as the comparison between the m. galloprovincialis and c. gallina is concerned, it is worth noting that despite an always higher percentage of cytotoxic animals in the c. gallina population, the trend in the clams over the year is very similar to that observed for m. galloprovincialis (fig. 2). this last observation points to a possible similarity between the time course of cytotoxic activity in bivalves from contiguous areas. fluctuations in biological activity during the year are not new for mussels (cao et al., 2007; novas et 51 fig. 2 comparison between the course of hemolymph cytotoxic activity of m. galloprovincialis (mg) and c. gallina (cg) during the year. al., 2007b). in particular, variations in immune reactivity or the production of immune-related molecules have been recorded (barcia and ramosmartinez, 2008). various stress factors have been linked to alterations in molluscan immune responsiveness, particularly of the cellular component, i.e. the hemocytes (pampanin et al., 2002; ballarin et al., 2003; canesi et al., 2006; cao et al., 2007; novas et al., 2007b; malagoli et al., 2007b, 2008). however, cytotoxic activity is part of the humoral component of the immune system and is of protein origin (hubert et al., 1997). this means that cytotoxicity is not susceptible to rapid modification (i.e. within hours) as a consequence of a sudden alteration in an environmental parameter, because the rate of protein turnover and biosynthesis are the main causes of fluctuations in cytotoxic activity in the hemolymph. it remains to be established whether the component determining the time course of cytotoxicity is primarily environmental or rather connected to the mollusc life-cycle. from the present and previous data (malagoli et al., 2006, 2007a), it seems probable that environmental conditions may modify a state that is based on the life/reproductive cycle. thus, the cytotxic activity of the hemolymph has to be recommended as a valid parameter to determine the status of immune surveillance and welfare, especially for regular checks of mollusc welfare. indeed, for this purpose, the evaluation of cytotoxcity seems more reliable than rapidly changing parameters such as phagocytosis and lysosomal membrane stability, the levels of which may be influenced by manipulations and fishing procedures (pampanin et al., 2002; ballarin et al., 2003; malagoli et al., 2007b, 2008). acknowledgment this work has been in part supported by the centro di ricerche marine (cesenatico, fc, italy) and in part by the regione emilia-romagnaservizio economia ittica (italy) and the consorzio gestione pesca molluschi bivalvi (rimini division, italy) (project: “actions in chamelea gallina to define management strategies to improve production”). the authors also wish to thank the centro trasfusionale of the policlinico (modena, italy) for making available the blood and mr m marangoni who kindly provided the mussels. references ballarin l, pampanin dm, marin mg. mechanical disturbance affects haemocyte functionality in the venus clam chamelea gallina. comp. biochem. physiol. 136a: 631-640, 2003. barcia r, ramos-martinez ji. effects of interleukin2 on nitric oxide production in molluscan innate immunity. inv. surv. j. 5: 43-49, 2008. barsiene j, lehtonen kk, koehler a, broeg k, vuorinen pj, lang t, et al. biomarker responses in flounder (platichthys flesus) and mussel (mytilus edulis) in the klaipeda-būtinge area (baltic sea). mar. pollut. bull. 53: 422-436, 2006. canesi l, betti m, ciacci c, lo russo lc, pruzzo c, gallo g. cell signalling in the immune response of mussel hemocytes. inv. surv. j. 3: 40-49, 2006. canesi l, ciacci c, lorusso lc, betti m, gallo g, pojana g, et al. effects of triclosan on mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms. comp. biochem. physiol. 145c: 464-472, 2007a. canesi l, lorusso lc, ciacci c, betti m, regoli f, poiana g et al. effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, mytilus galloprovincialis. chemosphere 69: 994-1002, 2007b. cao a, novás a, ramos-martínez ji, barcia r. seasonal variations in haemocyte response in the mussel mytilus galloprovincialis lmk. aquaculture 263: 310-319, 2007. hubert f, cooper el, roch p. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloproíincialis. biochim. biophys. acta 1361: 29-41, 1997. laffon b, rabade t, pasaro e, mendez j. monitoring of the impact of prestige oil spill on mytilus galloprovincialis from galicia coast. env. int. 32: 342–348, 2006. malagoli d, ottaviani e. cytotoxicity as a marker of mussel health status j. mar. biol. ass. u.k. 85: 359-362, 2005. malagoli d, casarini l, ottaviani e. effects of the marine toxins okadaic acid and palytoxin on mussel phagocytosis. fish shellfish immunol. 24: 180-186, 2008. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005. inv. surv. j. 3: 1-3, 2006. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2006: regular changes of cytotoxicity during the year. inv. surv. j. 4: 10-12, 2007a. malagoli d, casarini l, sacchi s, ottaviani e. stress and immune response in the mussel mytilus galloprovincialis. fish shellfish immunol. 23: 171-177, 2007b. 52 novas a, barcia r, ramos-martínez ji. after the prestige oil spill modifications in no production and other parameters related to the immune response were detected in hemocytes of mytilus galloprovincialis. aquat. toxicol. 85: 285-290, 2007a. pampanin dm, ballarin l, carotenuto l, marin mg. air exposure and functionality of chamelea gallina haemocytes: effects on haematocrit, adhesion, phagocytosis and enzyme contents. comp. biochem. physiol. 131a: 605-614, 2002. novas a, barcia r, ramos-martínez ji. nitric oxide production by haemocytes from mytilus galloprovincialis shows seasonal variations. fish shellfish immunol. 23: 886-891, 2007b. pellacani c, buschini a, furlini m, poli p, rossi c. a battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters. aquat. toxicol. 77: 1-10, 2006. 53 cytotoxic activity by the mussel mytilus galloprovincialis and the venus clam chamelea gallina in the adriatic sea in 2007 results and discussion the invertebrate blueprint of the connection between aging and immune neuroendocrine responses isj 6: 102-105, 2009 issn 1824-307x visions and perspectives the invertebrate blueprint of the connection between aging and immune neuroendocrine responses e ottaviani1, d malagoli1, c franceschi2 1department of animal biology, university of modena and reggio emilia, 41100 modena, italy 2department of experimental pathology, university of bologna, 40126 bologna, italy accepted july 13, 2009 abstract the present paper summarizes the main findings related to aging and immune neuroendocrine responses in invertebrates. in particular, the functional aspects and the genes involved are examined. a possible mechanism of correlation between aging and functioning of immune-neuroendocrinerelated genes is also discussed. key words: aging; longevity; immune neuroendocrine responses; gerontogenes introduction aging, or senescence, is a progressive somatic degeneration that provokes homeostatic modifications with damage to cellular functions and drastic inefficiency in survival. notwithstanding the presence of various stressors, such as bacteria, virus, radiations, heat, oxygen, and free radicals, the aging process is controlled by a variety of defense functions or anti-stress responses (franceschi et al., 2000). indeed, aging is a remodeling phenomenon that involves mainly the immune and neuroendocrine systems, while the aging phenotype can be interpreted as a global, adaptive response of the body to the age-related accumulation of nonrepaired damages, so allowing an optimal redistribution/utilization of the resources in order to survive (garinis et al., 2008; schumaker et al., 2009). in terms of mammalian immunosenescence, changes in adaptive and innate immunity have been observed. on the one hand, there is the involution of the thymus (nasi et al., 2006), a profound shrinkage in the t-cell repertoire (wack et al., 1998), and a massive increase of megaclones of memory cells directed against typical infections of old age (larbi et al., 2008). on the other, we see the activation of the macrophage that produces a large amount of cytokines and is responsible for the chronic inflammatory process in the aged organisms. this profound correlation between aging and inflammatory status has been described with the neologism “inflamm-aging” (franceschi et al., 2000). ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it overall, during aging the immune system appears to concentrate on the recognition of external and internal environmental antigens met during life, while neglecting new encounters. furthermore, immunosenescence seems to be the result of a chronic hyper-stimulation of both adaptive and innate immunity (fagiolo et al., 1993). the appearance of adaptive clonotypical immunity from lower vertebrates onwards has given rise to great variability in the molecular structures devoted to the recognition of epitopes. in this context, the macrophage, a cell that plays a central role in the innate immunity, has long been neglected by the immunologists and has only recently assumed its own, correct, immune role. several data indicate that the macrophage has a profound evolutionary root, and in both vertebrates and invertebrates, the phagocytic capacity of these cells has always played a primary role in the stress response and inflammation. these activities share cells and mediators from vertebrates to invertebrates, suggesting a common origin (ottaviani et al., 2007). this idea is not completely new, but rather a reappraisal and extension of the phagocytosis theory of immunity proposed by elie metchnikoff, who was the first to propose the existence of an evolutionary mechanism devoted to the protection of organisms (metchnikoff, 1901). the macrophage is able not only to perform phagocytosis, but also to respond to different stimuli, such as bacterial products, neuropeptides, neurohormones, cytokines, and to release proinflammatory cytokines, e.g., nitric oxide, biogenic amines and neuropeptides, among others (ottaviani et al., 1997). in this light, various features and mechanisms of aging are a reshaping of old and conserved 102 functions of innate immunity (macrophage-centered) with the new functions of anticipatory immunity (lymphocyte-centered) and the neuroendocrine system (ottaviani, 1992; panerai and ottaviani, 1995). invertebrates functional age-related data aging has been extensively studied in two invertebrate models, drosophila melanogaster and caenorhabditis elegans. as far as we know, the freshwater snail lymnaea stagnalis is one of the first invertebrate models in which the relationship between aging and immune response was examined. in particular, juvenile samples of l. stagnalis presented fewer and less well developed circulating amoebocytes with more limited phagocytic capacity compared to adult specimens, suggesting that the immune system of juvenile snails is less competent (dikkeboom et al., 1984, 1985). this scenario could also explain the greater susceptibility to trichobilharzia ocellata infection of juvenile snails compared to adult specimens (meuleman et al., 1982; dikkeboom et al., 1985). the response to bacteria and caloric restriction are the main protocols in studying the effects of aging in flies and worms, respectively. it should be underlined that in these investigations, the researchers focused on the longevity aspect. although these studies are intriguing, some contradictions have been reported. it has been demonstrated that aged flies transcribe more antimicrobial peptide diptericin than young flies when exposed to septic bacterial infection, suggesting that this behaviour is due to immune senescence (zerofsky et al., 2005). aging also reduces the capacity to survive bacterial infection (ramsden et al., 2008). different findings were reported by ren et al. (2007), who showed that aged drosophila can tolerate a significant bacterial load and mount a large immune response without compromising survival. other experiments have demonstrated that caloric restriction counteracts aging and extends the lifespan of various organisms, including d. melanogaster and c. elegans (pletcher et al., 2002; lee et al., 2006). genetic age-related data in both d. melanogaster and c. elegans several genes have been seen to change their expression with age. in particular, chico, a drosophila homolog of vertebrate irs1-4, that plays an essential role in the control of cell size and growth is involved in the extension of lifespan in the flies (böhni et al., 1999; clancy et al., 2001). indeed, the use of mutants, such as chico1 homozygotes has resulted in increased median and maximum lifespan in females of up to 48 % and 41 %, respectively. chico1 heterozigotes showed an increase in median lifespan of up to 36 % and 13 % in females and males, respectively (clancy et al., 2001). in restricted diet conditions, elevated superoxide dismutase (sod) activity has been associated with an extension of lifespan, while increased sod activity (cuzn sod, but not mn sod activity) has been detected in chico1 homozygotes (kabil et al., 2007). when grown on a diet of escherichia coli, mutant alleles of age-1 and daf-2 increase the lifespan of c. elegans by 65 % and 100 %, respectively (friedman and johnson, 1988; kenyon et al., 1993). a further increase in lifespan is observed (100 % and 200 %, respectively) when the worms are maintained in axenic culture, and this increase is correlated to metabolic rate, suggesting that genes impact on longevity and senescence by regulating metabolic activities (vanfleteren and de vreese, 1995). the c. elegans germ-line directly influences its lifespan by modulating the activity of an insulin/igf-1 pathway that is involved in the regulation of worm aging. mutants able to reduce the efficiency of daf-2 (an igf-1 receptor homologue) extend the worm’s lifespan, and this process requires daf-16 activity (hsin and kenyon, 1999). the gerontogenes are also involved in immuneneuroendocrine responses. the daf-2, age-1 and age-2 genes in c. elegans have been seen to increase resistance to bacterial pathogens (laws et al., 2004; evans et al., 2008). moreover, such mutations, which normally enhance life expectancy, were also able to increase resistance to death caused by bacterial pathogens such as pseudomonas aeruginosa and salmonella enterica. these experimental results suggest that longevity is associated with stress resistance and that immunological challenges are an integral part of the spectrum of environmental stressors. with regards the neuroendocrine response, it has been demonstrated that in drosophila serotonin controls stress behaviour by regulating the daf-2 insulin/igf-1 receptor signalling to the daf16/foxo transcription factor. two classes of serotonergic neurons that present distinct serotonergic receptors are able to influence specific aspects of daf-16/foxo functions (liang et al., 2006). a recent finding suggests that in c. elegans, a distinct daf-16-dependent signalling pathway is involved in the activation of genes that provide resistance to bacterial pathogens (miyata et al., 2008). these findings offer a molecular and genetic basis for our hypothesis that the immune response and stress (neuroendocrine response) are both highly interconnected and conserved over the course of evolution (ottaviani and franceschi, 1997; ottaviani et al., 2007). concluding remarks the data reported above points to a relationship between aging and the efficacy of immune and stress responses. however, it is not easy to understand how these events are correlated. deveale et al. (2004) raise two questions: 1) why does the immune response change with age? 2) is this change aging-related or does it contribute to aging? with regards invertebrates, we can surmize an explanation. we have postulated that the immune and neuroendocrine systems have a common origin in which the macrophage is an immuneneuroendocrine effector system integrating innate 103 fig. 1 a schematic representation of the macrophage (ø) as the witness of the common origin of the immune and neuroendocrine systems. a) the normal homeostatic condition; b) during aging immunity, stress and inflammation (ottaviani and franceschi, 1997; ottaviani et al., 2007). this defense network may be interpreted as an equilateral triangle whose angles represent immunity, stress response and inflammation, while the macrophage sits in the middle (fig. 1a). in this unitarian perspective, these phenomena can be seen as an integrated network, with a common objective, i.e., the maintenance of body homeostasis. however, in different situations, one phenomenon may prevail over the others. as previously reported, the proinflammatory status of aging (inflamm-aging) is the result of the chronic activation of the macrophage. in this condition, the prevalence of inflammation over the other two parameters (immunity and stress) induces a change in the geometric shape to a scalene triangle (fig. 1b) representing the state of senescence. as far as invertebrates are concerned, old d. melanogaster present an inflammatory status (pletcher et al., 2007), suggesting that the causes and consequences may be the same in metazoans. from an ecoimmunology point of view, resources in the immune and neuroendocrine systems must be redistributed during aging in order to minimize the cost in terms of energy expenditure (ottaviani et al., 2008). our hypothesis of a reduced immune response as a consequence of a persisting inflammatory status could represent an answer to the questions raised by deveale et al. (2004). references böhni r, riesgo-escovar j, oldham s, brogiolo w, stocker h, andruss bf, et al. autonomous control of cell and organ size by chico, a drosophila homolog of vertebrate irs1-4. cell 97: 865-875, 1999. clancy dj, gems d, harshman lg, oldham s, stocker h, hafen e, et al. extension of lifespan by loss of chico, a drosophila insulin receptor substrate protein. science 292: 104106, 2001. deveale b, brummel t, seroude l. immunity and aging: the enemy within? aging cell 3: 195-208, 2004. dikkeboom r, van der knaap wp, meuleman ea, sminia t. a comparative study on the internal defence system of juvenile and adult lymnaea stagnalis. immunology 55: 547-553, 1985. dikkeboom r, van der knaap wp, meuleman ea, sminia t. differences between blood cells of juvenile and adult lymnaea stagnalis. cell tissue res. 238: 43-47, 1984. evans ea, chen wc, tan mw. the daf-2 insulinlike signaling pathway independently regulates aging and immunity in c. elegans. aging cell 7: 879-893, 2008. fagiolo u, cossarizza a, scala e, fanales-belasio e, ortolani c, cozzi e, et al. increased cytokine production in mononuclear cells of healthy elderly people. eur. j. immunol. 23: 2375-2378, 1993. 104 franceschi c, bonafè m, valensin s, olivieri f, de luca m, ottaviani e, et al. inflamm-aging. an evolutionary perspective on immunosenescence. ann. ny acad. sci. 908: 244-254, 2000. friedman db, johnson te. a mutation in the age-1 gene in caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. genetics 118: 75-86, 1988. garinis ga, van der horst gtj, vijg j, hoeijmakers jhj. dna damage and ageing: new-age ideas for an age-old problem. nat. cell biol. 10: 12411247, 2008. hsin h, kenyon c. signals from the reproductive system regulate the lifespan of c. elegans. nature 399: 362-366, 1999. kabil h, partridge l, harshman lg. superoxide dismutase activities in long-lived drosophila melanogaster females: chico1 genotypes and dietary dilution. biogerontology 8: 201-208, 2007. kenyon c, chang j, gensch e, rudner a, tabtiang r. a c. elegans mutant that lives twice as long as wild type. nature 366: 461-464, 1993. larbi a, franceschi c, mazzatti d, solana r, wikby a, pawelec g. aging of the immune system as a prognostic factor for human longevity. physiology (bethesda) 23: 64-74, 2008. laws tr, harding sv, smith mp, atkins tp, titball rw. age influences resistance of caenorhabditis elegans to killing by pathogenic bacteria. fems microbiol. lett. 234: 281-287, 2004. lee gd, wilson ma, zhu m, wolkow ca, de cabo r, ingram dk, et al. dietary deprivation extends lifespan in caenorhabditis elegans. aging cell 5: 515-524, 2006. liang b, moussaif m, kuan cj, gargus jj, sze jy. serotonin targets the daf-16/foxo signaling pathway to modulate stress responses. cell metab. 4: 429-440, 2006. metchnikoff e. immunity in infective diseases, cambridge university press, cambridge, uk, 1901. meuleman ea, huyer ar, luub twj. the effects of the age o the host and the miracidial dose on the development of trichobilharzia ocellata in lymnaea stagnalis. trop. geogr. med. 34: 298, 1982. miyata s, begun j, troemel er, ausubel fm. daf16-dependent suppression of immunity during reproduction in caenorhabditis elegans. genetics 178: 903-918, 2008. nasi m, troiano l, lugli e, pinti m, ferraresi r, monterastelli e, et al. thymic output and functionality of the il-7/il-7 receptor system in centenarians: implications for the neolymphogenesis at the limit of human life. aging cell 5: 167-175, 2006. ottaviani e. some facts and speculation on the origin of the immunoneuroendocrine system and its correlation with aging. ann. ny acad. sci. 663: 331-334, 1992. ottaviani e, franceschi c. the invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems. immunol. today 18: 169-174, 1997. ottaviani e, franchini a, franceschi c. proopiomelanocortin-derived peptides, cytokines, and nitric oxide in immune responses and stress: an evolutionary approach. int. rev. cytol. 170: 79-141, 1997. ottaviani e. malagoli d, capri m, franceschi c. ecoimmunology: is there any room for the neuroendocrine system? bioessays 30: 868874, 2008. ottaviani e, malagoli d, franceschi c. common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidences to in silico approaches. trends immunol. 28: 497-502, 2007. panerai ae, ottaviani e. immunoendocrine reshaping with age. int. rev. immunol. 12: 7584, 1995. pletcher sd, kabil h, partridge l. chemical complexity and genetics of aging. annu. rev. ecol. evol. syst. 38: 299-326, 2007. pletcher sd, macdonald sj, marguerie r, certa u, stearns sc, goldstein db, et al. genome-wide transcript profiles in aging and calorically restricted drosophila melanogaster. curr. biol. 12: 712-723, 2002. ramsden s, cheung yy, seroude l. functional analysis of the drosophila immune response during aging. aging cell 7: 225-236, 2008. ren c, webster p, finkel se, tower j. increased internal and external bacterial load during drosophila aging without life-span trade-off. cell metab. 6: 144-152, 2007. schumaker b, hoeijmakers jh, garinis ga. sealing the gap between nuclear dna damage and longevity. mol. cell. endocrinol. 299: 112-117, 2009. vanfleteren jr, de vreese a. the gerontogenes age-1 and daf-2 determine metabolic rate potential in aging caenorhabditis elegans. faseb j. 13: 1355-1361, 1995. wack a, cossarizza a, heltai s, barbieri d, d'addato s, franceschi c, et al. age-related modifications of the human alphabeta t cell repertoire due to different clonal expansions in the cd4+ and cd8+ subsets. int. immunol. 10: 1281-1288, 1998. zerofsky m, harel e, silverman n, tatar m. aging of the innate immune response in drosophila melanogaster. aging cell 4: 103-108, 2005. 105 isj 3: 89-96, 2006 isj 3: 97-102, 2006 issn 1824-307x short communication morphological abnormalities in tribolium castaneum (herbst) and tribolium confusum duval due to cyromazine and pirimiphos-methyl treatments alone or in combination ahm kamaruzzaman1, ams reza2, kamsh mondal3, s parween2 1institute of biological sciences, rajshahi university, rajshahi 6205, bangladesh 2 department of zoology, rajshahi university, rajshahi 6205, bangladesh 3member, bangladesh public service commission, agargaon, dhaka, bangladesh accepted november 03, 2006 abstract newly hatched (24 h old) larvae of tribolium castaneum and t. confusum were allowed to feed on different doses of cyromazine or pirimiphos-methyl, or on a combined dose of both compounds up to pupation. all the treatments produced deformities at all the life stages. cyromazine produced a number of abnormalities in the larval stage (p < 0.001) of the two species. both the compounds produced similar type of deformities in the adults, but the effect was slightly more in the female t. confusum. the combined action (10 ppm cyromazine + 0.1 ppm pirimiphos-methyl) of the compounds also produced deformities at each stage; and the effects were more pronounced than the effect caused by a single dose of either 10 ppm cyromazine or 0.1 ppm pirimiphos-methyl, and this will produce less stress on the environment and human health. key words: abnormalities; tribolium; cyromazine; pirimiphos-methyl introduction insects surviving insecticidal treatments very often become variously deformed, and the formation of chimeric individuals and elytral deformities in adults are very common. the organophosphorous insecticide pirimiphos-methyl has been reported to produce morphogenetic abnormalities in treated insects including tribolium species (khan, 1981; mondal, 1984; rahman, 1992). the insect growth regulators (igrs) are also able to produce various morphological abnormalities in treated insects (stall, 1975). among the igrs, chitin synthesis inhibitors (csis) interfere with the formation of new cuticle (hajjar, 1985), and disturb the process of ecdysis. a number of these compounds affects moulting in insects (hajjar, 1985), among which the triazine compounds are effectively used to control dipteran insects in poultry (bloomcamp et al., 1987), animal house (el-oshar et al., 1985) and public health (awad and mulla, 1984; nelson et al., 1986). cyromazine, derived from azido-triazine herbicides (shen and flapp, 1990), is effective against dipteran larvae (fox, 1990); but was ___________________________________________________________________________ corresponding author: dr. a.m. saleh reza department of zoology, rajshahi university rajshahi 6205, bangladesh e-mail: salehbgd@yahoo.com found to be active against the larvae of colorado potato beetle, leptinotarsa decemlineata (say) (bishop et al., 1990; sirota et al., 1993; sirota and grafius, 1994). as a csi compound, cyromazine affects growth and development in different insect species, including t. castaneum (herbst) (mondal and port, 1995). the present study is aimed to assess the abnormalities produced in flour beetles tribolium castaneum (herbst) and t. confusum duval due to the activities of cyromazine and pirimiphos-methyl alone or in combination, using low doses of both the compounds for protection of the stored products destined for human consumption. materials and methods insects used laboratory strains of tribolium castaneum and tribolium confusum were used in the present experiment. the insects were collected from the ipm laboratory, institute of biological sciences, rajshahi university. the stock cultures of these species were maintained in the laboratory for the last 15 years. compounds used the tested compounds were a chitin synthesis 97 mailto:salehbgd@yahoo.com table 1 larval deformities produced by cyromazine or pirimiphos-methyl alone and by their combination in t. castaneum (n = 250) treatment (ppm) pupal recovery (%) no. of abnormal larvae no. of larvalpupal intermediates total no. of abnormal larvae (%) control (0) 241 (96.40) cyromazine 10 20 30 228 (91.20) 215 (86.00) 197 (78.80) 09±0.23a 14±0.04b 17±0.31b 04±0.64a 07±0.5a 09±0.33ab 13±0.42 (5.20)a 21 ± 0.67 (8.40)b 26±0.34 (10.40)bc pirimiphos-methyl 0.1 0.2 0.4 230 (92.00) 196 (78.40) 180 (72.00) 07±0.41a 15±0.36b 19±0.1b 00 04±0.06a 05±0.72a 07± 0.48 (2.80)a 19 ± 0.39 (7.60)b 24±0.05 (9.60)b cyromazine + pirimiphos-methyl 10 + 0.1 190 (76.00) 16±0.7 08±0.48 24±0.55 (9.60)b note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) inhibitor, cyromazine and an organophosphorous insecticide pirimiphos-methyl. cyromazine was kindly supplied by the ciba geigy as larvedex ca 98.4% wp formulation. pirimiphos-methyl was purchased from the local agrochemical shop. doses used doses were prepared by mixing required amount of each of the compound and mixed with standard food medium (19:1, whole wheat flour: brewers’ yeast) to obtain the doses in ppm unit. the doses prepared for cyromazine were 10, 20 and 30 ppm and those for pirimiphos-methyl were 0.1, 0.2 and 0.4 ppm. the single combined dose used was prepared as 10 ppm cyromazine + 0.1 ppm pirimiphos-methyl (lowest doses of both the compounds). the doses of cyromazine and pirimiphos-methyl chosen were based on the mortality tests of the compounds on the larvae of t. castaneum and t. confusum (kamaruzzaman et al., 1999; kamaruzzaman, 2000). experimentation newly hatched larvae (12 h old) of both species were collected separately from sub-cultures of the beetles. the larvae were released in food medium treated with different doses of either cyromazine or pirimiphos-methyl or cyromazine + pirimiphosmethyl. the larvae were reared up to pupation. after every three days the food material was replaced by a fresh one treated with same dose and compound. the pupal recovery (%) was recorded. the pupae were sexed according to halstead (1963) and kept separately for emergence of adults. adult recovery (%) was recorded. the morphologically abnormal individuals were separated. the deformed characters were studied, and the number of abnormal individuals was counted for each life stage. a set of control larvae was reared similarly on untreated food. fifty larvae of each species were used for each treatment, each dose and control. the experiments were carried at 30±1 0c in an incubator, without controlling light and humidity, and replicated five times. statistical analyses the abnormalities produced by different treatments alone or in combination and in different species of tribolium were tested for significance using analysis of variance (anova). the effect of the different doses of each treatment with respect to control was tested with duncan’s multiple range test (dmrt). results abnormal characters produced cyromazine treatment produced various types of abnormalities in the larvae and adults of both species. abnormalities produced by cyromazine were recorded as follows: a) larval abnormalities i) reduced body size, ii) swelling in the integument/cuticular lesions, iii) stiffness of the cuticle, iv) incomplete metamorphosis: larviform pupae, pupal head with larval body, and pupa with larval skin. 98 table 2 larval deformities produced by cyromazine pirimiphos-methyl alone and by their combination in t. confusum (n = 250) treatment (ppm) pupal recovery (%) no. of abnormal larvae no. of larvalpupal intermediates total no. of abnormal larvae (%) control (0) 240 (96.00) cyromazine 10 20 30 230 (92.00) 221 (84.40) 209 (83.60) 10±0.88a 15±0.53ab 21±0.55b 05±0.72a 06±0.6a 08±0.46a 15±0.49 (6.00)a 21±0.55 (8.40)b 29±0.32 (11.60)c pirimiphos-methyl 0.1 0.2 0.4 227 (90.80) 198 (79.20) 195 (78.00) 08±0.52a 12±0.62ab 14±0.55b 00 05±0.46a 06±0.31a 08±0.51 (3.20)a 17±0.33 (6.80)b 20±0.05 (8.00)bc cyromazine + pirimiphos-methyl 10 + 0.1 200 (80.00) 15±0.43b 07±0.06a 22±0.71 (8.80)bc note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) b) adult abnormalities i) bent abdomen, ii) incomplete elytra. pirimiphos-methyl alone or in combination with cyromazine produced a similar type of deformities in both species, as follows: i) reduced larval body size, ii) larval-pupal intermediates, iii) adultoids and incomplete elytra in adults. the abnormal individuals with incomplete metamorphosis at larval-pupal transformation were categorized as larval abnormality and the adultoids as pupal abnormality. percentages of abnormalities produced cyromazine produced a higher percentage of abnormal larvae in t. confusum than t. castaneum (tables 1, 2). effect of cyromazine on the normal growth of larvae was found to be dose related (p < 0.001, f = 87.219), and was similar in both species (p > 0.05, f = 0.981). the number of abnormal larvae was higher than the number of larval-pupal intermediates, in both species of tribolium. pirimiphos-methyl produced comparatively less number of abnormal larvae in both species (p > 0.05, f = 1.31), though the effect varied with the doses (p < 0.001, f = 84.283) (tables 1, 2). the combined effect of the igr and the insecticide produced a greater effect on the morphogenesis of the larvae of both species (p > 0.05, f = 0.4). the effects of the combined treatment was more than the effects recorded with the lower doses of either cyromazine or pirimiphosmethyl (p < 0.001, f = 211.6) (tables 1, 2). cyromazine produced similar abnormal adults at same extent in both sexes of t. castaneum (table 3), whereas the effect was slightly greater in the females of t. confusum (table 4). the percentages of abnormal adults produced were not significant between the species (p > 0.05, f = 0.067); but significant differences of the effect was observed between the doses in t. confusum (p < 0.05, f = 9.25). pirimiphos-methyl treatments also produced similar percentage of adult abnormalities in both the species (p > 0.05, f = 0.72), which did not vary between the sexes of t. castaneum (table 3), but the females were more affected than the males of t. confusum (table 4). the adult morphogenesis of the beetles slightly varied among the doses (p < 0.01, f = 10.47). the combined treatment of cyromazine (10 ppm) and pirimiphos-methyl (0.1 ppm) produced a greater effect on the adult morphogenesis than the single treatment with either cyromazine or pirimiphos 99 table 3 adult abnormalities produced by cyromazine or pirimiphos-methyl alone and by their combination in t. castaneum (n = 250) no. of abnormal individuals (%) treatment (ppm) total adult recovery (%) male (no.) female (no.) male female control (0) 237 (94.80) 115 122 cyromazine 10 20 30 218 (87.20) 204 (81.60) 188 (75.20) 117 109 100 101 95 88 09±0.06 (7.69)a 09±0.2 (8.26)a 09±0.11 (9.00)a 06±0.21 (5.94)a 07±0.13 (7.37)a 12±0.31 (13.64)a pirimiphos-methyl 0.1 0.2 0.4 209 (83.60) 189 (75.60) 168 (67.20) 115 95 96 94 94 72 08±0.21 (6.96)a 09±0.05 (9.47)a 09±0.21 (9.37)a 07±0.07 (7.45)a 09±0.23 (9.57)a 13±0.15 (18.05)b cyromazine + pirimiphos-methyl 10 + 0.1 185 (74.00) 107 78 19±0.25 (17.76)b 15±0.22 (19.23)b note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) methyl at the same dosage (tables 3, 4). the combined treatment produced similar effects in both species of tribolium (p > 0.05, f = 0.034). discussion the mode of action of cyromazine is different from that of pirimiphos-methyl. being a chitin synthesis inhibitor, cyromazine affects the mechanical properties of the insect cuticle and produces abnormalities in the skin, and resists moulting (fox, 1990). inhibition of moulting results in increase of the internal body pressure in the larvae (fox, 1990), producing swellings on the cuticle (cuticular lesions) (kotze and reynolds, 1993; sirota et al., 1993; sirota and grafius, 1994). so, larval deformities are common in csi treated insects. the abnormal characteristics as noted in the present experiment, have been also reported in cyromazine treated colorado potato beele (sirota and grafius, 1994) and triflumuron (a csi compound) treated t. castaneum (parween, 1998). due to csi activity insect cuticle often becomes stiff (fox, 1990). consequently feeding is often hampered, as reported by neuman and guyer (1988), soltani (1984) and parween (1996) in different insects. even is some could survive, the starved larvae were reduced in body size. pirimiphos-methyl had been reported to avoid feeding on the treated medium tribolium species, and as a result adults loose weight and length (mondal, 1984b; rahman, 1992). pirimiphos-methyl affected the larval-pupal transformation and produced intermediate forms along with adult abnormalities. both cyromazine and pirimiphos-methyl affected growth of the emerged adults, which were superficially normal but with deformed elytrae. the combined action of cyromazine and pirimiphos-methyl to some extent was greater on the morphogenesis of tribolium species, than the action of single treatment of either compound. all the abnormal larvae and larval-pupal intermediates failed to survive long, and the abnormal adults were found to be uncapable to mate or oviposite. cyromazine is easily miscible with most of the standard insecticides and fungicides (fox, 1990), and in the present study was found to reduce the doses of insecticide used, producing the same effect. so, it can be used combined along with traditional insecticides at minimal doses for the protection of the stored products against beetle infestation, which may produce less stress on the environment and human health. both t. castaneum and t. confusum have become resistant against most of the insecticides used. for insect management in the grain and cereal stores, high doses of these insecticides are needed, which affect the human health, the environment and its biota. to overcome this problem, cyromazine could be used with pirimiphos-methyl against tribolium species, since this association would be able to produce abnormal individuals. the abnormal larvae or adults either would die or fail to 100 table 4. adult abnormalities produced by cyromazine or pirimiphos-methyl alone and by their combination in t. confusum (n = 250) no. of abnormal individuals (%) treatment (ppm) total adult recovery (%) male (no.) female (no.) male female control (0) 237 (94.80) 116 121 cyromazine 10 20 30 219 (87.60) 207 (82.80) 197 (78.80) 114 110 105 105 97 92 08±0.21 (7.02)a 08±0.03 (7.27)a 11±0.21 (10.48)b 06±0.04 (5.71)a 10±0.22 (10.31)b 14±0.5 (15.22)bc pirimiphos-methyl 0.1 0.2 0.4 221 (88.40) 194 (77.60) 188 (75.20) 116 101 102 105 93 86 06±0.06 (5.17)a 10±0.22 (9.90)b 14±0.22 (13.72)bc 10±0.15 (9.52)b 11±0.21 (11.83)b 10±0.03 (11.63)b cyromazine + pirimiphos-methyl 10 + 0.1 195 (72.78) 103 92 21±0.15 (20.39)c 11±0.3 (11.96)b note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) develop further or to reproduce, and ultimately the population would be controlled. moreover, as very low doses of these compounds could be used, the residues may not create hazard to the stored commodities as well as to the environment. references awad ti, mulla ms. morphogenic and histopathogenic effects induced by the insect growth regulator cyromazine in musca domestica (diptera: muscidae). j. med. entomol. 21: 419-426, 1984. bloomcamp cl, patterson rs, koehler pg. cyromazine resistance in the housefly (diptera: muscidae). j. econ. entomol. 80: 352-357, 1987. el-oshar ma, motoyama n, hughes pb, dauterman wc. studies on cyromazine in the housefly, musca domestica (diptera: muscidae). j. econ. entomol. 78: 1207-1213, 1985. fox p. insect growth regulators. pjb punl. ltd., richmond, uk, 1990. hajjar np. chitin synthesis inhibitors as insecticides. in: hutson dh roberts tr (eds), insecticides, john wiley & sons., new york, pp. 275-310, 1985. halstead tjh. external sex differences in stored products coleopteran. bull. entomol. res. 54: 119-134, 1963. kamaruzzaman ahm. effect of cyromazine and pirimiphos-methyl on tribolium castaneum herbst and tribolium confusum duval. ph.d. thesis, university of rajshahi, bangladesh, 2000. kamaruzzaman ahm, mazid ma, parween s, mondal kamsh, islam w. dose-mortality responses of the flour beetles to triflumuron and cyromazine. tribolium inf. bull. 39: 298-307, 1999. khan ar. the combined action of organophosphorous insecticides and microsporidians on tribolium castaneum herbst. ph.d. thesis, university of newcastle upon tyne, 1981. kotze ac, reynolds se. effect of cyromazine on the mechanical properties of the larval integument of luciola cuprina (diptera: calliphoridae). bull. entomol. res. 83: 389-393, 1993. mondal kamsh, port gr. effect of cyromazine on larval growth and adult population of susceptibla and malathion resistant strains of tribolium castaneum herbst. j. biol. sci. 3: 1-10, 1995. mondal kamsh. effects of methylquinone, aggregation pheromone and pitrimiphos-methyl on tribolium castaneum herbst larvae. ph.d. thesis, university of newcastle upon tyne, 1984a. mondal kamsh. repellent effect of pirimiphosmethyl to tribolium castaneum herbst. inter. pest control. 26: 98-99, 1984b. nelson frs, holloway d, mohamed aka. a laboratory study of cyromazine on aedes aegypti and culex quingfasciatus and its sensitivity on selected predators of mosquito larvae. j. am. mosq. control assn. 2: 296-299, 1986. neumann r, guyer w. a new chitin synthesis inhibitor cga 112 113: its biochemical mode of action as compared to diflubenzuron. proc. 10th 101 int. congr. plant prot. brighton, pp. 445-451, 1988. parween s. distribution and food consumption of larval and adult tribolium castaneum herbst on baycidal treated medium. j. biol. sci. 4: 113119, 1996. parween s. symptoms of triflumuron in toxicication in larvae of tribolium castaneum herbst (coleoptera: tenebrionidae). tribolium inf. bull. 38: 268-270, 1998. rahman asms. combined action of pirimiphosmethyl, synthetic methyl quinine and botanicals on tribolium confusum duval. ph. d. thesis, university of rajshahi, 1992. sher j, plapp fw. cyromazine resistance in the house fly (diptera: muscidae). genetics and cross resistance to diflubenzuron. j. econ. entomol. 83: 1689-1697, 1990. sirota jm, grafius e. effects of cyromazine on larval survival, pupation and adult emergence of colorado potato beetle (coleoptera: chrysomolidae). j. econ. entomol. 87: 577582, 1994. sirota jm, grafius e, ferrari b, kolarik p, seriber b,simstead s, boylan-pett w. control of colorado potato beetle with trigard. acarucide tests. 18: 155-156, 1993. soltani n. effects of ingested diflubenzuron on the longevity and the peritrophic membrane of adult mealworms (tenebrio molitor l.). pestic. sci. 15: 221-225, 1987. stall gb. insect growth regulators with juvenile hormone activity. ann. rev. entomol. 20: 417460, 1975. 102 introduction materials and methods insects used compounds used doses used experimentation results abnormal characters produced references prof isj 6: s3-s8, 2009 issn 1824-307x review professor giuseppe reverberi and the ascidian school in palermo f de bernardi department of biology, university of milan, milan, italy accepted march 13, 2009 giuseppe reverberi was born near perugia on 1901. after being ordained as a priest, he graduated in theology in 1924 from the pontificio ateneo lateranense. in the same year, he went to the university of rome, where he took another degree in natural science in the lab directed by federico raffaele, working on amphibian and chick embryos, under the guidance of professor pasquale pasquini. after the degree, he became an assistant at the zoological institute where started his studies in the field of experimental embryology, carrying out researches on the chick embryo-eye, on the centrifugation of the amphibian egg and on the potentialities of the amphibian tail bud: these researches were then continued in palermo by some of his collaborators. in 1930 he qualified to teach general embryology and became professor of experimental psychology and biology at the ateneo lateranense. in 1939 he was appointed at the university of perugia as director of the zoological institute. in the meantime, he worked at the zoological station of naples, a great attraction pole of international level for marine biology and a breeding ground of scientists. here he became director of the biological center of the national council for research and started to study the first stages of development of the ascidian embryos. in 1948, as a winner of a public competition, he was appointed as professor of zoology at the university of palermo, where he was director of the zoological institute and professor of biology at the medical school. in 1957 he founded the magazine acta embryologiae et morphologiae experimentalis, a latin title for an international journal published in english, the first in italy with an international editorial board. it represented a person whose broad knowledge was not limited to biology, but also covered philosophy, literature and theology. his researches, together with those carried out by the many research groups led by his collaborators were addressed essentially to developmental biology of several animals, overall marine invertebrates: annelids, ___________________________________________________________________________ corresponding author: fiorenza de bernardi department of biology university of milan via celoria 26, 20133 milan, italy e-mail: fiorenza.debernardi@unimi.it mollusca (dentalium) ctenophora, amphioxus and ascidians. in 1971 he published a fundamental textbook experimental embryology of marine and fresh-water invertebrates (north holland) which received a lot of quotations. for the above mentioned reasons, i wish to speak about the monumental scientific activity of professor reverberi, which lasted more than 50 years, and of his outstanding human personality. unfortunately, i did not have the honour to be one of his pupils, but i can remember very well two meetings. the first one was in 1968, at the zoological station of naples, during an experimental embryology course for young researchers: his tall figure, full of charisma, his availability to meet young people coming from different european countries for a dinner party. he was there with the teachers of the school, all the most outstanding embryologists of the 20th century: john runnstrom, sven horstadius, jean brachet, tryggve gustafson, gerhard czihak. i was very impressed by the friendly terms between professor reverberi and all those distinguished embryologists. it was a very important signal of his international reputation in a period (40 years ago) when not all the italian universities used to be so well-known abroad. i remember the meeting of the italian embryological group in 1977, an atypical club born in 1952 from the idea of some italian scientists on the model of the institut international d’embryologie (iie). the founders were pasquale pasquini, silvio ranzi, alberto monroy, alberto stefanelli, mario benazzi and, obviously, reverberi himself, who was a member of the iie. during this meeting, professor reverberi gave a lecture on the latest results and the perspectives of the ultrastructure and the cellular biochemistry of developing embryos, in which he compared and discussed the most recent results obtained in the palermo lab and in many other labs all over the world . i was very impressed by the fervour of his speech about the communication between nucleus and cytoplasm, one of his favourite subjects. ten years later, in 1987, i entered the institute of zoology in the historical place of via archirafi 18: this building was still characterized by his strong personality, even if his illness kept him away for many years. in fact, most researchers were working on ascidians and this was the demonstration that a real school had been created there; a place where the s3 fig. 1 prof. giuseppe reverberi at work in his room at the institute of zoology of palermo in 1965. same animal model was studied under manifold point of view. this fact impressed me very much as i was coming from the zoological institute of milan, where the researches used to be more heterogeneous. it was then inevitable to be overwhelmed by the contagious enthusiasm of his first collaborator and outstanding successor, giuseppina ortolani, for the manipulation of the embryos and eggs of the ascidians: one day i told her the technical troubles encountered when highlighting some rna markers in opaque xenopus embryos, and she said to me:“i worked a lot with xenopus eggs: my advice is to work with phallusia, which has big, transparent and wonderful eggs. try it and you’ll see!” so, i have been working for 20 years with ascidian embryos and i am feeling, even indirectly, an alumna of the famous ascidian school of palermo! reverberi’s scientific activity in palermo mainly involved the experimental embryology field, but the development of ascidian eggs always gained first priority among his interests. in the following sections i will try to define the main research lines concerning the ascidian development. development of egg fragments and isolated blastomeres when reverberi started to work on the development of ascidians, the current information on such matter was that developed by conklin, who wrote in the conclusion of his famous paper published in 1905: “the development of ascidians is a mosaic work because there are definitely localized organ-forming substances in the egg; in fact the mosaic is one of organ-forming substances rather than of cleavage cells. the study of ctenophores, nemertines, annelids, mollusks, ascidians and amphibians (the frog) shows that the same is probably true of all these forms and it suggests that the mosaic principle may apply to all animals.” it is interesting to follow the evolution during the time in which reverberi formed his opinion about the mosaicism based on experimental results. in one of his first papers, in 1931, reverberi describes the results obtained from the fertilization of egg fragments obtained by gentle crushing or by a small incision of the chorion. the fragments until 1/22 collected by a needle tip developed “as a whole” and gave normal larvae. his conclusion was “the ciona egg has to be classified among the regulative eggs” (reverberi, 1931). 1933: blastomeres separated at stages 2,4,8: the animal 4/8 developed blastulae; the vegetal 4/8 developed also gastrulae. “the cleavage is strictly partial (=incomplete), even if some aspects could make suspect of a regulation” (reverberi, 1933). 1936: “the partial cleavage appears only from the two blastomere stage onwards…; before this stage in fertilized egg the cleavage is of total type” (reverberi, 1936). in a frequently quoted paper, even if published only in italian (reverberi and minganti, 1946), we can find some very beautiful drawings, much more explanatory than modern photos. such drawings showed the results of the selective removal of two s4 blastomeres each time at 8-cell stage and the histological sections of the larvae obtained from the combination of the animal quartet with the two vegetal anterior blastomeres. this was the first paper unambiguously proving the existence of a “brain evocator” in the two vegetal anterior blastomeres and of a “ brain inhibitor” in vegetal posterior blastomeres. these figures became well known as reported in the book of nori satoh “developmental biology of ascidians” (1994), a textbook widely diffused in the world of ascidian researchers. in another paper of the following year (reverberi and minganti, 1947), the systematic combinations of blastomeres were described and it was shown that vegetal anterior blastomeres do not “induce” any presumptive epidermis to become brain, but they only “evocate” the neural presumptive ectoderm to become brain. these works were continued and reconsidered with other collaborators in the following years. they used the knowledge obtained in the meantime by ortolani about the cell-lineage of the single blastomeres until 64-cell stage and systematically removed the cells committed to give notochord or endoderm from stage 8 to 64. the single cells obtained were then transplanted under an isolated animal quartet. this great bulk of experiments, singularly evaluated, definitely showed that “the formation of the neural system in the ascidians is strictly directed by the same laws which spemann discovered in the amphibians. only slight differences are to be noticed between the inductive system in the amphibian and in the ascidians: in the amphibians, the inductor is chorda-mesoderm, in the ascidians it is (part of) the chorda-entoderm”. the observation that in the ascidians the inducing power of the chorda-endoderm was more restricted than in the amphibian led to support the idea of the “evocative” power instead of an “inducing” power, enclosed in a bright discussion among the embryologists at that time. these results were reported in another frequently quoted paper “the causal formation of the brain in the ascidian larva” (reverberi et al., 1960). it is to be noted that this discussion is coming close to a solution only in recent years, after the genome sequencing and the knowledge of the sequences involved in determination of embryonic territories. development of sense organs at the same time of the study on the induction of the nervous system and in the same papers was also studied the development of the principal sense organs: the otholith, the ocellus and the palps. a very elegant result was the cytochemical detection of the dopa oxidase, the enzyme activating the melanin precursor, only after the induction process at neurula stage. twenty years later, the problem of the sense organ development was resumed and in two papers was described the ultrastructure of the so-called “third sense organ”, probably dedicated to the hydrostatic pressure detection (reverberi, 1975, 1979). this organ is formed by a few cells bearing bulbous projections projected inside the cavity of the sensory vesicle, it was observed in many species and its function was related to the different ability of the larvae to move vertically along the water column. this topic was also innovative at that time: in fact the third organ was recently studied in several labs, and other hypotheses were developed for its function (e.g., neurosecreting cells or secondary sensorial neurons, imai and mainhertzhagen, 2007), but no hypotheses were considered more convincing than the hypothesis originally proposed by reverberi. causal analysis of embryonic development in a paper of 1939, published in commentationes pontificiae academiae scientiarum, with a latin abstract, reverberi resumed the experiments of fertilization of egg fragments obtained by centrifugation of the egg. the centrifuged eggs were divided in two parts, one completely hyaline and the other full of granules. only the latter, after fertilization, can develop even into larval stage, but the hyaline fragment cannot start any cleavage. from these observation, reverberi inferred that the construction of an organism starting from the zygote is the result of an ordered series of biochemical events. during the development of the ascidian egg the different constituent of the egg become more strictly segregated (plasm segregation) (reverberi and pitotti, 1939). the further development of these researches, carried out with collaborators, led to the cytochemical study of the segregation of rna and dna. in a review of 1960, published in advances in morphogenesis, reverberi states to trust the evidence that differentiating tissues have a higher concentration of rna (e.g., neural tissue at neurula stage), but he is sceptical about a different concentration of dna. in fact, at that time, the dispute about the constancy of the dna content was still going on. reverberi suggested that “the strong coloration of the nuclei of the nervous cells is not necessarily due to a higher content of dna, but, more likely, to the fact that the nuclei are more condensed”. interest was also shown in the mitochondrial respiratory enzymes. through a very accurate nadireaction and through various cytochemical reactions, it was observed that many enzymes accumulate following mitochondrial segregation in the yellow crescent, in vegetal posterior blastomeres, and in the musculature of the larva (reverberi, 1957a). the role of the enzymes in ascidian morphogenesis was established by blocking their activity with specific inhibitor. the main results were to ascertain a causal relation between the enzymatic activity and the differentiation of the muscular system, rich in mitochondrial enzymes. in fact the larvae obtained from the treatment with inhibitors showed a normal trunk, and a tail with various anomalies, all referred to a defective differentiation of the muscular fibers (reverberi, 1957b). also related to the previous ones, were the results of the cytochemical reaction for cholinesterase which can be noticed at neurulation not in the neural plate, as previously reported by other researchers, but in the muscular territories. s5 after the publication of these pioneer results (durante, 1956), the acetylcholinesterase as a marker of muscular differentiation was used by many authors in many labs (e.g., in usa by r whittaker, who visited several times the zoological institute in palermo, and in japan by n satoh). heterospecific hybridizations a lot of work was dedicated by reverberi to the heterospecific hybridizations. the results highlighted that, in order to obtain heterospecific fertilization, the eggs have to be naked, without chorion and follicular cells. the latter not only favour the floating of the eggs, but have also a function in speciesspecific recognizing.. the viable hybrid andromerogons (an enucleated egg of ascidia malaca fertilized with a sperm of phallusia mammillata) have only one set of paternal chromosomes. the morphological characters of the larva are, however, matroclinous, as the cytoplasm was “conditioned” by its nucleus much earlier, perhaps in the ovary. it must be noted that similar results have been observed in other labs on echinoderms. some studies carried out from 1955 to 1960 tried to explain this phenomenon by developing the embryos in sea water additioned with thymidin, adenine etc, in order to identify possible errors in the syntesis of dna and rna. but these experiments were too advanced at that time and the transcriptomic and proteomic were still far. very advanced were also the experiments on the fusion of the eggs of the same species or of two different species and subsequent fertilization with an heterospecific spermatozoon: giant triploid larvae were obtained and some time these larvae metamorphosed in juveniles (farinella et al., 1969). in order to better understand the great interest of reverberi for these studies it is important to remember that three chapters of his textbook “introduzione all’embriologia sperimentale” (1967), are dedicated to the heterospecific hybridizations. all the problems connected with the suppression of one of the two sets of chromosomes or their maintenance and integration, the “conditioning” of the diploid nuclei from differentiated cells and transplanted in the eggs, were debated in the sixties and had an intellectual appeal that can be grasped in the textbook. it is clearly resumed in the definition “the egg is an equipotential armonic system”. in the first chapter of the above mentioned book, entitled “birth and development of the embryology” a complex and fascinating historical account is outlined with clarity of style and the numerous latin quotations demonstrate that reverberi was a very cultured and intellectual person. effects of extraneous substances through a fusion of the two main research fields, the classical experimental biology and the chemical embryology, reverberi also tried a new way of manipulating the embryos: instead of using the glass needles, he used some chemical substances. the first of these new research lines on ascidian embryos was carried out with a classical, historical substance: lithium chloride (farinella ferruzza, 1955). treatment of unfertilized eggs produced normal larvae, but a treatment at stage 2 blastomeres gave rise to larvae lacking the trunk and all ectodermal derivatives, like nervous system and palps. the treatment at stage 64 gave apparently normal larvae, but deprived of nervous system and palps. the well known “vegetalizing” action of lithium chloride produced the absence of the neural induction (reverberi and farinella ferruzza, 1961). this research line was further developed and extended by some collaborators who also studied the effects of the environmental pollutants. another research line was the treatment of the embryos with chromomycin, actinomycin d or with aminoacids and their analogs, in order to modify the quality of the aminoacid pool leading the embryos to produce abnormal proteins. in all these cases a normal development was obtained when the treatment was carried out before the first cleavage. treatments in further stages until gastrula produced larvae with various degree of abnormalities (bramachary and reverberi, 1964). the results obtained by reverberi, together with the other results obtained in those years on amphibians, sea urchins and chickens were fundamental to establish that the transcription of embryonic rna starts at the blastula-gastrula stage and that the ascidians, even if traditionally considered examples of the mosaic development, “have many regulative aspects” as reverberi already wrote in 1932. the ascidian embryo ultrastructure in the fifties, electron microscopy was still in a pioneer phase when the first papers on the ultrastructure of the oocytes, of the unfertilized egg and of follicular and test cells were published. vincenzo mancuso and his collaborators worked actively in this research line. bearing in mind the lines of the previous researches, we shouldn’t be surprised that the electron microscopy was utilised to study the fine structure of the egg and of its constituents separated by centrifugation, in order to study directly what was inferred by cytological and cytochemical methods (reverberi and mancuso, 1960; mancuso, 1963). once established the unequal distribution of principal constituents of the cytoplasm, their segregation was observed during the cleavage in lineage of blastomeres defined by their morphogenetic commitment. in particular, the cell-lineage of the muscular cells, already outlined in the experiments of ortolani, was confirmed by the electron microscopy, from the richness in mitochondria of the committed blastomeres . i would like to mention a series papers on some “peroxidase cells”, so identified by ries through cytochemical reactions. these cells are present in two rows, between the ventral surface of the pharynx and the epidermis, in the mature larvae only in the species carrying a heavy tunic, such as a. malaca, p. mammillata, ascidiella aspersa. they have been considered of mesodermal origin, but materazzi and ortolani (1969) demonstrated that s6 they develop from the anterior vegetal blastomeres, an endodermal territory. these cells cross the ectoderm and join the tunic, where are probably active in the synthesis of mucopolissaccharids of the tunic. reverberi reported the electron microscopy observation of these cells, just sorted from the endoderm and rich in golgi apparatus and vesicles (reverberi, 1971). one of my first papers on the ascidians, carried out with the enthusiastic advice of ortolani, was about the cytoskeletal modifications of these “button cells” studied by immunofluorescence and confocal microscope while they penetrate the ectoderm (sotgia et al., 1993). immunobiology ascidian immunobiology has recently become very important in the department of animal biology in palermo but, surprisingly there are no studies on this matter in the papers of reverberi. precisely, this research line began with an idea of nicolò parrinello who decided to carry out the study on serum proteins through immunological techniques in order to solve some taxonomy problems. professor reverberi understood immediately the potentiality of this line and encouraged him to continue. in fact, one of the duties of a real master is also to support self-esteem and to encourage autonomy in young researchers! naturally, speaking of reverberi involves mentioning his collaborators and his school as well. in fact, all his students soundly developed their analytical skills thanks to their master, following and sometimes being ahead of the new research orientations suggested by the technical progress in biological research. in 2003, during the first international urochordate meeting, nori satoh, in the opening lecture “let’s move on ascidian biology with new ideas”, made an historical review on the ascidian researches and recalled the major scientists: chabry (1887), conklin (1905), reverberi (1931) and his collaborators ortolani and minganti. the meaning of this historical parade was clearly explained during the lecture: the school of palermo was leader in the experimental embryology for more than 50 years, mainly studying the ascidian development: its heraldic animal. references brahmachary rl., reverberi g. on the action of chromomycin on the eggs and embryos of ciona intestinalis. experientia 20: 621623,1964. conklin eg. mosaic development of ascidian eggs. j. exper. zool. 2: 146-223, 1905 durante m. cholinesterase in development of ciona intestinalis, experientia 12: 307-310, 1956 farinella ferruzza n. lo sviluppo embrionale delle ascidie dopo trattamento con licl pubbl. staz. zool. napoli 26: 42-54, 1955 farinella ferruzza n, reverberi g, single giant larvae of ascidia malaca double eggs. experientia 25: 651-652, 1969. imai jh, meinertzhagen ia. neurons of the ascidian larval nervous system in ciona intestinalis: i. central nervous system. j comp neurol 501: 335-352, 2007 mancuso v. distribution of the components of normal unfertilized eggs of ciona intestinalis examined at the electron microscope . acta embryol. morphol. exper. 6: 260-274, 1963. mancuso v. an electron microscopy study of the test cells and follicle cells of ciona intestinalis during oogenesis. acta embryol. morphol. exper. 8: 239-251, 1965. materazzi g, ortolani g. a study of the origin of the cells containing sulphated acid mucopolysaccharides in the cephalic portion of the larvae of phallusia mammillata and ascidia malaca. dev. biol. 20: 378-385, 1969. reverberi g. studi sperimentali sull’uovo di ascidia.i il comportamento e lo svilppo dei frammenti d’uovo di ciona nei diversi momenti compresi tra la deposizione e la segmentazione. pubbl. staz. zool. napoli 11: 170-193, 1931. reverberi g. studi sperimentali sull’uovo di ascidia. ii la segmentazione dei blastomeri isolate dell’uovo di ciona e di phallusia. pubbl. staz. zool. napoli 12: 385-406, 1933. reverberi g. la segmentazione dei frammenti dell’uovo non fecondato di ascidia. pubbl. staz. zool. napoli 15: 198-216, 1936. reverberi g. the embryology of ascidians. in adv. morphogen. 1: 55-101, 1960. reverberi g. introduzione all’embriologia sperimentale feltrinelli, milano, 1967. reverberi g. la citocromo-ossidasi lungo lo sviluppo delle ascidie.risultati che conseguono alla sua inibizione con azide sodico. pubbl. staz. zool. napoli 29:187-212, 1957a. reverberi g. some effects of enzymes inhibitors on the ascidian development . acta embryol. morphol. exper. 1: 12, 1957b. reverberi g. experimental embryology of marine and fresh-water invertebrates, north holland publ.co., 1971. reverberi g. osservazioni sulle cellule “a perossidasi” di alcune larve di ascidia col microscopio elettronico rend. acc. naz. lincei 50:795-798, 1971. reverberi g. on the third sensory receptor of ascidian tadpole. rend. acc. naz. lincei 58: 948-954, 1975. reverberi g. on the third sensorial organ of ascidian tadpoles., acta embryol. morphol. exper. 91-99, 1979. reverberi g, farinella ferruzza n. lithium ions and brain formation in the ascidian larvae. acta embryol. morphol. exper. 4: 239-249, 1961. reverberi g, mancuso v. the constituents of the egg of ciona intestinalis as seen at the electron microscope. acta embryol. morphol. exper. 3: 221-230, 1960. reverberi g, minganti a. fenomeni di evocazione nello sviluppo dell’uovo di ascidie. pubbl. staz. zool. napoli 20: 201-252, 1946. reverberi g, minganti a. la distribuzione delle potenze nel germe di ascidie allo stadio di otto blastomeri, analizzata mediante la combinazioni e i trapianti di blastomeri. pubbl. staz. zool. napoli 21:1-35, 1947. s7 reverberi g, ortolani g, farinella ferruzza n. the causal formation of the brain in the ascidian larva. acta embryol. morphol. exper. 3:296336, 1960. satoh n. developmental biology of ascidians. cambridge university press, cambridge, uk, 1994. sotgia c, fascio u, ortolani g, de bernardi f. behavior of endodermal “button cells” during metamorphosis of ascidian larvae. int. j. dev. biol. 37: 547-553, 1993. reverberi g, pitotti m. differenziazioni fisiologiche nell’uovo delle ascidie commentationes pont. acad. scientiarum 3: 471-488, 1939. s8 isj 3: 111-xx, 2006 isj 4: 13-17, 2007 issn 1824-307x short communication the effect of bmnpv infection on protein metabolism in silkworm (bombyx mori) larva k etebari 1, l matindoost 1, sz mirhoseini 1, mw turnbull 2 1dept. of sericulture, faculty of natural resources, university of guilan, somehe sara 1144, iran 2department of entomology, soils, and plant sciences, clemson university, 114 long hall, clemson, sc 296340315, usa accepted february 02, 2007 abstract grasseri is one of the most important diseases of silkworm with significant yield loss, which is caused by nuclear polyhedrosis viruses (npv). in the present research the effect of this disease on changes of biochemical compounds which are related to protein metabolism in 5th instar larvae were studied. the larvae that showed the grasseri symptoms after contamination with 5.5×10-4 polyhedral/ml were assumed as infected treatment. the hemolymph of infected and uninfected larvae in 3 and 5 days after 4th molting were collected and its total protein, urea, alanine aminotransferase (alt) and aspartate aminotransferase (ast) were measured. the results showed that the amount of all the compounds except urea were considerably different in both groups. total protein had decreased in infected larvae but activity level of two aminotransferases significantly increased. therefore, grasseri has a considerable effect on protein metabolism. key words: silkworm; nuclear polyhedrosis virus; protein metabolism; grasseri __________________________________________________________________________________________ introduction silkworm is susceptible to many diseases caused by viruses and this is one of the main problems in sericulture every year. generally, 70 % of damages caused by diseases are due to viruses (anonymous, 1976). among viruses, nuclear polyhedrosis viruses (npv) in recent years have caused the highest damage to silkworm (bombyx mori) in tropical regions (sivaprasad et al., 2003; biabani et al., 2005 a, b). bmnpv affect midgut epithelial cells, trachea system, hemolymph cells, fat body, etc. the nuclear of middle and inner cells of silk gland are also sometimes invaded by this virus (khurad et al., 2004). baculovirus infection starts when inclusion body is taken in by the sensitive insects. midgut fluid of lepidopteran larvae is totally alkali which digests the viral occlusion bodies. consequently, virions are released into alimentary system and cross the peritrophic membrane. they combine with midgut ___________________________________________________________________________ corresponding author: kayvan etebari dept. of sericulture, college of natural resources, university of guilan, somehe sara 1144, iran e-mail: etebari@guilan.ac.ir epithelial cells and enter nuclei starting the first cycle of viral production and replication. these processes cause many biochemical changes in larvae which respond to these biological phenomena with changing many of its metabolisms to defend against pathogen invasion. understanding and identifying these biochemical changes will be very important in discussing many biological stresses. additionally, silkworm and bmnpv make a good model for studying the interaction between insect and virus. so, determination of the biochemical responses in silkworm against bmnpv could facilitate the control of agricultural pests (gao et al., 2006). biochemical compounds of silkworm have been investigated under many stress conditions as an appropriate marker. serratia marcescens as one of the factors causing flasheri disease has been studied for the biochemical changes in silkworm hemolymph after infection and considerable decrease has been reported in total protein, carbohydrate and lipid (sam devdas et al., 1994). rami reddy et al. (1992) analyzed the effects of usi fly (exorista sorbillans) parasitism on activity levels of aminotransferases and showed that stress increases the activity of the enzymes in silkworm. the researches have shown that infection with npv does not have considerable effect on carbohydrates 13 mailto:etebari@guilan.ac.ir up to third day but with the time flow, the amount of carbohydrates is enhanced in infected larvae. however total lipid showed significant increase in the hemolymph from the first day of infection (sarma et al., 1994). the effects of npv on midgut enzymes of spodoptera litura were investigated by nathan et al. (2005) and it was demonstrated that alkaline phosphatase is decreased after infection by virus. conversely, matindoost (2006) showed that bmnpv had caused a considerable decrease in activity of this enzyme in silkworm after infection of a cell line established from silkworm embryo (bm-ek1). together with a drop in alkaline phosphatase activity, also many culture medium components such as cholesterol, urea and glucose showed considerable decrease. these findings confirm that viral infection has a significant effect on many cellular metabolisms. due to the importance of protein metabolism and its crucial involvement in many compensative and reductive processes involving biological stresses, in this study the effects of bmnpv infection on changes of some biochemical compounds which are related to protein metabolism was investigated. materials and methods bmnpv inoculum preparation larvae infected with grasseri were collected from a field in north of iran and their hemolymph was extracted into microtubes. the bmnpv in the larval hemolymph was confirmed by light microscopy of polyhedra. bmnpv polyhedra were purified as described by sugimori et al. (1990). samples were centrifuged at 10,000 rpm for 10 min to pelletize the bmnpv polyhedra. the polyhedra were then suspended in distilled water and quantified by hemocytometry as initial inoculums (biabani et al., 2005). silkworm rearing and hemolymph preparation the eggs of a japanese line, 103, were obtained from iran sericulture research centre (rasht, iran) and reared in the laboratory with standard rearing techniques under 25 ºc with rh of 75±5 % and photoperiod of 16l:8d (krishnasawam, 1978). after 3rd molting, the larvae were divided into two groups including bmnpv infected larvae and uninfected ones. in each treatment 500 larvae were reared in two part of rearing room and both groups were fed by the leaves of shinichenoise mulberry variety. a group of larvae were orally treated with 5.5×10-4 polyhedral/ml of bmnpv at the first day of 4th instar. since disease symptoms were not observed in 4th instar in order to be certain that larvae have been infected, for each treatment 20 random samples were selected from 5th instar larvae in their 3rd and 5th days. larval proleg was cut and hemolymph was collected in microtubes. one milligram phenylthiourea was added immediately to the tubes to prevent melanization. the samples were centrifuged at 14,000 rpm for 10 min. the supernatant was removed and kept in –20 ºc for analysis. biochemical analysis the method of lowry et al. (1951) was used for the total protein estimation. hemolymph was diluted with distilled water and was added to alkaline copper reagent in microtubes. after 10 min 0.5 ml of folin ciocalteu’s reagent was added to the mixture and microtubes were shaken thoroughly. the tubes were kept 20 min in room temperature for color development. the readings were taken on the spectrophotometer at 650 nm. for the reference, standard bsa was used. the concentration of urea was determined by measuring ammonia produced from urea, using a commercial urea assay kit (chemenzyme co., iran). alanine aminotransferase (alt) (ec 2.6.1.2) and aspartate aminotransferase (ast) (ec 2.6.1.1) were measured utilizing thomas (1998) procedure. the t-test in sas software was performed for determination of significant differences between the data in infected and healthy groups (sas, 1997). results and discussion the results of this study have been demonstrated in figs 1-4. as it is shown in fig. 1, total protein considerable decreased in infected larvae compared to control. this reduction was observed in both sampling times in a way that total protein in the third day of 5th instar was 36.5 mg/ml in healthy larvae while in the same day the amount of protein in infected larvae was 7.5 mg/ml. this trend continued through fifth day of 5th instar and the amount of total protein in infected larvae decreased to 50 % of the control (fig. 1). it was previously reported that the total protein of hemolymph decreases in npv infected silkworm larvae from the first to the seventh day of infection. this difference in the last larval day was more than 35 units decrease (sarma et al., 1994). reduction of protein caused by npv infection, which is called hypoproteinemia, not only occurs in silkworm but also it has been reported from many other lepidopteran species such as mamestra brassicae, trichoplusia ni, lymantria dispar, etc (young and lovell, 1971; pawar and ramakrishnan, 1977; mazzone, 1985). fig. 1 the changes of total protein in infected and healthy groups of larvae (mg/ml) 14 reduction of hemolymph protein could be due to the decrease of protein synthesis. it is assumed that virus activity is followed by the disruption of fat body cell and the lack of protein release to the hemolymph, hence, protein levels drop in hemolymph. in insects, the most important place for protein synthesis is fat body that is also the most sensitive tissue to npv in silkworm. on the other hand, protein decrease could be the consequence of absorption process in midgut, because after entrance of virus to larval body, epithelial cells of midgut are invaded and therefore absorption process is interrupted. although there are some researches that show there are not considerable changes in the quantity of some compounds in the insects infected by virus (miao, 2002), often biochemical studies of silkworm larvae show that most of the stresses drop the amount of total protein in their hemolymph. it is assumed that silkworm compensates the deficiency of energy caused by the stress with intensive breakdown of proteins to amino acids and entering them to tca cycle as a keto-acid (nath et al., 1997; etebari and matindoost, 2004a, b; etebari et al., 2006). it has been reported that high doses of baculovirus could increase the total protein in the cell culture of silkworm embryonic tissue. matindoost (2006) suggested that the reason for this could be due to the bursting of cell membrane after cell death and release of cellular proteins, however, other factors have also been outlined. baculoviruses cause intensive interruption in hormonal regulation of larvae by having egt gene. this gene codes ecdysteroid udp-glycosyl transferase and its expression inhibits in time ecdysis of larvae (liu and hou, 1985). this is while utilizing ecdyson and its derivatives affects protein metabolism in larvae infected by npv. liu and hou (1985) demonstrated that compounds containing this hormone when treated on npv infected larvae prevent intense changes in the amount and type of proteins within hemolymph. fig. 2 the changes of urea in infected and healthy groups of larvae (mg/ml) fig. 3 the changes of ast activity in infected and healthy groups of larvae (iu/l) hemolymph urea did not have significant changes in two experiment times although it showed little difference (fig. 2). urea fluctuated between 4.37 to 6 mg/ml in uninfected and infected larvae. urea as an excretory compound plays an important role in silkworm physiology and changes of its concentration in hemolymph of silkworm larvae are dependent to many factors such as larval stage, diet, etc (sumida et al., 1993). urea changes are directly related to nitrogen metabolism and amino acids (hirayama et al., 1996). in this study not only no difference was observed between infected and uninfected larvae in the amount of urea but also no difference was evident in two sampling times. two amino transferases, ast and alt, showed considerable increase in infected larvae (figs 3, 4). the mean activity of ast was more than five times higher than that in uninfected larvae. in detail, at third and fifth day of 5th instar the activity reached to 2,458.8 and 2,632.2 iu/l, respectively, while the activity in control at the same days was 294.7 and 474.5 iu/l. the changes of alt follow the same pattern and its activity in infected larvae was more than triplicate. it has been shown that silkworm larvae under different stress factors like parasitism by parasitoid flies, and/or after treatment with phosphorus pesticides and juvenile hormone analogues present fluctuations in the activities of amino transferase enzymes (rami reddy et al., 1992; nath et al., 1997; singh et al., 1997). etebari et al. (2005) used the activity levels of these two enzymes as an appropriate biochemical marker to study the biodiversity of silkworm strains. the transaminases are the important components of amino acid catabolism; which is mainly involved in transferring an amino group from one amino acid to another keto acid. the ast and alt serve as a strategic link between the carbohydrate and protein metabolism and are known to be altered during various physiological and pathological conditions (etebari et al., 2005). 15 fig. 4 the changes of alt activity in infected and healthy groups of larvae (iu/l) generally alt activity is mentioned as an index for breakdown of amino acids and ast as a sign for entrance of amino acid to glucogenesis process. glucogenesis is a main path for sugar synthesis from non carbohydrate substrates (lehninger, 1982). the carbon sources for glucogenesis in these series of reactions are amino acids and the activation of this metabolic pathway is usually associated by intensive decrease of free amino acids in fat body and hemolymph, because often with alt activity, alanine transforms to pyruvate and enters the above pathway for energy supply. although it has been reported that many factors such as larval strain, larval age, light period or rearing season and leaf type affect the changes of alt activity levels (khanikor et al., 1998; devi and sarma, 2000) viral infection is far more effective than other factors. references anonymous. fao agricultural services bulletin, manual of sericulture, fao of the united nations, rome, 1976. biabani mr, seydavi ar, gholami mr, etebari k, matindoost l. evaluation of resistance to nuclear polyhedrosis virus in commercial hybrids of silkworm (bombyx mori) formosan entomol. 25: 103-112, 2005. biabani mr, mirhoseini sz, etebari k, matindoost l. the effects of nuclear polyhedrosis virus infection in 9 commercial hybrids of silkworm (bombyx mori l.) xxth congress of the international sericultural commission, 15-18 dec. 2005 bangalore, india, 2005. devi d, sarma dk. aminotransferase activity of muga silkworm (antheraea assama ww.) proceedings of the third international conference on wild silk moths, india, 246-248, 2000. etebari k, matindoost l. a study on the effects of larval age on biochemical macromolecules abundance of haemolymph in silkworm bombyx mori l. (lep. bombycidae). j. entomol. soc. iran 24: 1-16,2004a. etebari k, matindoost l. effects of hypervitaminosis of vitamin b3 on silkworm biology. j. biosci. 29: 417-422, 2004b. etebari k, mirhoseini sz, matindoost l. study on interaspecific biodiversity of eight groups of silkworm (bombyx mori) by biochemical markers. insect sci. 12: 87-94, 2005. etebari k, bizhannia ar, sorati r, matindoost l. biochemical changes in haemolymph of silkworm larva due to admiral residue. pestic. biochem. physiol., 2007 (in press). gao l, chen k, yao q, chen h. bmnpv infection enhances ubiquitin conjugating enzyme e2 expression in the midgut of bmnpv susceptible silkworm strain. int. j. indust. entomol. 13: 3135, 2006. hirayama c, konno k, shinbo h. utilization of ammonia as a nitrogen source in the silkworm, bombyx mori, j. insect physiol. 42: 983-988, 1996. khanikor d, unni bg, rai ak, baruah r. biochemical aspects of protein biosynthesis in the fat body of muga silkworm antheraea assama westwood during larval and spinning period. ad. bios. 17: 89-98, 1998. khurad am, mahulikar a, rathod mk, rai mm, kanginakudru s., nagaraju j. vertical transmission of nucleopolyhedrovirus in the silkworm, bombyx mori l. j. inver. phatol. 87: 8-15, 2004. krishnaswami s. new technology of silkworm rearing. bull. no. 2, central sericultural research & training institute (csrti) press, mysore, india, pp 1–23, 1978. lehninger al. principles of biochemistry, worth publishers, inc., new york, 1982. liu hs, hou r.f. studies on inhibition of infection with nuclear polyhedrosis virus by an ecdysteroid-containing agent in the silkworm bombyx mori l. chinese j. entomol. 5: 23-30, 1985. lowry h, rosebrough nl, far al, randal rj. protein measurement with folin phenol reagent, j. biol. chem. 193: 265 – 275, 1951. matindoost l. establishment and characterization of a newcell line from embryonic tissue of bombyx mori and its susceptibility to baculovirus (bmnpv). m.sc. thesis on entomology, the univ. guilan, iran, 2006. mazzone hm. pathology associated with baculovirus infection. in: maramorosch k, sherman ke (eds), viral insecticides for biological control, academic press, london, 1985. miao y. studies on the activity of the alkaline phosphatase in the midgut of infected silkworm, bombyx mori l. j. appl. entomol. 126:138–142, 2002. nath bs, suresh a, mahendra varma b, kumar rp. changes in protein metabolism in hemolymph and fat body of the silkworm, bombyx mori l., in responce to organophosphorus insecticides toxicity. ecotoxicol. envirn. saf. 36: 169-173, 1997. nathan ss, kalaivani k, chung pg. the effects of azadirachtin and nucleopolyhedrovirus on midgut enzymatic profile of spodoptera litura fab. (lepidoptera: noctuidae). pestic. biochem. physiol. 83: 46-57, 2005. 16 pawar vm, ramakrishnan n. biochemical changes in larval haemolymph of spodoptera litura (fabricius) due to nuclear polyhedrosis virus infection. indian j. exp. biol. 15: 755-758, 1977. rami reddy kv, benchman kv, remadevi ok. metabolic profiles of the haemolymph and fat body the silkworm, bombyx mori, in response to parasitation by the uzifly exorista sorbillans (dipt. tachnidae), during the final instar. sericologia 32: 227-233, 1992. sarma bj, samson mv, sivaparsad v, venkatsubbaiah mb, data rk. biochemical changes in the haemolymph of the silkworm bombyx mori l during the progressive infection of nuclear polyhydrosis virus (bmnpv). sericologia 34: 539-541, 1994. sas institute sas/stat user’s guide for personal computers, cary, nc: sas institute, 1997. sam devdas c, sasidharan to, samson mv, sivarpasad v,. datta rk. infectivity of serratia marcescens to the silkworm, bombyx mori l. (lep.: bombycidae), and its effect on certain biochemical constituents in the haemolymph and gut. sericologia 34: 275-281, 1994. singh pk, singh pn, prasad b. biochemical changes in the haemolymph of healthy and uzifly infested larvae of antheraea proylei jolly (lep.: saturnidae). sericologia 37: 465-472, 1997. sivaprasad vc, chandrasekharaiah c, misra s, kumar kpk, rao yum. screening of silkworm breeds for tolerance to bombyx mori nuclear polyhedrosis virus (bmnpv) int. j. indust. entomol. 7: 87-91, 2003. sugimori h, nagamine t, kobayashi m. analysis of structural polypeptide of bombyx mori nuclear polyhedrosis virus. appl. entomol. zool. 25: 6777, 1990. sumida m, haga k, tanaka y, shimabukuro j, ichida m, matsubara f. developmental changes in urea in the haemolymph (determined by a urease-indophenol method) in hybrid strains of the silkworm, bombyx mori and the effect of starvation in the fifth instar larvae, fed an arteficial diet, on urea level in subsequent development. comp. biochem. physiol. 105a: 563-570, 1993. thomas l. clinical laboratory diagnostics. 1st ed. th books verlasgesellschaft, frankfurt, 1998. young sy, lovell js. hemolymph proteins of trichoplusia ni during the course of a nuclear polyhedrosis virus infection. j. invert. pathol. 17: 410-418, 1971. 17 bmnpv inoculum preparation << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice microsoft word isj192 isj 6: 138-143, 2009 issn 1824-307x minireview origins and functions of annelide immune cells: the concise survey v vetvicka1, p sima2 1university of louisville, department of pathology, 511 s. floyd, louisville, ky 40202, usa 2institute of microbiology, department of immunology and gnotobiology, czech academy of sciences, videnska 1083, 142 20 prague 4, czech republic accepted october 13, 2009 abstract immune cells and structures of annelida represent a complex subject that was studied for over 120 years. an overwhelming mountain of data have been accumulated during these studies, most of them being the subject of many excellent reviews and books. in the this paper we focused on a brief survay of old studies and some reflection on the findings during recent years. key words: annelida; cells; immunocytes; defense introduction annelids are considered to be the ancestral key assemblage in which important evolutionary novelties have originated. these animals represent virtually a new, highly progressive monophyletic group in which the emergence of new structures brought about better dynamics of locomotion and more effective functional and immune capabilities (clark, 1964). this allowed these animals an extensive adaptive radiation into all biocenoses of the earth with exception of the air. subsequently, evolutionary pressures within these different environments resulted in high diversification leading to approximately 9,000 estimated species which are ordinarily divided in two superclasses-the aclitellata with major group of polychaeta, and the clitellata consisting of oligochaeta and hirudinea (see for details sima, 1994a). compared to their predecesors, the annelids exhibit all three major advanced structural features, such as the metameric body arrangement, the secondary body cavity (the coelom) filled by coelomic fluid constituting the hydrostatic skeleton, and the blood-vascular system (in some taxa already closed). this means that all necessary requirements for more advanced eucoelomate basic body plan were finally present from space for development of more complex and hierarchized organs, to increased regional differentiation, and finally, to the ability to change duplicated parts in ways that might be advantageous for survival. all ___________________________________________________________________________ corresponding author: vaclav vetvicka department of pathology university of louisville 511 s. floyd, louisville, ky 40202, usa e-mail: vetvickavaclav@netscape.net these hallmarks enabled a more active way of life and a higher adaptive plasticity which are not found in any earlier bilateralian acoelomate animals. both the coelom and the closed vascular system are important for emergence of a new type and more effective immune strategy. this also contributed toward their successful survival from an evolutionary point of view. a celomic cavity is composed of paired chambers lying within the third germinal layer the mesoderm which represents a general source of all types of immunocompetent cells found in all eucelomate taxa including deuterostomia. morphological separation of celom from the vascular system allowed cytogenesis of mutually independent cell lineages and their functional specialization and higher degree of functional cooperation. consequently, a high number of free and sessile cell types can be found both in the celomic and vascular fluids. cell types differ in morphology among various annelide species. to this date, no clean-cut classification has been formed and widely accepted. thus, morphofunctional criteria still remain basic for distinguishing various annelide immunocyte lineages. roughly speaking, these cells are usually characterized as amoebocytes, eleocytes, erythrocytes and hemocytes (polychaeta), celomocytes, amoebocytes, vascular lymphocytes, eleocytes and macrophages (oligochaeta) and amoebocytes and chloragocytes in hirudinea. current literature on annelide colemocytes is vast and far beyond the scope of the review. therefore, we will focus on a survey of the main immunocyte categories in three annelide subclasses: polychaetes, oligocheates, and hirundineans. 138 table 1 main categories of polychaete amoebocytes (from dhainaut and porchet-henneré, 1988) granulocyte morphology responding to presence in families type i fusiform, spindleshape, granules/microfilaments in cytoplasm adherent hyaline fusiform leukocyte (1) arenicolodae, ophelidae, glyceridae, nephtyidae, nereidae type ii* spindleshape, granular, cytoplasm with vacuoles and without microfilaments adherent hyaline leukocyte (1) arenicolodae, nereidae, terebellidae, capitellidae type iii small, few granules, large nucleus small lymphocyte (1) precursor cell? in many families type iv typical macrophage common macrophage found in immunized animals type v large cytoplasmic spherules, stacks of rough endoplasmic reticulum eosinophil granulocyte (1) many families *similar cells also in sipunculids, arthropods and molluscs (1): romieu (1923) polychaeta the primary and most numerous defense cells are the amoebocytes that represent a rather heterogeneous population of cells. amoebocyes are present in almost all the polychete species studied. the most common nomenclature used for amoebocyte is granulocyte, convenient term proposed by baskin (1974). according dhainaut and porchet-henneré (1988), polychete granulocytes are divided into five types (table 1). it is important to note that not all the these call types are present in every polychaeta. despite decades of research, the relationship among the individual types is still not established. still unknown is whether they represent different stages of a single cell lineage or discrete series, or if ameobocytes have a common origin with other cell types, e.g., the eleocytes. similarly, we are not certain of their origin. in some species, such as aphrodite, the distinct “lymph glands” have been described (fordham, 1925), whereas in sabelliade, the longshaped extension of nephridia has been suggested as a possible source. discret tissues derived from celomic peritoneum in glycera might play similar role. in other polychaete species, the mesenchymal tissue agglomerations around the vessels and nephridia might also serve for hemopoiesis (dehorne, 1922). the major defense functions of these cells consist of phagocytosis, waste removal and pathogen clearing. they are involved in antibacterial defense and encapsulation, which was a detailed study in nereis (porchet-henneré et al., 1987). the high degree of the cooperation by individual subtypes of granulocytes during encapsulation reaction was documented via means of monoclonal antibodies (porchet-henneré, 1990). a second population of free cells within a polychaete celom is represented by the eleocytes (rosa, 1896), the relatively large cells (appr. about 40 μm) contained in their cytoplasm nutrition reserves (mainly lipids and glycogen). they have often been described as agranular lymphocytes, trephocytes, or adipo-spherular cells. eleocytes were found in a row of families belonging to both polychaete subclasses (the errantia and the sedentaria) but not in arenicolidae, glyceridae, nephyidae, and syllidae. celomic lining epithelia covering blood vessels and somatic peritoneal epithelium were documented as a source of eleocytes in nereidae (eckelbarger, 1976) and terebellidae (dhainaut, 1970). conversely, other authors supposed that they develop from phagocytic amoebocytes (romieu, 1923; dales and dixon, 1981). their role is mainly trophic, even if their active but non-defensive pinocytosis used for removing of damaged tissues has been documented (dhainaut and porchet-henneré, 1988). third cell type found in polychetes constitutes hemoglobin-containing-cells termed erythrocytes (rarely hemocytes, red celomocytes or blood cells). they are found in capitellidae, cirratulidae, glyceridae, and opheliidae, exclusively in their celomic cavity, (goodrich, 1898). according to dhainaut and porchet-henneré (1988) erythrocytes form two lineages differing in morphology and functional specialization. in general, eleocytes and erythrocytes are primarily involved in regeneration and respiration. as with other immunocytes in this phylum, the origin of these cells is unclear and probably differs among species. while the role of eleocytes in defense is most probably limited, hemoglobin-containing erythrocytes are actively phagocytosing (goodrich, 1898). a more questionable population of cells represent hemocytes (blood cells) closed in the vascular systems of various representatives of polychete families such as terebellidae, sabellidae, arenicolidae, and nereidae. the hemocytes resemble celomocytes in morphology but they are relatively small, around 3-10 μm. with the exception 139 of magelona papilliocornis (boilly, 1974), they never contain hemoglobin. their origin is unresolved and, for decades, they were considered to be an immigrant cell population. only later, electron microscopic studies (dhainaut and porchethenneré, 1988) provided clearer knowledge and allowed classification into three categories (types i to iii, from which the type iii comprises sessile or attached cells and may be morphologically analogized to blood folicle cells of oligochaetes). their role in defense reactions remains unknown. oligochaeta from a practical perspective, the oligochaeta, and the earthworms (of the family lumbricidae) in particular, have by far been the most studied annelids and have become the object of interest in various scientific fields such as evolutionary biology and invertebrate immunology as well as a variety of environmental diciplines (agriculture, pedology, environmental pollution, epidemiology, etc.). extensive studies accomplished during last 50 years have made them one of the most popular models of invertebrate immunity (bilej, 1994; cooper and roch 1994, 2003). due to the intense study of the earthworm species, our knowledge of cell population composition in this category is superior. current classification of free cells is based on old research (kukenthal, 1885; rosa, 1896). for detailed information on origin, classification and formation, the reader is asked to refer to the detailed reviews written by sima (1994b) and cooper and roch (1994). again, the primary source of most information were the lumbricus terrestris and eisenia foetida models and, on rare occasions, from limited representatives of other lumbricid species. therefore, they might not be universally valid for all oligochaete taxa. the rather complicated classification can be simplified into two basic categories: the celomocytes (amoebocytes and eleocytes) and the hemocytes (blood cells). amoebocytes can be, according their morphology, further subdivided into granulocytes (acidophils type i and type ii) and non-granular hyalocytes (basophils, amoebocytes forming a 70 % majority of free cells and a monolite population of neutrophils). granular amoebocytes are present in all representatives of lumbricids. some nomenclatures subdivide them further based on size or type of granules. it was generally supposed that, at least in lumbricids, free celomocytes originated from mesodermal peritoneal cells comprising the visceral or parietal epithelial lining of the coelom (splanchnopleure and somatopleure) (fischer, 1993; sima and slipka, 1995; sima et al., 1995). other possible sources are epithelial lining of the blood vessels and septa forming specialized poietic structures (liebmann, 1942; valembois, 1971). the third eventuality, the origin from specialized hemopoietic organs named blood glands or blood folicles described in several representatives of the lumbricullus sp. and genera sparganophilus, maoridrilus, pheretima and pontodrilus, must be taken into account as well (see for review sima, 1994b). it was further observed that losts of celomocytes after their depletion induced by irritation (via dorsal pores in the body wall) are followed by extensive cell proliferation in coelomic lining in the typhlosole and metanepridial regions (homa et al., 2008). the mesodermal origin of all subpopulations of lumbricid free cells was recently confirmed using different mammalian antigenspecific monoclonal antibodies reacting with distinct coelomocyte surface markers (engelmann et al., 2002, 2005). both types of amoeboid celomocytes form an extremely important part of the immune system of annelids. as with most invertebrates, they are involved in non-self recognition, transplantation reaction, cytotoxicity, encapsulation, endocytosis and enzymatic digestion of engulfed material. in addition, they actively participate in regenerative processes and wound healing. phagocytosis alone has been studied in earthworms since metchnikoff (1891). due to the availability, this model has been extensively studied and, in the last several decades, a vast amount of data has been obtained. those seeking detailed information should read these excellent reviews (cameron, 1932; davis, 1978; bilej, 1994; cooper and roch, 1994, 2003; salzet et al., 2006). two main different subpopulations among earthworm immunocompetent free celomocytes were identified by flow cytometric analyses (cossarizza et al., 1996; engelmann et al., 2002, 2005). a subpopulation of large celomocytes (25-50 μm in diameter) is active in phagocytosis and encapsulation of bacteria but no surface markers were identified using monoclonal antibodies. small celomocytes (10-25 μm) reacting with anti cd11a, cd45ra, cd45ro, cdw49b, cd54, cd90, and beta 2-microglobulin are cytotoxic. non-granular coelomocyte subpopulation recognize and neutralize foreign antigens. these cells exprime surface markers thy-1 (cd90) and beta 2microglobulin, both molecules belonging to the immunoglobulin superfamily (shalev et al., 1981; roch et al., 1983). eleocytes are again a heterogenous group of cells, usually divided into ergastoplasmic chloragocytes (which are probably the stem cells of this group), chloragocytes and free eleocytes. their morphology can vary with oval, round or oblong cell´s shape 10-60 μm in diameter. a majority of eleocytes possess spherical granules (1-3 μm) and chloragosomes within their cytoplasm. chloragocytes play a role in nutrition, excretion and osmotic balance. the defensive roles of these cells are less studied but they are certainly less pronounced. these cells proved inactive in phagocyting bacteria or other particles (dales and kalaç, 1992). in addition, they differ among individual species, e.g., they phagocytose in eisenia but not in lumbricus. with respect to hemocytes, the information is scarce and the current information has not substantially improved since the last century. other oligochaete families if someone gained the impression that the situation concerning the cells and cellular structures in earthworms is confusing, an even more complicated 140 table 2 main categories of coelomic cells of the theromyzon tessulatum (hirudinea) (from lefebvre et al., 2008) coelomocyte morphology responding to function large coelomic cell (chloragocyte) 100-150 μm free or attached to mesothelia granules in cytoplasm oligochaete chloragocyte non-phagocytic presence of recognition surface molecules encapsulation? vitellin production? granular amoebocyte 30-70 μm granules in cytoplasm numerous pseudopodia oligochaete granular amoebocyte chemotactic phagocyting g+ and gbacteria small coelomic cell 7-12 μm without cytoplasmic granules hyaline cytoplasm rich endoplasmic reticulum oligochaete hyaline cell non-phagocyting situation can be found in other oligochaeta families. even when these members commonly possess the same basic cell types, additional, more-or-less specialized or distinguished cell types have been described. pontodrilus bermudensis serve as an example (wamper and jamieson, 1986). on the other hand, genera such as achaeta, analycus, or grania, have only one type of celomocytes. buchholzia possesses two types (brinkhurst and jamieson, 1971). hirudinea general body anatomy responds to annelide basic body plan. however, these animals endured substantial modification when separate coleomic and vascular spaces were fused together into common hemocoel. these modifications of the vascular system and coelomic cavity led to the flow of hemocoelomic fluid through a highly complicated network of coelomic sinuses and channels. hirudidae can be characterized by the presence of the botryoidal tissue located within the parenchyma adjacent to the body wall. besides being involved in angiogenesis, this celothelium-derived tissue display myelo/erytroid function. it also can change its shape from a solid cord of cells to a prevascular structure when groups of closely associated cells became evident in the centre of the immature lumen. as the vessel growth, the precursors of circulating cells loose the cell-cell attachment and move freely within the lumen. unfortunately, cytology of hirudineans has attracted little attention, so our knowledge of the origin and function of their free cells are still incomplete. cell populations within the hemocel are commonly divided to amoebocytes and chloragogen cells, similar to the previous annelide classes. amoebocytes denominated also leukocytes or lymphocytes are usually of homogeneous size and morphology, exerting phagocytosis as the main defense activity (sawyer and fitzgerald, 1981). these cells are either free or attached to the hemocel wall lining. hypotheses about their origin are rather speculative. the size of chloragogen cells differs among individual species. their origin is clear, they arise from the hemocelomic epithelia (oka, 1894) and from the cilio-phagocytic organ of the nephridia (abeloos, 1925). recently, using human monoclonal antibodies, three cell populations, the macrophage-like, nk-like, and granular cells, were identified in glossiphonia complanata (de eguileor et al., 2000). similarly, immunohistochemical and ultramicroscopic studies demonstrated more precisely the presence of three basic cell populations in theromyzon tessulatum (lefebvre et al., 2008) (table 2). we must be also aware that free cells may be derived from different tissue sources in different species. on the contrary to family hirudidae, the celothelial botryoidal tissue in some species of the glossiphoniidae plays besides the angiogenetic functions the important role in production of free hemocelomic cells. prevascular structures are tightly associated with solid cell cords, from which circulating cells release loosing mutual contacts and pass freely into the vascular lumen (de eguileor et al., 2001). an increasing body of evidence indicates that in the leech hirudo medicinalis the angiogenic process is regulated and coordinated by the botryoidal tissue. hirudo medicinalis subjected to an angiogenic stimulus (e.g., wounding) responds with an extensive angiogensis that is accompanied by the production of free cells. these processes, moreover, could be influenced by means of mammalian activators of vascular cell growth, antiangiogenic peptides (angiostatin and endostatin) and even proliferation-inducing mitomycin. the surprising degree of similarity of invertebrate angiogenesis with neovascularization in vertebrates, both at the biochemical and cellular levels, suggests that both involve similar growth factors and their receptors, and common cell-cell or cell-extracellular matrix interactions (grimaldi et al., 2006, grimaldi et al., 2008). the reviewed data confirm phylogenetic kinship between hirudinean and vertebrate processes in wound healing. it suggests that cytogenesis of free cells within the vascular system in such remote phyla as protstomian annelids and deuterostomian chordates may share evolutionary common basic mechanisms. 141 summary defense reactions of annelids may be clearly defined as the two compound systems developed from and housed in the coelom in which cooperate the cellular (celomocytes) and humoral (celomic and vascular fluid) part of immunity. individual types of celomocytes play a role similar to vertebrate lymphocytes, leukocytes and macrophages. together, both parts are sufficiently effective in disposing of foreign material and pathogenic invaders. references abeloos m. recherches histochimique et physiologiques sur le parenchyme et les nephridies des huridinees rhynchobdelles. bull. biol. fr. belg. 59 : 436-456, 1925. baskin dg. the coelommocytes of nereid polychaets. contemp. topics immunol. 4: 5564, 1974. bilej m. cellular defense mechanisms. in: vetvicka v, sima p, cooper el, bilej m, roch p (eds), immunology of annelids, crc press, boca raton, pp 167-200, 1994. bilej m. humoral defense mechanisms. in: vetvicka v, sima p, cooper el, bilej m, roch p (eds), immunology of annelids, crc press, boca raton, pp 245-261, 1994. boilly b. ultrastructure des hématies anuclés de magelona papillicornis f. müller (annélide polychète). j. microsc. 19: 47-67, 1974. brinkhurst ro, jamieson bgm. aquatic oligochaeta of the world. oliver and boyd, edinburgh, 1971 cameron gr. inflammation in earthworms. j. pathol. bacteriol. 35: 933-972, 1932. clark rb. dynamics in metazoan evolution, claredon press, oxford, 1964. cooper el, roch p. immunological profile of annelids: transplantation immunity. in: vetvicka v, sima p, cooper el, bilej m, roch p (eds), immunology of annelids, crc press, boca raton, pp 201-243, 1994. cooper el, roch p. earthworm immunity: a model of immune competence. pedobiologia, 47: 676688, 2003. cossariza a, cooper el, suzuki mm, salvioli s, capri m, gri g, et al. eartworm leucocytes that are non phagocytic and cross-react with several human epitopes can kill huma tumor cell lines. exp. cell res. 224: 174-182, 1996. dales rp. the basis of graft rejection in the earthworms lumbricus terrestris and eisenia foetida. j. invertebr. pathol. 32: 264-368,1978. dales rp, dixon lrj. polychaetes. in: invertebrate blood cells 1. ratcliffe na, rowley, af (eds), acad. press, london, pp 35-74, 1981. dales rp, kalaç, y. phagoytic defense by earthworm against pathogenic bacteria. comp. biochem. physiol. 101a: 487-490, 1992. de eguileor m, grimaldi a, tettamani g, vasvassori r, cooper el, lanzavecchia g. lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers. tissue cell 32: 437-445, 2000. de equileor m, grimaldi a, tettamanti g, congliu t, protasoni m, reguzzoni m, et al. ultrastructure and functional versatility of hirudinea botryoidal tissue. tissue cell 33: 332341, 2001. dehorne a. histolyse et phagocytose musculaire dans le coelome des nerédiens a maturité sexuelle. c. r. acad. sci. (paris) 164: 10431063, 1922. dhainaut a, porchet-henneré a. xiii. haemocytes and coelomocytes. in: the ultrastructure of polychaeta, microfauna marina 4, westheide w, hermans co (eds), gustav fischer verlag, stuttgart, pp 215-236, 1988. eckelbarger kl. origin and development of amoebocytes of nicolea zostericola (polychaeta: terebellidae) with a discussion of their possible role in oogeneses. mar. biol. 35: 169-182, 1976. engelmann p, pal j, berki t, cooper el, németh p. earthworm leukocytes feact with different mammalian antigen specific monoclonal antibodies. zoology 105: 257-265, 2002. engelmann p, cooper el, németh p. anticipating innate immunity without a toll. mol. immunol. 42: 931-942, 2005. fischer e. the myelo-erytroid nature of the chloragogenous–like tissues of the annelids. comp. biochem. physiol. 106a: 449-453, 1993. fordham mgc. aphrodie aculeata. l.m.b.c. memoir, 27, proc. liverpool. biol. soc. 40: 121131 1925. goodrich s. on the nephridia of polychaeta. ii. on glycera and goniada. q. j. microsc. sci. 41: 439-458,1898. grimaldi a, tettamanti g, perletti g, valvassori r, de eguileor m. hematopoietic cell formation in leech wound healing. curr. pharm. des. 12: 3033-3041, 2006. grimaldi a, bianchi c, greco g, tettamanti g, noonan dm, valvassori r, et al. in vivo isolation and characterization of stem cells with diverse phenotypes using growth factor impregnated biomatrices. plos one.3:e1910, 2008. homa j, bzowska m, klimek m, plytycz b. flow cytometric qauntification of proliferating coelomocytes non-invasively retrieved from earthworm, dnedrobaena veneta. dev. comp. immunol. 32: 9-14, 2008. kukenthal w. über die lymphoiden zellen der anneliden. jen. z. naturwiss. 18: 319333,1885. lefebvre c, vandenbulcke f, bocquet b, tasiemski a, desmons a, verstraete m, et al. cathepsin l and cytostatin b gene expression discriminates immune coelomic cells in the leech theromyzon tessulatum. dev. comp. immunol. 32: 795-807, 2008. liebmann e. the role of the chloragogue in regeneration of eisenia foetida (sav.). j. mophol. 70:151-187,1942. metchnikoff e. lectures on the comparative pathology of inflammation. starling fa, starling eh (eds), dover press, new york, pp 67-73, 1891. 142 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22grimaldi%20a%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22tettamanti%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22perletti%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22valvassori%20r%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22de%20eguileor%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'curr%20pharm%20des.'); http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22grimaldi%20a%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22bianchi%20c%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22greco%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22tettamanti%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22noonan%20dm%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22valvassori%20r%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'plos%20one.'); oka a. beitrage zuz anatomie der clepsine. z. wiss. zool. 58: 79-98,1894. porchet-henneré e. cooperation between different coelomocyte populations during the encapsulation response of nereis diversicolor, demonstrated using monoclonal antibodies. j. invertebr. pathol. 56: 353-361, 1990. porchet-henneré e, m’beri m, dhainaut a, porchet m. ultrastructural study of the encapsulation. response of the polychaete annelid nereis diversivolor. cell tissue res. 248: 463-471, 1987. roch p, cooper el, eskinazi dp. serological evidences for a membrane structure related to human beta 2-microglobulin expressed by certain earthworm leukocytes. eur. j. immunol. 13: 1037-1042, 1983. romieu m, recherches histophysiologiques sur le sang et le corps cardiaque des annélides polychétes. contribution a l´histologie comparée du sang. arch. morph. gener. exp. 17: 1-17, 1923. rosa d. les lymphocytes des oligochaetes, arch. ital. biol. 25: 455-475, 1896. salzet m, tasiemski a, cooper e. innate immunity in lophotrocozoans: the annelids. curr. pharm. des. 12: 3043-3050, 2006. sawyer rt, fitzgerald sw. hirudineans, in: invertebrae blood cells, vol. 1, ratcliffe na, rowley af (eds), academic press, london, pp 141-159, 1981. shalev a, greenberg ah, logdberg l, bjorck l. beta 2-microglobulin-like molecules in low vertebrates and inverteebrates. j. immunol. 127: 1186-1191, 1981. sima p. a survey of the evolution of fundamental body construction in relation to immunological phenomena. in: vetvicka v, sima p, cooper el, bilej m, roch p (eds), immunology of annelids, crc press, boca raton, pp 27-39, 1994a. sima p. annelid coelomocytes and hemocytes: role in cellular immune reactions. in: vetvicka, v, sima, p, cooper, el, bilej, m, roch p (eds), immunology of annelids, crc press, boca raton, pp 115-165, 1994b. sima p, slipka j. the spleen and its coelomic and enteric history. adv. exp. med. biol. 371a: 331334, 1995. sima p, bilej m, slipka j. perienteral chloragogen tissue and its role in defense in lumbricid worms. adv. exp. med. biol. 371a: 327-329, 1995. valembois p. etude ultrastructurale de coelomocytes du lombricien eisenia foetida (sav.). bull. soc. zool. (france) 96: 59-65, 1971. wampler je, jamieson bgm. cell bound luminescence from pontodrilus bermudensis and its similarities to other earthworm bioluminiscence. comp. biochem. physiol. 84a: 81-87, 1986. 143 isj 7: yyy-xxx, 2010 isj 7: 149-156, 2010 issn 1824-307x review relationships between innate immunity in bivalve molluscs and environmental pollution mi girón-pérez laboratorio de inmunotoxicología, universidad autónoma de nayarit, boulevard tepic-xalisco s/n, cd de la cultura amado nervo s/n, 63190 tepic, nayarit, mexico accepted may 25, 2010 abstract the immune system of invertebrates, such as molluscs consists of innate mechanisms very effective against antigens commonly present in the environment. however, these defense strategies could be altered by pollutants. this review is focused mainly on the effect of metals, pcb, pesticides, pahs, and others environmental pollutant on immune response of molluscs. key words: molluscs; immune system; environmental pollution; metals; pesticides; pahs; pcb introduction immune system is strongly influenced by environmental conditions. successful host resistance is a major determinant in whether a pathogen will result in a disease outbreak. altered environmental conditions can affect immunity directly, by changing the concentration and efficiency of components including cytokines, cytokine receptors and cells of the immune response, or indirectly by inducing general stress response. subsequently, the relationship immunityenvironment is complex, but is an essential comprehended mechanistic aspect of it, and thus allow predictions on the potential effect of environmental factor on immune response (mydlarz et al., 2006). the bivalve molluscs have characteristics such as high distribution worldwide, sedentary and filter-feeding habits; hence these organisms accumulate large number of bacteria and chemical pollutants, which are both a source of nourishment and an immune challenge (bernal-hernandez et al., 2010). the immune response of molluscs has an important defense function against bacteria, fungi, and parasites. the immune system is constituted for a first line defense including physicochemical barriers as the cuticle, shell and mucus layer. moreover, in bivalves, cellular and humoral components ___________________________________________________________________________ corresponding author: manuel iván girón pérez laboratorio de inmunotoxicología, c.a. contaminación y toxicología ambiental, universidad autónoma de nayarit, bd tepic-xalisco s/n, 63190 tepic, nayarit, méxico e-mail: ivan_giron@hotmail.com are present and operate in a coordinated way (galloway and depledge, 2001). cellular response is carried out by circulating hemocytes that can kill microbes through phagocytosis and citotoxic reactions that include the release of lysosomal enzymes and anti-microbial peptides, and the respiratory burst which involves the production of oxygen metabolites, meaning superoxide anion, hydrogen peroxide, and intermediated compounds with high bactericidal activity (pruzzo et al., 2005). hemocytes are also involved in other physiological functions, such as wound and shell repair, digestion and transport of nutrients. hemocytes classification is controversial, but has hypothesize the existence of two circulating hemocytes cells: granulocytes (containing many citoplasmatic granules) and hyalinocytes (containing few or no granules). granulocytes are generally the most abundant cell type with higher phagocytic activity, while hyalinocytes are usually smaller than granulocytes, and have a high nucleus/cytoplasm ratio (hine et al., 1999; matozzo et al., 2007). the humoral components present in hemolymph are lectins, lysosomal enzymes and antimicrobial peptides. the presences of lectins have been shown in marine bivalves such as mussels, oyster, and clams. the role of lectins is induced agglutination of bacteria and act as a molecular bridge between the surface of bacteria and hemocytes (pruzzo et al., 2005). in spite of the efficiency of the immune system of molluscs in normal conditions, it may be altered by external factors (fig. 1). thus, this review is focused mainly on the effect of metals, pcb, pesticides, pahs, and others environmental pollutants on immune response of molluscs. 149 fig. 1 effect of the main contaminants in aquatic ecosystems on the immune system of molluscs xenobiotics and immune system of molluscs the presence of chemical contaminants in water is a major subject of concern, since many of these molecules are potent immunosupressors, even at a low concentration (table 1). a possible consequence for immunodeficient oyster could be an increased susceptibility to parasites and other pathogenic microorganisms (auffret et al., 2002). metal effects studies performed to understand the relationship between metals and immunotoxicity have been showed that in vitro cd exposure of hemocytes at sub-lethal concentrations up to 15 μm cdcl2 induce significantly increase in metallothionein (mt) and inhibition of ros generation (butler et al., 2000). studies realized with oyster (ostrea edulis) showed that exposure to cdcl2 (1, 10, 50, 100 µm) and co-exposure to cdcl2 and cucl2 (0.75 µm), induced non significant changes in the serum total protein level. moreover, the level of serum acid phosphatase, and hydrolytic enzymes remained unaltered. but a dose-dependent increase in total hemocytes was found in oyster exposed to cdcl2. on the other hand, the exposure to 1, 10 or 50 µm cdcl2 resulted in a dose-dependent decreased in the cell membrane potential, probably related to membrane alterations. phagocytic activity of o. edulis exposed to 1 or 10 µm cdcl2, or to 1 µm cdcl2/0.75 µm cucl2 showed a severe decrease compared with control group (auffret et al., 2002). the effect of copper exposure (0.02 and 0.05 ppm), alone or simultaneously with vibrio tubiashii at different temperature, was evaluated on mussels (mytilus edulis). results showed that 0.05 ppm copper induced a significant reduction on cellular content in hemolymph. when co-exposed mussels to bacterial challenge, the reduction in cell number was higher, compared with the effect of metal alone. in addition, intracellular superoxide decreased significantly by exposure to 0.02 and 0.05 ppm of copper to 10 °c. however, there was an increase of this parameter when analyzed at 15 °c. while, phagocytosis was increment by exposure at 0.02 ppm compared with control group (parry and pipe, 2004). another study evaluated the effect of cadmium, copper on ros production, and hemocyte viability from mytilus galloprovincialis. results showed a significantly decrease on viability of hemocytes (according xtt test) exposed to cd (1,120x10-5 μg/ml). exposure to cu (12.72 μg/ml) also induced 150 table 1 immunotoxic effect of pollutants on molluscs xenobiotic effect species reference metals hemocyte counts ↑ cd cell membrane potential ↓ ostrea edullis auffret et al., 2002 cd alone or cd/cu phagocytic activity ↓ ostrea edullis auffret et al., 2002 cd (in vitro) cd, and cu cu cu methalothionein↑ ros ↑ hemocyte viability ↓ phagocytosis ↓ hemocyte counts ↓ superoxide anion ↓ phagocytosis ↑ crassostrea virginica mytilus galloprovincialis elliptio complanata mytilus edulis butler et al., 2000 gómez-mandikute et al., 2003 gangé et al., 2008 parry et al., 2004 gas cellular viability ↓ o3 cox-activity ↑ elliptio complanata gangé et al., 2008 no2 ↑ estrogenic substances nonylphenol, monoethoxilate carboxylate, and 17α-ethynyl estradiol 17β-estradiol pharmaceutical drugs lysosomal enzyme release ↑ phagocytosis ↑↓ phagocytosis ↓ mytilus galloprovincialis corbicula fluminea canesi et al., 2007 champeau et al., 2006 benzafibrinate, gembibrozil, and trimetophin phagocytosis ↑ elliptio complanata gagné et al., 2006 novobiocin, and morphin phagocytosis ↓ elliptio complanata gagné et al., 2006 zulfamethazole, novobiocin, gemfibrocil, benzafibrate, and carbamazepine esterase activity ↓ elliptio complanata gagné et al., 2006 oxytetracycline, novobiocine, naproxen cell adherence ↓ elliptio complanata gagné et al., 2006 gemfibrozil, bezafibrate cell adherence ↑ elliptio complanata gagné et al., 2006 novobiocine,and sulfapyridine lipoperoxidation ↑ elliptio complanata gagné et al., 2006 coprostanol, and naproxen lipoperoxidation ↓ elliptio complanata gagné et al., 2006 151 pahs benzo[a]pyrene, and phenanthrene granulocyte cell (%) ↑ cell mortality, esterase and lysosome-positive cells ↓ crassostrea gigas gagnaire et al., 2006 benzo[a]pyrene benzo[a]pyrene phenanthrene phenanthrene hemocytes viability ↓ lysozyme activity ↓ phagocytic activity ↓ adhesion capability ↓ hemocyte mortality ↑ phagocytic cells ↓ superoxide generation ↓ hemocyte number ↑ cell membrane stability↓ phagocytosis↓ mytilus galloprovincialis chamelea gallina cerastoderma edule pecten maximus gómez-mandikute et al., 2003 matozzo et al., 2009 wootton et al., 2003 hannam et al., 2010 pcb pcb 77 lysosome-positive cells ↓ crassostrea gigas gagnaire et al., 2006 pesticides 2.4d cell mortality ↑ crassostrea gigas gagnaire et al., 2006 paroxon esterase and lysosome positivecell (%) ↓ crassostrea gigas gagnaire et al., 2006 ros positive-cell (%) ↑ chlorothalonil cell mortality and granulocyte (%)↑ crassostrea gigas gagnaire et al., 2006 pesticide mixture (atrazine, gliphosate, alachlor, metalachlor, fosetylaluminum, terbuthilazine, diuron, carbaryl) phagocytosis ↓ cell mortality, and ros production ↑ genes relationship with immune response (e.g., lyzozyme, defensines) ↓ crassostrea gigas gagnaire et al., 2007 susceptibility to bacteria challenge ↑ paraquat other hemocytes viability mytilus galloprovincialis gómez-mandikute et al., 2003 fuel oil no. 6 cellular viability↓ pinctada imbricate nusseti, et al. 2004 gst and cat↑ 4-nonylphenol gpx ↓ lysozyme concentration ↓ apoptitic index ↑ tapes philippinarum matozzo, et al. 2005 symbol: reduction (↓), increased (↑), biphasic effect (↑↓) 152 decrease in this parameter. the superoxide anion production (using nbt reduction test) in hemocytes was evaluated. the results indicated that cd exposure induced no changes, but cu exposure decrease the nbt reduction (gómez-mandikute et al., 2003). the studies have been showed that metals could modulate different immunologic parameters on molluscs. however, the immunomodulation is influenced by metal concentration, and other factors such as presence of potential pathogens and environmental variables, like temperature for instance. estrogenic substances effects others pollutant substances frequently present in aquatic ecosystem are estrogenic chemical. in this context, canesi, et al. (2007), evaluated the in vitro effect of endocrine disruptor compounds on mytilus hemocytes. the results showed that hemocytes incubated during 30 minutes, with estrogenic compounds, such as nonylphenol monoethoxylate carboxylate (np1ec) and 17αethynyl estradiol, increased the lysosomal enzyme release in 65 % and 45 %, respectively compared with control hemocytes. on the other hand, a biphasic effect was observed on phagocytosis, thus to lower concentrations (0.1 5 µm) a significant stimulations was detected, while to 25 100 µm a inhibitory effect was observed. the effect of 17β-estradiol (20, 200 and 2000 ng/l) was evaluated on asian clam corbicula fluminea, exposure during 15 or 30 days. results showed that this estrogenic substance did not affect the cell viability. however, the exposure to 200 and 2000 ng/l significantly inhibit the phagocytosis, in both evaluated times (champeau et al., 2006). the effects of endocrine disrupters, such as natural or synthetic steroids, on immune system of molluscs are not well known yet, in part by limited knowledge on invertebrate endocrine system and immunendocrine network. however, results suggest that immune system represents an important target of estrogenic compounds. thus, the study of these compounds and their effect on invertebrate physiology is necessary. pharmaceutical products effects municipal effluents represent a major source of pollution. these effluents could contain pharmaceutical products, xenobiotics that could modulate immune response of aquatic organisms. studies on mussels (e. complanata) hemocytes exposure in vitro to pharmaceutical drugs (benzafibrate, carbamazepine, fluoxetine, gemfibrozil, morphine, naproxen, novobiocin, oxytetracycline, sulfamethazole, sulfapyridine and trimethoprim) and urban waste (coprostanol, caffeine, cotinine) at 0, 2.5, 25, 50 and 100 μm, showed that some products as benzafibrate, gemfibrozil and trimethoprim, increased phagocytosis, while novobiocin and morphine reduced its activity. intracellular esterase activity was reduced with sulfamethazole, novobiocin, gemfibrozil, benzafibrate and carbamazepine. cellular adhesion was decreased by oxytetracycline, novobiocin and naproxen, and increased by gemfibrozil, bezafibrate and sulfapyridine. exposure to these products also modulated lipoperoxidation (lpo) in hemocytes. coprostanol and naproxen were more potent to reduce lpo while novobiocin and sulfapyridine were the most potent to induce lpo. on the other hand, on a parallel experiment, mussels were placed in aeration lagoons for the treatment of domestic wastewaters during 60 days. in mussels, a decrease of intracellular esterase and phagocytic activity was observed (gagne et al., 2006). pahs effects polycyclic aromatic hydrocarbons (pahs) are a ubiquitous class of organic contaminants generated as results of anthropogenic sources or natural constituents of crude oil. the effect of phenanthrene (50, 100, 200 or 400 µg/l) on immunological parameters of mytilus edulis, cerastoderma edule, and ensis siliqua, were evaluated after exposure during 7 and 14 days. phenanthrene exposure at 400 µg/l resulted in 100 % mortality of c. edule after 14 days exposure, while total mortality of e. siliqua was observed 7 days after exposure. nevertheless, no mortality on m. edulis was reported. in general terms, results of immunologic parameters showed that acid phosphatase concentration was increase in m. edulis after 7 days exposure to phenanthrene 50, 100 and 200 µg/l, but a diminish in this parameter was observed 14 days after exposure at same concentrations. phagocytic cells percentage and superoxide generation were significantly reduce in c. edule after 14 days exposure to 100 and 200 µg/l. comparative analysis between three different species suggests that e. siliqua is less sensible to alterations by exposure to phenanthrene (wootton et al., 2003). studies realized with scallop pecten maximus, showed that exposure during 7 days at 100 and 200 µg/l phenanthrene, increase hemocyte number. nevertheless, cell membrane stability, and phagocytosis were reduced when organism were exposed to 200 µg/l. in addition, oxidative stress parameters were evaluated, indicating that 200 µg/l phenanthrene provoke diminish of gsh activity, but significantly increased lipoperoxidation index (hannam et al., 2010). another pah is benzo(a)pyrene, effect of 0.5 mg/l of this substance was evaluated on immune response of clam chamelea gallina. exposure to this xenobiotic during 7 and 12 days significantly decreased lysozyme activity, phagocytic activity and adhesion capability (matozzo et al., 2009). another study showed that benzo(a)pyrene did not have significant effect on viability of hemocytes of mytilus galloprovincialis. but this substance induced a significant increased in superoxide anion production (gómez-mandikute et al., 2003). studies made with pacific oyster crassostea gigas exposed in vitro to benzo(a)pyrene and phrenanthrene showed that these substances significantly increased granulocyte percentage, but decreased cell mortality and esterase and lysosome-positive cells at doses of 200 and 300 μmol/l, respectively (gagnaire et al., 2006). atlantic pearl oyster (pinctada imbricata) exposure to fuel oil no 6 during 7 days, showed no 153 significantly changes in immunological parameters, such as hemocyte number, phagocytosis, and lysozyme concentration. however, cellular viability was reduced when oyster were exposed to this xenobiotic. antioxidant enzymes such as, glutathione s-transferase (gst), and catalase (cat) were significantly higher in the digestive gland. while in mantle, an increase of glutathione peroxidasa (gpx), and decrease gst activity was detected. this report suggests that these enzymes should be considered as potential tools for biomonitoring marine environmental contamination (nusetti et al., 2004). other studies have focused their efforts to study the immune system in organisms living at low temperature (-1 to 5 °c). camus et al. (2002), evaluated the effect of benzo(a)pyrene on oxyradical scavenging capacity (tosc), and cell membrane stability of hemocytes from arctic scallops (chlamys islandicus). results indicated a reduction of tosc, and cellular membrane stability when benzo(a)pyrene was administrated at 74 and 90.6 mg/kg. these alterations should be negative for the cellular immunity of bivalves by reducing the phagocytosis ability of hemocytes. pahs exposure could increase susceptibility to infections. some studies suggest that reduction in immunocompetence is related to stimulation of ros production induced by pahs. pesticides effects pesticides are often used in successful agriculture. however, the pesticide use leads to severe environmental pollution. this way, aquatic organisms are frequently affected by these xenobiotics. studies realized with pacific oyster crassostea exposure to pesticide, showed that 2,4dichlorophenoxyacetic acid (2,4d), increased cell mortality at 450 μmol/l after a 4 h incubation period. while, paroxon exposure induced decrease in percentage of esterase-positive cells after 4 and 24 h of incubation at 400 μmol/l. but paroxon at 40 and 400 μmol/l, after 24 h incubation period, decreased lysosome-positive cells percentage; in contrast ros-positive cells were significantly increased at 400 μmol/l after 4 h incubation period. the fungicide chlorothalonil at 2 μmol/l, significantly increased cell mortality and granulocyte percentage at 200 μmol/l after 4 h incubation period. in addition, a pesticide mixture (alachlor, metolachlor, terbutilazina, glyphosate, diuron, atrazine, carbaryl, and fosteyl aluminium) was realized, but interestingly enough none of this eight compounds generated significantly effect when tested individually on c. gigas, but the mixture indeed decreased phagocytic activity (gagnaire et al., 2006). other studies carried out with pesticides, showed that paraquat on mytilus galloprovincialis hemocytes, showed a significantly decrease on viability of hemocytes (according xtt test) exposed to paraquat (10 μg/ml). on the other hand, paraquat exposure, induced a significantly increase in superoxide anion (gómez-mandikute et al., 2003) in order to know the effect of pesticide on bacteria challenge, pacific oyster c. gigas were exposed to a mixture of pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetylalumimium, terbuthylazine, diuron and carbaryl) at environmental relevant concentration over a 7-days period. as a first step, hemocyte parameters (cell mortality, enzymes activities, and phagocytosis) were evaluated. the results showed that phagocytosis was significantly reduced, while cell mortality, esterase and ros production, were not altered. however, real-time pcr analyses showed that 19 genes (involved with cell signaling, cytoskeleton function, phagocytosis and other defense mechanisms) were down-regulated in treated animals. moreover an increased susceptibility to a bacteria challenge was observed. as a second step, the interaction between pesticide exposure and bacteria challenge (vibrio splendidus, 4x107 ufc/oyter) was evaluated. in this coexposure condition, was observed that 10 of 19 genes (focolin, galectin, lbp, c-src, ankyrin, procl, sod, tmp, lysozyme, defensin) was up-regulated. the authors suggested that up-regulated genes could induce damage in host-tissue (gagnaire et al., 2007). ozone effects on the other hand, the municipal effluents then sometimes undergo disinfection. a common process involved is ozonation. however, ozone treatment might generate toxic products. studies carried out by gagné et al. (2008), showed that freshwater mussels (elliptio complanata) exposed to ozone (range 1 20 %), in laboratory condition for 7 weeks, significantly diminished phagocytosis and cellular viability. however, cell adherence suffered no changes when compared to control group. in contrast, cox-activity and nitrite levels were significantly increased. according to results, o3, at concentration evaluated, reduce microbial loading and completely remove citotoxicity, but increased inflammatory properties of the effluents. the observed effect could be related to the formation of carboxylic acid, aldehydes, and ketones which modifies the redox status of treated wastewaters. others substances the effect of 4-nonylphenol (np), final product of nonylphenol eyhoxylates, substances used as stabilizer, was evaluated on clam tapes philippinarum. results showed that exposure at sublethal concentrations (0.05 0.2 mg/l) during 7 days, significantly reduced lysozyme concentration and sod activity. in contrast, apoptotic index was increasing at same concentrations (matozzo et al., 2005). in another research, hemocytes from the pacific oyster crassostea gigas were exposed in vitro to polyclorinated byphenyls (pcb), such as pcb 77. the results showed that this substance significantly decreased lysosome-positive cells at 6 and 60 μmol/l after 4 h incubation (gagnaire et al., 2006). field studies laboratory studies showed some advantages, the principal being the experiments performed in controlled conditions. however, field researchers in ecotoxicology, permit to analyze parameters 154 according to conditions present at one particular moment, and with all the factors that have influence over an ecosystem. furthermore, these type of studies are more suited to distinguish a correlationship between factors present on specific sites. recently, our research group evaluated the presence and concentration of pahs (pyrene, naphtalene, and benzo(a)pyrene), metals (cu, pb, zn, mn, as, fe), and organophosphorus pesticide (acetylcholine inhibition) on mexican pacific estuarine zone, and the relationship of pollutants with immunological and oxidative stress parameters in oyster crassostrea corteziensis. results indicated that the main xenobiotic detected were cu and naphthalene. furthermore, the acethylcholine inhibition tests, suggest the presence of organophosphorus pesticide in the estuary. microbicidal activity was not altered, but a significantly decrease in hemocyte number was detected. on oxidative stress parameters, an increase of superoxide anion, hydrogen peroxide, catalase activity and lipoperoxidation were observed in gills from oyster (unpublished data). in other studies, a positive correlation between xenobiotic concentration presents in ecosystem and increase in defense mechanisms of molluscs has been reported (fisher et al., 2000; oliver et al., 2003).thus, experiment designing have been made to show if the deployment of eastern oyster (crassostrea virginica) from uncontaminated through contaminated sites would increase immune response parameters and vice versa. the results showed that hemocytes count and bactericidal activity were significantly elevated after 12-week deployment at contaminated sites (metals, pahs and pcb) from florida. however, when similar experiment were realized inverted, the results were ambiguous, thus lysozyme concentration was reduced, but hemocyte activities (principally bacteria killing index and hemocyte count) were not challenged. authors suggest that these results could be indicative of an acclimatization response with adaptative consequences of oyster to chronically polluted sites (fisher et al., 2003). conclusion the data presented here suggests that all groups of pollutants may be hazardous to molluscs defense system. in general terms, there are many examples of links between xenobiotic and susceptibility to diseases in wildlife species, principally vertebrates with economical importance. also laboratory tests allowed identifying potential hazard, mainly anthropogenic chemicals with immunosupressor properties. however, invertebrate organisms have ecological relevance besides only economical importance, as they represent around 95 % of all animal species. in this matter is very important to understand the immunologic mechanisms invertebrate as molluscs and relationship with environmental condition. this will allow acknowledging the susceptibility of each species to antigen challenges, mainly infectious agents, and if such conditions affects the intrinsic resistance of each organism. acknowledgments this review was conducted under fomix (conacyt-nayarit government) project (number 131614), and promep-sep founding. thanks to msc c rodríguez-cervantes for english correction style, and md student i parrao for figure design. references auffret m, mujdzic n, corporeau ch, moraga d. xenobiotic-induced immunomodulation in the european flat oyster, ostrea edulis. mar. environ. res. 54: 585-589, 2002. bernal-hernández yy, medina-díaz im, robledomarenco ml, velázquez-fernández jb, girónpérez mi, ortega-cervantes l, et al. acetylcholinesterase and metallothionein in oysters (crassostrea corteziensis) from a subtropical mexican pacific estuary. ecotoxicol. 19: 819-825, 2010. butler r, roesijadi g. metallothionein (mt) gene expression and cadmium-induced immunotoxicity in haemocytes of the eastern oyster crassostrea virginica. mar. environ. res. 50: 465-472, 2000. camus l, jones mb, borshet jf, grosvik be, regoli f, depledge mh. total oxyradical scavenging capacity and cell membrane stability of haemocytes of the arctic scallop, chlamys islandicus, following benzo(a)pyrene exposure. mar. environ. res. 54: 425-430, 2002. canesi l, lorusso l, ciacci c, betti m, rochi m, pojana g, et al. immunomodulation of mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies. aquat. toxiol. 81: 36-44, 2007. champeau o, narbonne jf. effects of tributyltin and 17β-estradiol on immune and lysosomal systems of the asian clam corbicula fluminea (m.). environ. toxicol. pharmacol. 21: 323-330, 2006. fisher w, oliver l, winstead j, volety a. stimulation of defense factors for oysters deployed to contaminated sites in pensacola bay, florida. aquat. toxicol. 64: 375-391, 2003. gagnaire b, gay m, huvet a, ves daniel j, saulnier d, renault t. combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of pacific oyster, crassostrea gigas (thunberg). aquat. toxicol. 84: 92-102, 2007. gagnaire b, thomas-guyon h, burgeot th, renault t. pollutant effects on pacific oyster, crassostrea gigas (thunberg), hemocytes: screening of 23 molecules using flow cytometry. cell. biol. toxicol. 22: 1-14, 2006. gagné f, andré c, cejka p, hausler r, fournier m, blaise c. immunotoxic effects on freshwater mussels of a primary-treated wastewater before and after ozonation: a pilot plant study. ecotoxicol. environ. safety 69: 366-373, 2008. gagné f, blaise c, fournier m, hansen pd. effects of selected pharmaceutical products on phagocytic activity in elliptio complanata mussels. comp. biochem. physiol. 143c: 179186, 2006. 155 galloway t, depledge m. immunotoxicity in invertebrates: measurement and ecotoxicological relevance. ecotoxicol. 10: 5-23, 2001. goméz-mandikute a, cajaraville mp. comparative effects of cadmium, copper, paraquat and benzo[a]pyrene on the actin cytoskeleton and production of reactive oxygen species (ros) in mussel haemocytes. toxicol. in vitro. 17: 539546, 2003. hannam ml, bamber s, galloway t, moody aj, jones m. effects of the model pah phenanthrene on immune function and oxidative stress in haemolymph of the temperature scallop pecten maximus. chemosphere 78: 779-784. 2010. hine pm. the inter-relationship of bivalves haemocytes. fish shellfish immunol. 9: 367385, 1999. matozzo v, marin mg. 4-nonylphenol induces immunomodulation and apoptotic events in the clam tapes philippinarum. mar. ecol. prog. ser. 285: 97-106, 2005. matozzo v, rova g, marin m. haemocytes of the cockle cerastoderma glaucum: morphological characterisation and involvement in immune responses. fish shellfish immunol. 23: 732-746, 2007. matozzo v, monari m, foschi j, cattani o, serrazanetti gp, marin mg. first evidence of altered immune response and resistence to air exposure in the clam chamelea gallina exposed to benzo(a)pyrene. arch. environ. toxicol. 56: 479-488, 2009. mydlarz ld, jones le, harvell d. innate immunity, environmental drivers, and disease ecology of marine and freshwater invertebrates. annu. rev. ecol. evol. syst. 37: 251-88, 2006. nusetti o, marcano l, zapata e, esclapés m, nusetti s, lodeiros c. respuestas inmunológicas y de enzimas antioxidantes en la ostra perla pinctada imbricate (mollusca: pteridae) expuesta a niveles subletales de fuel oil no 6. interciencia 29: 324-328, 2004. oliver l, fisher w, volety a, malaeb z. greater hemocyte bactericidal activity in oysters (crassostrea virginica) from a relatively contaminated site in pensacola bay, florida. aquat. toxicol. 64: 363-373, 2003. parry h, pipe r. interactive effects of temperature and copper on immunocompetence and disease susceptibility in mussels (mytilus edulis). aquat. toxicol. 69: 311-325, 2004. pruzzo c, gallo g, canesi l. persistence of vibrios in marine bivalves: the role of interactions with haemolymph components. environ. microbiol. 7: 761-722, 2005. wootton ec, dyrynda ea, pipe rk, ratcliffe na. comparisons of pah-induced immunomodulation in three bivalve molluscs. aquat. toxicol. 65: 13-25. 2003. 156 identification and preliminary characterisation of a haemagglutinin in the coelomic fluid of sipunculus nudus isj 7: 221-227, 2010 issn 1824-307x short communication identification and preliminary characterization of a ca2+-dependent hemagglutinin in the celomic fluid of sipunculus nudus l ballarin, m del favero department of biology, university of padua, padua, italy accepted october 5, 2010 abstract a soluble agglutinin was purified by affinity chromatography of the celomic fluid of the marine worm sipunculus nudus. this agglutinin requires metal cations for its activity and is specific for derivatives of d-galactose. it resulted lightly thermostable, with a ph optimum around 7.5. on sdspage, it was resolved in two bands, of 33 and 31 kda in reducing conditions and 29 and 26 kda in non-reducing conditions. this behavior is probably due to the presence of disulfide bridges between cysteine residues, which are required for the correct functioning of the hemagglutinin, as βmercaptoethanol completely abolish the agglutinating activity of cell-free celomic fluid. the purified lectin can influence in vitro phagocytosis of yeast by celomic leukocytes: in the presence of the molecule, ingestion of foreign particles results significantly decreased and yeast cells agglutinate and forms rosettes around the celomocytes. this suggests a role of the molecule in immunosurveillance. key words: sipunculus; invertebrates; celomic fluid; hemagglutinin; ca2+-dependent lectin introduction lectins are carbohydrate-binding proteins widely distributed in living organisms, animals included (barondes, 1981; yeaton, 1981a). animal lectins fulfill a variety of functions (yoshizaki, 1990; drickamer and taylor, 1993; gabius, 1997; dodd and drickamer, 2001) and many of them act as recognition molecules within the immune system, implicated in direct first-line defense against pathogens, cell trafficking and immune regulation (yeaton, 1981b; yoshizaki, 1990; drickamer and taylor, 1993; gabius, 1997). as regards the last point, lectins appear to participate in the tagging and exclusion of foreign organisms by invertebrate immunocytes, which are covered with different carbohydrate receptors (yeaton, 1981b; yoshizaki, 1990; gabius, 1997; kilpatrick, 2002). in the last two decades, the study of animal lectins has greatly increased and today we know that lectin activity in animals is found in association with a wide variety of primary structures which enable us to distinguish at least 12 families of sugar-binding proteins and a series of "orphan" lectins belonging to either some unknown lectin family or well-established protein families with the majority of the members unable to ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padua, via ugo bassi 58/b, 35100 padua, italy e-mail: loriano.ballarin@unipd.it bind sugars. in addition, many animal lectins also bind structures other than carbohydrates via proteinprotein, protein-lipid or protein-nucleic acid interactions (gabius, 1997; dodd and drickamer, 2001; kilpatrick, 2002; loris, 2002). sipunculans are marine worms devoid of circulatory system and with a large celomic cavity filled with celomic fluid containing various types of celomocytes, the majority of which are represented by hemerythrocytes, nucleated cells containing the red pigment hemerythrin (valembois and boiledieu, 1980; dybas, 1981). in addition, the celomic fluid contains wandering ciliated urn cell complexes, which secrete mucus able to trap foreign particles or cells (bang and bang, 1976, 1980; dybas, 1976; nicosia and sowinski, 1995), and leukocytes involved in defense responses against non-self materials. leukocytes are classically classified as granulocytes and hyalinocytes on the basis of the presence or absence of acidophilic or basophilic cytoplasmic granules (dybas, 1981). both granulocytes and hyalinocytes can engulf non-self material (brown and winterbottom, 1969; matozzo et al., 2001) and phagocytosis represents the main cell-mediated defense mechanisms against microbes in these organisms. both the cell types share a similar content of hydrolytic lysosomal enzymes and can produce superoxide anions as a consequence of the activation of a phagocytosisassociated respiratory burst. in addition, they contain lysozyme which can be released as a 221 mailto:loriano.ballarin@unipd.it consequence of a bacterial challenge (matozzo et al., 2001). as regards other humoral immune responses, the presence of various factors, such as lysins, agglutinins, opsonins, and antibacterial molecules has been reported in the celomic fluid of various sipunculan species (bang, 1966; weinheimer et al., 1970; evans et al., 1973; matozzo et al., 2001). however, up to now, no clear characterization of soluble molecules involved in immune responses has been carried out. with the aim of contributing to fill this gap, a search for soluble molecules with hemagglutinating activities in the celomic fluid of sipunculus nudus was undertaken. this reports presents some preliminary results on the identification and partial characterization of a ca2+dependent lectin with hemagglutinating activity from the celomic fluid of s. nudus with specificity for dgalactosides and able to influence phagocytosis. materials and methods animals specimens of sipunculus nudus were collected by hydraulic dredging in the sandy bottom (5-6 m depth) of the west coast of the northern adriatic sea, off the lagoon of venice (italy). they were transferred in 15 l aquaria containing abundant sand on the bottom and filtered (5 µm filter) seawater (fsw) at a temperature of 19 °c. celomic fluid collection celomic fluid (cf) was collected from the celomic cavity with a 1 ml plastic syringe. cfs from 10 animals were pooled and centrifuged at 780xg for 10 min and the supernatant was referred as cellfree celomic fluid (cfcf). hemagglutination (ha) assay rabbit erythrocytes were washed three times by centrifugation at 500xg for 10 min in tris-buffered saline (tbs: tris-hcl 50 mm, nacl 150 mm, ph 7.4) and incubated for 30 min at 37 °c in 0.1 mg/ml trypsin in tbs (ballarin et al., 1999). they were then washed again and resuspended in tbs containing 0.2 % gelatin to get a 1 % (v/v) solution. fifty µl of cfcf were serially diluted two-fold with tbs in the wells of u-bottomed microtiter plates and an equal volume of erythrocyte suspension was added to each well; fsw was used in controls. tbs containing 5 mm egta or 5 mm cacl2 (tbs-ca) was also used to assess the ca2+-dependency of the reaction. in another experimental series, cfcf was incubated for 30 min with 1 % rabbit erythrocyte to control the specificity of the interaction: the suspension was then centrifuged at 780xg for 10 min and the erythrocyte-absorbed supernatant was collected and used in the ha assay. plates were gently shaken, incubated for 1 h at 37 °c and then kept at 4 °c. the ha titer (ht), i.e., the reciprocal of the highest dilution giving positive ha was then evaluated. ha inhibition assay the following sugars were assayed for their effects on the agglutination of trypsinised erythrocytes: d-galactose, d-glucose, dgalactosamine, d-glucosamine, n-acetyl-dgalactosamine, n-acetyl-d-glucosamine, methyl-αd-galactopyranoside, methyl-β-dgalactopyranoside, methyl-α-d-glucopyranoside, 2deoxy-d-galactose, d-mannose, d-fucose, lrhamnose, d-melibiose, d-sucrose, d-lactulose, dlactose. they were purchased from sigma (st louis, mo, usa) and added to tbs-ca to yield 400 mm storage solutions. a total of 25 ml of cfcf were then added to an equal volume of two-fold serial dilutions of carbohydrates in the wells of ubottomed microtiter plates which were incubated for 30 min at 37 °c. erythrocytes were then added and, after a further incubation of 60 min at 37°c, the lowest carbohydrate concentrations able to inhibit agglutination were evaluated (modified from parrinello and canicattì, 1982). effects of periodate and β-mercaptoethanol on hemagglutinating activity to evaluate the importance of hemagglutininconjugated carbohydrates on ha, cfcf was incubated for 2 h at 4 °c with an equal volume of 0.08 m sodium meta-periodate in 0.2 m citrate buffer, ph 5.4 acid in order to oxidize sugars (millar and ratcliffe, 1987). the mixture was then dialyzed against tbs-ca for 3 h to remove periodate and used in ha assay. the importance of disulphide bridges in hemagglutinating activity was assessed by incubating cfcf with 20 mm of β-mercaptoethanol. the mixture was then dialyzed as described above and used in ha assay. lectin purification and characterization affinity chromatography of cfcf on acidtreated sepharose cl-6b (pharmacia, uppsala, sweden) was carried out as described by parrinello and canicattì (1982, 1983). the column (7 x 1.6 cm) was previously equilibrated with phosphate-buffered saline (pbs: 0.8 % nacl, 0.02 % kcl, 0.02 % kh2po4, 0.115 % na2hpo4, ph 7.2) containing 5 mm cacl2, loaded with 40 ml of cfcf, and washed with a solution of nacl 1 m and cacl2 5 mm. the flow rate was kept constant at 20 ml/h and 2-ml fractions were collected, the absorbance of which was measured, at 280 nm, with a kontron uvikon 930 uv-vis spectrophotometer. when absorbance resulted stable, at values close to zero, the column was eluted with 0.2 m d-galactose in 0.1 m nacl. a single absorbance peak was usually obtained after elution with d-galactose, and fractions corresponding to the peak were collected, dialyzed overnight at 4 °c against distilled water, lyophilized with a savant vacuum centrifuge, and stored at -20 °c until use. protein concentration was evaluated according to bradford (1976) using bovine serum albumin as standard. sds-page (12 % separating gel) of purified lectin was performed according to laemmli (1970). samples of lyophilized lectin were diluted to 1.0 mg/ml in sample buffer (0.5 m tris-hcl, ph 6.8, 10 % glycerol, 10 % sds, 0.5 % bromophenol blue with or without 5 % β-mercaptoethanol, for reducing and non-reducing conditions, respectively. proteins treated with β-mercaptoethanol were also boiled for 5 min. gels were calibrated with low molecular 222 weight marker proteins (biorad laboratories, hercules, ca, usa), run at a constant current of 18 ma/gel for approximately 3.5 h and stained with coomassie blue. effects of temperature and ph on hemagglutinating activity to study the effects of temperature on hemagglutinating activity, the sipunculus agglutinin (sa), at a concentration of 2.0 mg/ml in distilled water, was incubated for 30 min at 4, 25, 37, 60 and 80 °c, and then used in the ha assay as previously described. the stability of the lectin, at the above concentration, was tested by assaying its agglutinating activity, in tbs-ca, after incubation at room temperature for 0, 30, 60, 90, 120 and 180 min. fig. 1 presence and absence of hemagglutinating activity of s. nudus cfcf incubated in fsw (a) or fsw containing 5 mm egta (b). ht, hemagglutination titer fixed for 30 min at 4°c in a solution of 1 % glutaraldehyde and 1 % sucrose in fsw, and stained with 10 % giemsa for 5 min. at least 300 hemocytes per coverslip were observed under a leitz dialux 22 light microscope, in ten optical fields, at a magnification of 1250x, to determine the percentage of hemocytes with ingested yeast cells. each experiment was repeated three times with three different celomocyte pools. data are expressed as mean ± sd and were compared using the χ2 test. the effect of ph was evaluated using the following buffers in the ha assay, in the presence of 5 mm cacl2: 0.2 m tris-maleate (ph 6.0, 6.6, 7.0, 7.6, 8.0), 0.2 m glycine-naoh (ph 8.6, 9.0, 9.6, 10.0). data are expressed as mean ± sd. phagocytosis assay sixty µl of cf, collected as described above, were placed in the centre of culture chambers, prepared as described by ballarin et al. (1994) and cells were left to adhere to coverslips for 30 min at room temperature (rt). after adhesion, slides were repeatedly washed by dipping in a large volume of fsw in order to remove hemerythrocytes, which do not adhere to glass, and the remaining celomocytes were incubated with 60 ml of a suspension of yeast cell (yeast: hemocyte ratio = 10 : 1) in fsw in the presence or in the absence of 0.5 mg/ml of sa in fsw. in another series of experiments, yeast was previously incubated with sa (0.5 mg/ml in fsw) for 30 min before the assay. slides were then washed in fws to remove uningested yeast and cells were results cfcf shows hemagglutinating activity cfcf can agglutinate trypsinized rabbit erythrocytes (ht: 64; fig. 1a). ha was almost absent in erythrocyte-absorbed cfcf. it was inhibited in presence of various monosaccharides and disaccharides at various concentrations. galactose resulted the most powerful inhibiting sugar, showing the lowest minimum effective concentration, followed by other galactosides and galactose-containing disaccharides (table 1). table 1 effects of different sugars in hemagglutinating activity of s. nudus cfcf sugar minimum effective concentration (mm) d-galactose 50 d-glucose 200 d-galactosamine > 400 d-glucosamine > 400 n-acetyl-d-galactosamine — n-acetyl-d-glucosamine — methyl-α-d-galactopyranoside 100 methyl-α-d-glucopyranoside 200 methyl-β-d-galactopyranoside > 400 2-deoxy-d-galactose 400 d-fucose — l-rhamnose — d-mannose — d-sucrose — d-raffinose — d-lactulose 200 d-lactose 200 d-melibiose 400 cfcf, cell-free celomic fluid 223 fig. 2 affinity chromatography of s. nudus cfcf on acid-treated sepharose cl-6b. arrows indicate addition of galactose. the hemagglutinating activity required bivalent cations, as indicated by the absence of ha in the presence of egta (fig. 1b). treatment with periodate and β-mercaptoethanol completely abolished the ability of cfcf to agglutinate rabbit erythrocytes. hemagglutinin purification and physico-chemical characterization of the lectin sugar specificity was exploited to purify soluble sa by affinity chromatography on acid-treated sepharose cl-6b. we obtained a single peak showing hemagglutinating activity towards trypsinized rabbit erythrocytes (fig. 2). when sa was incubated at rt, its ht remained stable at the value of 64 after 60 min, but dropped to 16 after 120 min and conserved this residual activity in the following 4 h. the agglutinin resulted lightly thermostable as the ht remained stable at the value of 64 after 30 min exposure at temperatures ranging from 4 to 25 °c; decreased to 8 after 30 min at 37 °c and retained residual detectable activity (ht: 2) after 30 min at 80 °c (fig. 3a). the lectin was stable within ph ranging from 7.0 to 9.5, with maximum activity around 7.5 (fig. 3b). after sds-page, of the affinity-purified material, two bands were obtained with apparent molecular weight of 33 and 30 kda and 29 and 26 kda under reducing and non-reducing conditions, respectively (fig. 4). fig. 3 effects of ph (a) and temperature (b) on hemagglutinating activity of s. nudus cfcf. ht, hemagglutination titer 224 purified lectin can influence phagocytosis most of the s. nudus leukocytes can phagocytose foreign cells or particles (fig. 5a, table 2). incubation of celomocytes and yeast with the purified lectin resulted in a significant decrease of in vitro yeast phagocytosis (table 2). yeast cells appeared agglutinated and frequently form rosettes or clumps adherent to celomocyte surface without being ingested (figs 5b, c). when yeast was preincubated with the purified lectin, washed and incubated with celomocytes in fsw, no significant variation in the fraction of phagocytozing cells with respect to controls was observed (table 2). discussion in the present study, we have identified a lectin with agglutinating properties in the celomic fluid of s. nudus. d-galactose shows the highest inhibiting power and the molecule resulted specific for derivatives of d-galactose, which share the c4 hydroxyl in β position, whereas limited or no effects on ha were observed in the presence of glucosides, mannose, and rhamnose. the c6 hydroxyl is important for sugar-lectin interaction as its absence in fucose resulted in the lack of inhibition. the inhibition of agglutination is influenced by the addition of a methyl group to c1 hydroxyl: it leads to a light decreases if in position α (in methyl-α-dgalactopyranoside ),  but to a higher decrease when in position β (in methyl-β-d-galactopyranoside). the absence of c2 hydroxyl (in 2-deoxy-d-galactose) reduces the inhibitory power which is further decreased by its substitution as in d-galactosamine and n.acetyl-d-galactosamine: in this case the degree of inhibition depends on the steric hindrance, the latter compound being less effective than the former. among disaccharides, those containing galactose, i.e., lactulose and lactose, show inhibition of ha although to a less extent than the monosaccharide. fig. 4 sds-page of s. nudus purified lectin (b, c) and cfcf (d). lane a) molecular weight markers; lanes b and c) non reducing and reducing conditions, respectively. this excludes its belonging to galectin family, that includes the majority of ca2+-independent lectins, none of which are glycoproteins (kasai and hirabayashi, 1996); ii) the absence of any inhibitory effect on ha of rhamnose and melibiose. this indicates that sa is not a member of rhamnosebinding lectins, another family of sugar-binding proteins which do not require divalent cations for their activity (jimbo et al., 2007; terada et al., 2007; gasparini et al., 2008). two bands were present in the electrophoretic pattern of the affinity-purified material. similar patterns were reported for other purified lectins (parrinello and arizza, 1989; arizza et al., 1991; gasparini et al., 2008) and they can be interpreted as the consequence of the presence, in the pooled samples, of lightly different isoforms of the same molecule. the presence of different isoforms in the pooled hemolysate has been recently demonstrated the observed dependency on divalent cations suggests that the identified lectin belongs to the clectin family. this is further supported by: i) the nature of the protein which carries glycoconjugates required for the interaction with ligands, as indicated by the absence of ha after treatment with periodate. table 2 effect of the preincubation and incubation of yeast cells in the affinity-purified lectin on yeast phagocytosis by sipunculus leukocytes preincubation medium incubation medium percentage of phagocytosing cells fsw fsw (control) 54.8 ± 8.9 fsw sa 39.5 ± 5.3 ** sa fsw 47.1 ± 5.6 fsw, seawater; sa, sipunculus agglutinin ** = p < 0.01 225 fig. 5 a) phagocytes filled with ingested yeast cells (arrowheads); b) yeast cells clumped around a leukocyte (arrow) forming a rosette; c) agglutinated yeast cells in the proximity of a leukocyte (arrow) with ingested yeast cells (arrowheads). bar = 10 µm. in the compound ascidian botryllus schlosseri (gasparini et al., 2008). the apparent molecular weight of the two protein bands of 29 and 26 kda under non-reducing and 33 and 31 kda under reducing conditions suggests the presence of intramolecular disulphide bridges which keep the proteins in a highly folded form in non-reducing conditions so that they can move more rapidly in page. this is supported by the observation that βmercaptoethanol completely abolish the agglutinating capability of cfcf, which stress the importance of the disulphide bonds for the correct functioning of the hemagglutinin. in addition, similar electrophoretic behavior has been reported for various ascidian lectins (parrinello and arizza, 1988; cammarata et al., 2007; gasparini et al., 2008). despite the huge number of published papers on invertebrate lectins, their biological role is still a matter of debate. in most cases, they are thought to be involved in immune recognition, although only in few cases it has been clearly demonstrated. our lectin is involved in cell-cell interactions between: i) yeast cells enabling their agglutination and hindering their phagocytosis; ii) celomocytes and yeast cells leading to the formation of rosettes. the formation of large aggregates of foreign cells, although preventing their phagocytosis, may stimulate their encapsulation and subsequent melanization by wandering celomocytes, leading to the final formation of brown bodies, present in the hemolymphatic or celomic cavities of many invertebrates (hetzel, 1965; valembois et al., 1992, 1994; jans et al., 1996; pagliara et al., 2003). we have no direct evidences, at the moment, that this can occur in the case of yeast cells, but numerous brown bodies were frequently found in the celom of the animal used in the present experiments. future efforts are, therefore, required to better understand the role of the lectin, identify the source of the protein and complete its sequence for comparative and functional studies. acknowlegements the authors wish to thank dr a ferrarese and dr a faggin for technical help. this work was supported by the italian miur grant. references arizza v, parrinello n, schimmenti s. in vitro release of lectins by phallusia mamillata hemocytes. dev. comp. immunol. 15: 219-226, 1991. ballarin l, cima f, sabbadin a. phagocytosis in the colonial ascidian botryllus schlosseri. dev. comp. immunol. 18: 467-481, 1994. ballarin l, tonello c, guidolin l, sabbadin a. purification and characterization of a humoral opsonin, with specificity for d-galactose, in the colonial ascidian botryllus schlosseri. comp. biochem. physiol. 123b: 115-123, 1999. bang fb. serologic response in a marine worm, sipunculus nudus. j. immunol. 96: 960-972, 1966. bang bg, bang fb. the mucous secretory apparatus of the free urn cell of sipunculus nudus. cah. biol. mar. 17: 423-432, 1976. bang bg, bang fb. the urn cell complex of sipunculus nudus: a model for study of mucusstimulating substances. biol. bull. 159: 571581, 1980. barondes sh. lectins: their multiple endogenous cellular functions. ann. rev. biochem. 50: 207231, 1981. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. anal. biochem. 72: 248-254, 1976. brown ac, winterbottom r. the fate of thorium dioxide injected into the coelom of the sipunculid, golfingia capensis. j. invertebr. pathol. 13: 229-234, 1969. cammarata m, benenati g, odom ew, salerno g, vizzini a, vasta gr, et al. isolation and 226 characterization of a fish f-type lectin from gilt head bream (sparus aurata) serum. biochim. biophys. acta 1770: 150-155, 2007. nicosia sv, sowinski jm. cytological analysis of the urn cell complex of sipunculus nudus before and after serum-induced secretion. biol. bull. 188: 267-280, 1995. dodd rb, drickamer k. lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity. glycobiology 11: 71r-79r, 2001. pagliara p, candia carnevali md, burighel p, ballarin l. the spherule cells of holothuria polii delle chiaie, 1823, (aspidochirata, holothuroidea) during brown body formation: an ultrastructural study. j. submicrosc. cytol. pathol. 35: 295-301, 2003. drickamer k, taylor me. biology of animal lectins. ann. rev. cell biol. 9: 237-264, 1993. dybas l. 1976. a light and electron microscopic study of the ciliated urn of phascolosoma agassizii (sipunculida). cell tissue res. 169: 67-75, 1993. parrinello n, canicattì c. carbohydrate binding specificity and purification by biospecific affinity chromatography of ascidia malaca traust hemagglutinins. dev. comp. immunol. 6: 53-64, 1982. dybas l. sipunculans and echiuroids. in: ratcliffe na, rowley af (eds), invertebrate blood cells, vol. 1, academic press, new york, london, pp 161-188, 1981. parrinello n, canicattì c. lactose-binding haemagglutinins from the ascidian phallusia mamillata (cuv.). biol. bull. 164: 124-135, 1983. evans ee, cushing je, evans ml. comparative immunology: sipunculid bactericidal responses. infect. immun. 8: 355-359, 1973. parrinello n, arizza v. d-galactose binding lectins from the tunicate ascidia malaca: subunit characterization and hemocyte surface distribution. dev. comp. immunol. 12: 495-507, 1988. gabius h-j. animal lectins. eur. j. biochem. 243: 543-576, 1997. gasparini f, franchi n, spolaore b, ballarin l. novel rhamnose-binding lectins from the colonial ascidian botryllus schlosseri. dev. comp. immunol. 32: 1177-1191, 2008. parrinello n, arizza v. sugar specific cellular lectins of phallusia mamillata hemocytes: purification, characterization and evidence for cell surface localization. dev. comp. immunol. 13: 113-121, 1989. hetzel hr. studies on holothurian coelomocytes. ii. the origin of coelomocytes and the formation of brown bodies. biol. bull. 128: 102-111, 1965. terada t, watanabe y, tateno h, naganuma t, ogawa t, muramoto k, et al. structural characterization of a rhamnose-binding glycoprotein (lectin) from spanish mackerel (scomberomorous niphonius) eggs. biochim. biophys. acta 1770: 617-629, 2007. jans d, dubois p, jangoux m. defensive mechanisms of holothuroids (echinodermata): formation, role, and fate of intracoelomic brown bodies in the sea cucumber holothuria tubulosa. cell tissue res. 283: 99-106, 1996. jimbo m, usui r, sakai r, muramoto k, kamiya h. purification, cloning and characterization of egg lectins from the teleost tribolodon brandti. comp. biochem. physiol. 147b: 164-171, 2007. valembois p, boiledieu d. fine structure and functions of haemerythrocytes and leucocytes of sipunculus nudus. j. morphol. 163: 69-77, 1980. kasai k, hirabayashi j. galectins: a family of animal lectins that decipher glycocodes. j. biochem. 119: 1-8, 1996. valembois p, lassègues m, roch p. formation of brown bodies in the coelomic cavity of the earthworm eisenia fetida andrei and attendant changes in shape and adhesive capacity of constitutive cells. dev. comp. immunol. 16: 95101, 1992. kilpatrick cd. animal lectins: a historical introduction and overview. biochim. biophys. acta 1572: 187-197, 2002. laemmli uk. cleavage of structural protein during the assembly of the head of the bacteriophage t4. nature 277: 680-685, 1970. valembois p, seymour j, lassègues m. evidence of lipofuscin and melanin in the brown body of the earthworm eisenia fetida andrei. cell tissue res. 277: 183-188, 1994. loris r. principles of structures of animal and plant lectins. biochim. biophys. acta 1572: 198-208, 2002. weinheimer pf, acton rt, cushing je, evans ee. reactions of sipunculid coelomic fluid with erythrocytes. life sci. 9: 145-152, 1970. matozzo v, cima f, perin l, ballarin l. phagocytic and enzymatic activities of cells and urn cell complexes in the coelomic fluid of the marine worm sipunculus nudus (sipuncula). ital. j. zool. 68: 273-280, 2001. yeaton rw. invertebrate lectins: i. occurrence. dev. comp. immunol. 5: 391-402, 1981a. yeaton rw. invertebrate lectins: ii. diversity of specificity, biological synthesis and function in recognition. dev. comp. immunol. 5: 535-545, 1981b. millar da, ratcliffe na. activity and preliminary characterization of a hemagglutinin from the hemichordate saccoglossus ruber. dev. comp. immunol. 11: 309-320, 1987. yoshizaki n. functions and properties of animal lectins. zool. sci. 7: 581-591, 1990. 227 isj 3: 97-102, 2006 isj 3: 118-124, 2006 issn 1824-307x minireview cytoskeletal proteins and morphogenesis in planarians a fagotti, f simoncelli, i di rosa, r pascolini department of cellular and environmental biology, university of perugia, perugia, italy accepted november 29, 2006 abstract regeneration processes employ a series of differentiative events related to various embryonic morphogenetic phenomena in which the cytoskeleton plays a fundamental role. planarians are an excellent model to study development mechanism because they show an exceptional physiological morphogenetic plasticity that is also at the basis of their extraordinary regenerative ability. in this paper we discuss results concerning two cytoskeleton components, actin and tubulin, during morphogenetic processes of the planarian schmidtea polychroa. comparative studies on cytoskeleton function during morphogenetic processes in evolutionary-distant animal models can offer important new insights in the fields of stem cell biology and regenerative medicine. key words: planarian morphogenesis; actin; tubulin __________________________________________________________________________________________ introduction the cytoskeleton of eukaryotic cells plays a basic role in many dynamic processes that are controlled by a network of cytoskeletal fibers constituted by three types of filaments: microfilaments made up of various actin isoforms, microtubules consisting of αand β-tubulin, and intermediate filaments. these cytoskeletal components are associated with hundreds of proteins that regulate a rich variety of structural or dynamic machineries, crucial for many essential cellular functions including cell migration, cell division, maintenance of cell shape, cell signalling, chromosome and organelle movements. the cytoskeleton also plays a pivotal role in cellsubstrate and cell-cell adhesions, supplying the structural support necessary for these contacts as well as providing the focal point for signal transductions. important advances have been made in the last years not only in characterizing the molecular and functional mechanisms of cytoskeleton but also in defining connections between cytoskeleton disorders and pathological conditions. cytoskeletal protein abnormalities are frequently the origin of many pathological conditions ___________________________________________________________________________ corresponding author: rita pascolini department of cellular and environmental biology university of perugia via a. pascoli 1, 06123 perugia, italy e-mail: ritapasc@unipg.it including several cardiovascular, muscle, neurodegenerative and skin diseases and cancer (ramaekers and bosman, 2004). many studies in various animal developmental models have also demonstrated the importance of cytoskeletal proteins in controlling cellular behavior during the morphogenetic and developmental processes. tissue building which occurs during both embryo development and wound-healing events, involves a series of cellular dynamics and movements that are driven by similarly orchestrated cytoskeletal machinery (martin and parkhurst, 2004; redd et al., 2004). embryonic epithelial wound repair shows striking similarities in cytoskeletal remodelling with morphogenetic events of dorsal closure in drosophila and ventral enclosure in caenorhabditis embryos (martin and parkhurst, 2004; wood et al., 2002). therefore, the study of cytoskeleton changes during wound repair can offer insights about evolutionary conserved cytoskeleton functions during embryo morphogenetic processes and viceversa. in this review we report and discuss results concerning the involvement of cytoskeletal proteins during the morphogenetic events underlying regeneration in freshwater planarians. planarians are a fascinating model for studying the development processes because they show a great physiological morphogenetic plasticity that is also at the basis of their exceptional regenerative ability. there is a high degree of molecular and functional conservation throughout the phylogeny in the basic 118 mechanisms of the developmental phenomena: the elucidation of molecular mechanisms underlying morphogenetic plasticity in animal models such as planarians will contribute to clarify key issues in the fields of wound healing and regenerative medicine. developmental plasticity in planarians the biological peculiarity of the planarian model is characterized by a continuous status of morphogenesis; its tissues are physiologically subjected to a continuous cell renewal. a striking example is its ability to either grow or de-grow depending on nutritional availability: the significant modifications in body size to which planarians are subjected always maintain the correct proportions and are reversible (baguñà and romero, 1981). the great developmental plasticity is also directly related to extraordinary regenerative power; it is known that planarians are able to rebuild a complete organism from a very small body region. the source of this morphological plasticity and regenerative properties is a stable population of totipotent stemcells, called “neoblasts”, that, in the adult, are the only proliferating cells (baguñà, 1976a, b). these stem cells are able to generate all different cell types present in the adult and are responsible for physiological homeostasis and tissue renewal. regeneration in planarians regeneration is widely distributed among metazoans: a large number of species have the capacity to replace injured or lost body parts and, in few cases, to rebuild the entire organism from a tiny portion of the body. models of regeneration are provided by cnidarian, platyhelminthes and echinodermata, that can regenerate a complete organism by bi-directional regeneration, and by urodele amphibians that can rebuild a complete appendage. morgan (1901) described two major types of regeneration: 1) morphallaxis, that involves remodelling and re-patterning of pre-existing tissues into newly organized structures as occurs in hydra, the classical example of this form of regeneration and 2) epimorphosis, that requires active cell proliferation at the wound site and the formation of a new growth zone from which the missing structure will be regenerated and correctly patterned. this mode of regeneration can involve the dedifferentiation of cells that then proliferate and redifferentiate to form new structures, such as occurs in amphibians, and/or the activation of preexisting stem cell populations to form a new stump, the blastema, that then differentiates into missing body parts. planarians are capable of blastemabased epimorphic regeneration. they are triploblastic acoelomates belonging to the platyhelminthes clade and are included as members of the lophotrochozoa (adoutte et al., 2000). although the epimorphosis is the prevalent regenerative process in planarians, morphallactic events also contribute to the restoration throughout the re-establishment of the exact body proportion and symmetry. the two phenomena can contribute differently to regeneration process depending on the starting-conditions. as mentioned before, epimorphic fig. 1 electron micrograph of a cluster of neoblasts. bar = 2 μm. regeneration in planarians is based mainly on a population of undifferentiated cells, the neoblasts. they are small (5-8 µm in diameter) with large nuclei and very little cytoplasm and are distributed throughout the parenchyma or mesenchyme along the whole body (fig. 1) (reddien and sànchez alvarado, 2004). when a planarian is injured a strong muscular contraction occurs immediately to minimize the wound area that is then rapidly covered by a thin layer of epithelium (pascolini et al., 1984; 1988a, b; baguñà et al., 1994; sànchez alvarado and newmark, 1998). sequentially, the neoblasts proliferate close to the site of injury and migrate distally accumulating under the wound epithelium. they form a regenerative blastema, the unpigmentated structure in which missing tissues regenerate in a week (dubois, 1949; baguñà, 1976b; saló and baguñà, 1984). once inside the blastema, the neoblasts no longer divide (saló and baguñà, 1984). there are two principal hypotheses concerning the source of blastema-forming cells: one is based on a transdetermination event that is supported by evidence that somatic cells can originate from undifferentiated cells committed to becoming germ cells (gremigni and miceli, 1980); the second is based on the totipotency of preexisting stem cells that proliferate in response to injury. the evidence that blastema is constituted by pre-existing stem cells came from experiments of xray irradiation after which planarians completely lost the ability to regenerate and died within several weeks. the irradiated animals only regained their regenerative ability when injected with an enriched fraction of neoblasts (brøndsted, 1969; baguñà et al., 1989). moreover, brdu labelling experiments of neoblasts confirmed that planarian stem cells contribute to the regeneration process through an active migration towards the wound region (newmark and sànchez alvarado, 2000). new molecular and genetic approaches have allowed to identify molecular markers of neoblasts and developmental genes important for both stemcell biology and regeneration. recently, proteins belonging to the argonaute/piwi family, smedwi-1 and smedwi-2, with a regulative role in the production of neoblast progeny have been isolated in schmidtea mediterranea (reddien et al., 2005). djpum, a member of evolutionary conserved puf 119 fig. 2 a) schematic representation of the head amputation in planarian. the blastema area at various times of regeneration is used for immunoblotting. b, c) immunoblotting of planarian blastema area at 0, 1, 2, 5, 7 days of regeneration decorated with anti-αsm-1 (b) and anti-total actin (c) mabs. family of rna-binding proteins, homologue of drosophila puf gene pumilio, also plays an essential function in dugesia japonica neoblast maintenance by supporting their mitotic proliferation (salvetti et al., 2005). the analysis of several neoblast markers, such as pcna protein and djpiwi-1 in dugesia japonica, have suggested that a neoblast population could be heterogeneous (ito et al., 2001; rossi et al., 2006). the molecular processes underlying regeneration in planarians are largely unknown. it is likely that a combination of chemical and mechanical cues stimulate blastema growth such as the triggers originating from wound epithelium, dorsal/ventral (d/v) pattern information and epidermis-mesenchyme interactions (chandebois, 1979; baguñà et al., 1988; kato et al., 1999, 2001). cytoskeletal proteins in planarians there is a high degree of molecular and functional conservation of the cytoskeletal proteins during animal evolution (doolittle, 1995). knowledge about the role of cytoskeleton proteins has increased due to immunochemical and molecular and genetic studies on animal models at different evolutionary levels such as yeast, dictyostelium, caenorhabditis, drosophila and mice. this paper discusses data concerning actin and tubulin, the most conserved cytoskeletal components, during morphogenetic processes of the planarian schmidtea polychroa. actin actin is the major component of the microfilament system of eukaryotic organisms and plays a central role in cell shape and motility processes. in most organisms it is encoded by a multigene family encoding different isoforms regulated in a tissue-, cell type-, and contextdependent fashion (vandekerckhove and weber, 1984; rubenstein, 1990; herman, 1993). the high evolutionary conservation reflects a stringent functional and structural constraint to interact with itself, as well as with a variety of other proteins (hennessey et al., 1993). among the higher vertebrates, six isoforms are present and grouped into two classes, muscle (α-skeletal, α-cardiac, αsmooth muscle, and γ-smooth muscle) and cytoplasmic (β and γ) actins (vandekerckhove and weber, 1978). the distinction between muscle and non-muscle actins is a characteristic of higher animals and insects; in other invertebrate the actin isoforms are mainly similar to cytoplasmic type. it has been hypothesized that muscle actins have appeared from non-muscle actins by gene duplication and divergence twice during animal evolution (mounier et al., 1992).the major differences among actin isoforms are essentially their nh2-terminal sequences. it has been hypothesized that this specific domain could be responsible for specialized functions due to the binding to specific actin-binding proteins. many expressing studies have demonstrated that actin isoforms show typical modulations during cell differentiation events characteristic of both celland tissue development and wound repair (sawtell and lessard, 1989; ruzicka and schwartz, 1988; darby et al., 1990; gunning et al., 1997). it has been demonstrated that in mammals α-smooth muscle isoactin is a marker of differentiation of both vascular smooth muscle cell during embryogenesis and regeneration, and myofibroblasts in wound healing (darby et al., 1990; serini and gabbiani, 1999). the development of striated muscle cells also shows in myotome the transient expression of the alpha-vascular smooth actin (woodcock-michell et al., 1988; sawtell and lessard, 1989). α-skeletal and α-cardiac actin isoforms are involved in cardiomyogenesis (ruzicka and schwartz, 1988). by using immunochemicaland pcr-based approaches various actin isoforms have been characterized in the planarian s. polychroa (pascolini et al., 1988a, b, 1992a, b; fagotti et al., 1998). a specific cytoplasmic isoform has been shown to be involved in cell differentiation and 120 fig. 3 immunoelectron micrographs of migrating neoblast stained with anti-αsm-1 mab. the labelling is mainly localized at the level of pseudopodia and filopodia (arrow). n, nucleus. bar = 0.5 μm (modified from pascolini et al., 1992b). morphogenesis (pascolini et al., 1992b; di rosa et al., 1994a). this isoactin was revealed by using the anti-αsm-1 mab that selectively recognized the nh2-terminus sequence ac-eeed, the specific domain of the endothermic vertebrate α-smooth muscle actin (skalli et al., 1986; chaponnier et al., 1995). in normal planarians, this anti-αsm-1 reactive actin is localized in cytoplasmic domains of migrating undifferentiated neoblasts as well as in epithelial and nerve cells (pascolini et al., 1992b; di rosa et al., 1994b). interestingly, when a planarian is induced to regenerate, the expression of antiαsm-1 reactive actin is up-regulated in the restricted region of blastema. there is a significant increase of this protein after two days of regeneration, it increases gradually to reach a maximum expression at about 5-6 days and then gradually decreases (fig. 2) (pascolini et al., 1992b). fine analysis of blastema has shown that the anti-αsm-1 reactive actin localization is associated with cytoskeletal re-arrangement that occurs during the activated neoblast migration and differentiation (pascolini et al., 1992b; di rosa et al., 1994a). in migrating neoblasts, whose activation is amplified during blastema growth, the labelling was mainly localized at the level of the filamentous structures of pseudopodia and filopodia (fig. 3). differentiating myoblasts, that are the neoblasts committed to originate muscle fibers, also transiently express this isoactin which disappears when the cellular phenotype is differentiated and the myofibers are organized (fig. 4). it is interesting to note that this specific expression pattern resembles those described for α-smooth muscle actin during the myogenesis process in higher vertebrates (woodcock-michell et al., 1988). the involvement of anti-αsm-1 reactive actin has also been well documented in the re-epithelialization process. the planarian epidermis consists of a single layer of columnar cells linked to one another in the apical portion by septate junctions and anchored to the basement membrane by hemidesmosomes (fig. 5) (hori, 1989; di rosa et al., 1994a). the basal region of epidermal cells form the characteristic processes, called ‘feet’, that are rich in intermediate filaments and microfilaments that are selectively labelled by an anti-αsm-1 mab (pascolini et al., 1992b; di rosa et al., 1994a). fig. 4 immunoelectron micrographs of differentiating myoblast stained with anti-αsm-1 mab. the labelling is not detected in the organized myofibers (arrow). bar = 0.5 μm (modified from pascolini et al., 1992b). because of the inability of epidermal cells to divide, renewal depends on neoblasts that migrate across basement membrane from the parenchyma (di rosa et al., 1994a; newmark and sanchez alvarado, 2000). this phenomenon is amplified during wound repair. in response to an injury, the wounded surface is first quickly covered by the cellular spreading of the old differentiated epidermal cells that lose their organized structure (hori, 1991; pascolini et al., 1984, 1988a, b; baguñà et al., 1994; sánchez alvarado and newmark, 1998). subsequently, the wound epidermis is renewed in about 1 week by the active migration and differentiation of stem cells from the blastema (newmark and sanchez alvarado, 2000). during their migration through basal lamina, the epidermal cell precursors over-express anti-αsm-1 reactive actin. they undergo a spatial and temporal cytoskeletal remodelling that involves the formation of stress-fibers in both lateral and basal cellular domains, that are specifically labelled by anti-αsm-1 (fig. 6) (pascolini et al., 1992b; di rosa et al., 1994a). fig. 5 electron micrograph of intact planarian monolayered epidermis (ep) showing basal ‘feet’ (arrow) anchored to the basement membrane (b m) and a differentiating epidermal cell ( ). m, myofiber. bar = 2 μm. 121 fig. 6 immunoelectron micrograph of regenerating planarian epidermis (ep) decorated with anti-αsm-1 mab: a differentiating epidermal cell ( ) appears markedly labelled. b m, basement membrane. bar = 1 μm (modified from pascolini et al., 1992b). these results demonstrate the modulation of anti-αsm-1 reactive actin in various planarian morphogenetic phenomena, such as blastema formation, myogenesis and re-epithelialization and strongly support the hypothesis of its specific role in cell differentiation. in particular, this specific actin is involved in neoblast activation and motility through a characteristic expression. tubulin microtubules are a ubiquitous cytoskeletal component present in all eukaryotic cells. they are formed by the self-assembly of αand β-tubulin heterodimers and are involved in various cellular functions such as cell division, intracellular transport, maintenance of cell shape and flagellar and ciliary motility. the variety of functions can probably be attributed to the expression of different genes that are selectively expressed in specific cell types or at specific stages of development. a tubulin cdna (sptub-1) has been isolated in planaria s. polychroa. sptub-1 encodes for a tubulin protein which shares the highest degree of similarity with the known α-tubulin sequences (simoncelli et al., 2003). the study of transcript expression shows that sptub-1 is selectively restricted to testis tissues that consist of many elliptical follicles organized in clusters that are located on the dorsal side of the animal (fig. 7a). our results show that sptub-1 mrna was expressed in the testis at the level of spermatogenetic cells as spermatogonia, spermatocytes and spermatids (fig. 7b). no signal was detected in the spermatozoa found inside the testis. the expression pattern suggests that sptub1 is a tubulin isotype specific of undifferentiated and differentiating germinal cells with a possible role in spermatogenesis. conclusions and perspectives morphogenesis includes processes in which cytoskeleton and cell migration are strongly involved. many studies on the molecular mechanisms of physiological and pathological processes, such as embryonic morphogenesis, wound healing, immune surveillance and cancer metastasis, indicate that a coordinated organization of cytoskeletal proteins is central to cell behavior, cell migration, intracellular signalling and cell-cycle control processes. our studies on planarian morphogenesis have demonstrated that cytoskeletal proteins are strongly implicated in the differentiation processes, that also occur in higher animals. in particular the findings reported in planarian show that: 1) a specific actin isoform is a marker of neoblast activation and differentiation. its modulation during morphogenetic phenomena suggests that this isoactin is necessary for regeneration. future studies should investigate the molecular dynamic of the anti-αsm-1 reactive actin. studies are in progress to confirm its specific involvement in cell locomotion processes. rna interference studies should also analyze the effects of loss-of-function of this isoactin gene during both physiological and regenerative conditions; fig. 7 a) in situ hybridization of sptub-1 mrna in planarian male gonad: the transcript is exclusively expressed in testis (te). bar = 10 μm (modified from simoncelli et al., 2003). b) in situ hybridization of sptub-1 mrna in planarian male gonad: higher magnification showing that the signal is restricted to spermatogonia, spermatocytes and spermatidis but not in the spermatozoa (sp). bar = 10 μm (modified from simoncelli et al., 2003). 122 2) a tubulin isoform is selectively expressed during spermatogenesis. further study should focus on the mechanisms underlying germ cell fate determination. the role of this tubulin should also be studied in relation to the expression of other genes implicated in planarian gametogenesis, such as vasa-like genes. greater knowledge about the cytoskeletal components and their function during morphogenetic phenomena in planarian sheds light the fundamental process of cell differentiation during animal evolution. comparative studies of molecular and functional cytoskeletal regulation among different animal models can provide important new insights on cytoskeleton function and reveal conserved mechanisms. acknowledgements we thank dr. nancy hutchinson for critical reading of the manuscript. references adoutte a. the new animal phylogeny: reliability and implications. proc.natl. acad.sci. usa 97: 4453-4456, 2000. baguñà j. mitosis in the intact and regenerating planarian dugesia mediterranea n. sp. i. mitotic studies during growth, feeding and starvation. j. exp. zool. 195: 53-64, 1976a. baguñà j. mitosis in the intact and regenerating planarian dugesia mediterranea n. sp. ii. mitotic studies during regeneration and a possible mechanism of blastema formation. j. exp. zool. 195: 65-80, 1976b. baguñà j and romero r. quantitative analysis of cell types during growth, degrowth and regeneration in the planarians dugesia mediterranea and dugesia tigrina. hydrobiologia 84: 181-194, 1981. baguñà j, saló e, collet j, auladell mc, ribas m. cellular, molecular and genetic approaches to regeneration and pattern formations in planarians. fortschr. zool. 36: 65-78, 1988. baguñà j, saló e, auladell c. regeneration and pattern formation in planarians. iii. evidence that neoblasts are totipotent stem cells and the source of blastema cells. development 107: 7786,1989. baguñà j, salò e, romero r, garcia-fernandez j, bueno d, muñoz-marmol am. regeneration and pattern formation in planarians: cells, molecules and genes. zool.sci. 11: 781-795, 1994. brøndsted hv. planarian regeneration. pergamon press, oxford, uk, 1969. chandebois r. the dynamics of wound closure and its role in the programming of planarian regeneration. i. blastema emergence. dev. growth differ. 21: 195-204, 1979. chaponnier c, goethals m, janmey pa, gabbiani f, gabbiani g, vandekerckhove j. the specific nh2-terminal sequence ac-eeed of alphasmooth muscle actin plays a role in polymerization in vitro and in vivo. j. cell biol. 130: 887-895, 1995. darby i, skalli o, gabbiani g. α-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. lab.invest. 68: 21-29, 1990. di rosa i, panara f, fagotti a, simoncelli f, contenti s, gabbiani g, et al. the modulation and spatial distribution of an anti-αsm-1 reactive actin during planarian wound repair. j. submic. cytol. pathol. 26: 293-299, 1994a. di rosa i, fagotti a, simoncelli f, gabbiani g, panara f, pascolini r. immunoelectron microscopic localization of an anti-αsm-1 reactive actin in differentiated tissues of planarian dugesia lugubris s.l.. contributions to animal biology, 1994b. dolittle rf. the origins and evolution of eukaryotic proteins. phil. trans. r soc. lond. b 349: 235240, 1995. dubois f. contribution à l’etude de la migration des cellules de regeneration chez les planaries dulcicoles. bull.biol. fr. belg. 83: 213-283, 1949. fagotti a, gabbiani g, pascolini r, neuville p. multiple isoform recovery (mir)-pcr: a simple method for the isolation of related mrna isoforms. nucleic acids res. 26: 2031-2033, 1998. gremigni v, miceli c. cytophotometric evidence for cell ‘transdifferentiation’ in planarian regeneration. wilhelm rou’s arch. 188: 107113, 1980. gunning p, weinberger r, jeffrey p. actin and tropomyosin isoforms in morphogenesis. anat. embryol. 195: 311-315, 1997. hennessey es, drummond dr, sparrow jc. molecular genetics of actin function. biochem. j. 282: 657-671, 1993. herman jm. actin isoforms. curr. opin. cell biol. 5: 48-55, 1993. hori i. observations on planarian epithelization after wounding. j. submic. cytol. pathol. 21: 307315,1989. hori i. differentiation of epidermal cells in the regenerating planarian dugesia japonica. hydrobiologia 227: 19-24, 1991. ito h, saito y, watanabe k, orii h. epimorphic regeneration of the distal part of planarian pharynx. dev. genes evol. 211: 2-9, 2001. kato k, orii h, watanabe k, agata k. the role of dorsoventral interaction in the onset of planarian regeneration. development 126: 1031-1040, 1999. kato k, orii h, watanabe k, agata k. dorsal and ventral positional cues required for the onset of planarian regeneration may reside in differentiated cells. dev. biol. 233: 109-121, 2001. martin p, parkhurst sm. parallels between tissue repair and embryo morphogenesis. development 131: 3021-3034, 2004. morgan th. regeneration. the macmillan co., new york, usa, 1901. mounier n, gouy m, mouchiroud d, prudhomme jc. insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins. j. mol. biol. 34: 406-415, 1992. newmark pa, sánchez alvarado a. bromodeoxyuridine specifically labels the 123 regenerative stem cells of planarians. dev. biol. 220: 142-153, 2000. pascolini r, tei s, vagnetti d, bondi c. epidermal cell migration during wound healing in dugesia lugubris. cell tissue res. 236: 345349, 1984. pascolini r, lorvik s, camatini m. cell migration during wound healing of dugesia lugubris s.l. fortschr. zool. 36: 103-109,1988a. pascolini r, lorvik s, maci r, camatini m. immunoelectron microscopic localization of actin in migrating cells during planarian wound healing. tissue cell. 20: 157-163, 1988b. pascolini r, panara f, di rosa i, fagotti a, lorvik s. characterization and fine structural localization of actinand fibronectin-like proteins in planaria (dugesia lugubris s.l.). cell tissue res. 267: 499-506, 1992a. pascolini r, di rosa i, fagotti a, panara f, gabbiani g. the mammalian anti-α-smooth muscle actin monoclonal antibody recognizes an α-actin-like protein in planaria (dugesia lugubris s.l.). differentiation 51: 177-186, 1992b. ramaekers fc, bosman ft. the cytoskeleton and disease. j. pathol. 204: 351-354, 2004. redd mj, cooper l, wood w, stramer b, martin p. wound healing and inflammation: embryos reveal the way to perfect repair. phil trans r. soc. lond. b 359: 777-784, 2004. reddien pw, sánchez alvarado a. fundamentals of planarian regeneration. annu. rev. cell dev. biol. 20: 725-757, 2004. reddien pw, oviedo nj, jenning jr, jenkin jc, sánchez alvarado a. smedwi-2 is a piwi-like protein that regulates planarian stem cells. science 310: 1327-1330, 2005. rossi l, salvetti a, lena a, batistoni r, deri p, pugliesi c, et al. djpiwi-1, a member of the paz-piwi gene family, defines a subpopulation of planarian stem cells. dev. genes evol. 216: 335-346, 2006. rubenstein pa. the functional importance of multiple actin isoforms. bioessays 12: 309315,1990. ruzicka dl, schwartz rj. sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation. j. cell biol. 107: 2575-2586, 1988. salò e, baguñà j. regeneration and pattern formation in planarians. i. the pattern of mitosis in anterior and posterior regeneration in dugesia (g) tigrina, and a new proposal for blastema formation. j. embryol. exp. morphol. 83: 63-80, 1984. sánchez alvarado a, newmark pa. the use of planarians to dissect the molecular basis of metazoan regeneration. wound rep. regener. 6: 413-420, 1998. salvetti a, rossi l, lena a, batistoni r, deri p, rainaldi g, et al. djpum, a homologue of drosophila pumilio, is essential to planarian stem cell maintenance. development 132: 1863-1874, 2005. sawtell nm, lessard jl. cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alphavascular but not the gamma-enteric isoform in differentiating striated myocytes. j. cell biol. 109: 2929-2937,1989. serini g, gabbiani g. mechanisms of myofibroblast activity and phenotypic modulation. exp. cell res. 250: 273-283, 1999. simoncelli f, sorbolini s, fagotti a, di rosa i, porceddu a, pascolini r. molecular characterization and expression of a divergent α-tubulin in planarian dugesia polychroa. biochem. biophys. acta 1629: 26-33, 2003. skalli o, ropraz p, trzeciak a, benzonana g, gillessen d, gabbiani g. a monoclonal antibody against α-smooth muscle actin: new probe for smooth muscle differentiation. j. cell biol. 103: 2787-2796,1986. vandekerckhove j, weber k. at least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. j. mol. biol. 126: 783-802, 1978. vandekerckhove j, weber k. chordate muscle actins differ distinctly from invertebrate muscle actins. j. mol. biol. 179: 391-413, 1984. wood w, jacinto a, grose r, woolner s, gale j, wilson c, et al. wound healing recapitulates morphogenesis in drosophyla embryos. nat. cell biol. 4: 907-912, 2002. woodcock-michell j, mitchell jj, low rb, kieny m, sengel p, rubbia l, et al. α-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. differentiation 39: 161-166, 1998. 124 acknowledgements isj100.pdf 60 isj 2: 60-68, 2005 issn 1824-307x research report early suppression of immune response in heliothis virescens larvae by the endophagous parasitoid toxoneuron nigriceps r ferrarese1*, m brivio1, t congiu2, p falabella4, a grimaldi1, m mastore1, g perletti3, f pennacchio4, l sciacca1, g tettamanti1, r valvassori1, m de eguileor1 1department of structural and functional biology, university of insubria, varese, italy 2department of human morphology, university of insubria, italy 3department of structural and functional biology, university of insubria, busto arsizio, italy 4dipartimento di biologia, difesa e biotecnologie agro-forestali, università della basilicata, potenza, italy accepted april 28, 2005 abstract toxoneuron nigriceps is an endophagous parasitoid of larval stages of the noctuid moth heliothis virescens. as all parasitoids, this wasp avoid host immune reaction by a combination of several passive and active mechanisms. secretions injected by ovipositing females, which contain venom, calyx fluid and polydnaviruses, are the most probably factors actively disrupting heliothis virescens immune system. this paper describes the main alterations of the host immune response observed shortly after oviposition by t. nigriceps. a transient block of prophenoloxidase activity is registered along with changes in hemocyte number, adhesion and structure, which suggest the occurrence of apoptosis. in contrast, the host plasmatocytes appear structurally unaltered, but unable to produce a capsule in vitro. key words: insects; parasitoid; immune defenses introduction foreign objects entering insect hemocoel are recognized as non-self and elicit a variety of defense reactions, which are somewhat arbitrary divided into humoral and cellular responses (hoffman, 1995; gillespie et al., 1997; lavine and strand, 2002). humoral responses include the production of antibacterial/antifungal peptides (boman et al., 1991; hoffmann et al., 1993; hultmark, 1993; cociancich et al., 1994; lowenberger, 2001) and of reactive intermediates of oxygen or nitrogen (bogdan et al., 2000; vass and corresponding author: roberto ferrarese department of structural and functional biology, university of insubria, j.h. dunant 3, 21100 varese, italy e-mail: roberto.ferrarese@uninsubria.it nappi, 2001), as well as in the activation of enzymatic cascades regulating coagulation or melanization of hemolymph (gillespie et al., 1997). host cellular immune responses, like phagocytosis, nodulation and encapsulation (schmidt et al., 2001; lavine and strand, 2002) are mediated by different types of hemocytes, but the regulatory molecular mechanisms involved are much less understood than those controlling humoral responses (lavine and strand, 2002). endophagous parasitoids, entering the host body, have to protect themselves from these defense barriers (schmidt et al., 2001). non-permissive hosts typically eliminate endoparasitoids by encapsulation, which usually involves the binding of overlapping layers of hemocytes to the surface of parasitoid. larval endoparasitoids evade host immune defenses either passively, or by active suppression of the host immune system, or by a combination thereof (schmidt et al., 2001; lavine and strand, 2002). female secretions injected at the oviposition are the most common factors 61 actively disrupting the host immune response. these secretions include venom and calyx fluid, which may contain different types of viruses and virus-like particles, often involved in the suppression of the immune response (schmidt et al., 2001). among these viruses, the polydnaviruses are by far the most studied and their molecular characterization in different systems has allowed to shed new light on their role in the host regulation process (kroemer and webb, 2004; webb and strand, 2005). toxoneuron nigriceps (hymenoptera, braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, heliothis virescens (lepidoptera, noctuidae). the bracovirus associated with this wasp (tnbv) is currently being studied and several genes expressed in parasitized host larvae have been isolated and their possible role partly elucidated (varricchio et al., 1999; pennacchio et al., 2001; falabella et al., 2003; malva et al., 2004; provost et al., 2004; lapointe et al., 2005). most of the isolated genes are actively expressed in the hemocytes of parasitized hosts and, then, probably involved in disruption and/or suppression. however, to better analyze the role played by these tnbv genes, and by other female secretions injected at the oviposition along with the egg, we need a better understanding of the alterations of the immune responses occurring in naturally parasitized hosts. this information is largely lacking, while for the passive evasion of host immune response it appears that the egg fibrous layer may play an important role (davies and vinson, 1986). the present paper aims at filling this gap by providing data on the most relevant precocious alterations associated with the suppression of host immune response observed in tobacco budworm larvae parasitized by t. nigriceps. materials and methods insect rearing toxoneuron nigriceps was reared in the laboratory according to the methodology described by vinson et al. (1973). heliothis virescens larvae were maintained on a modified artificial diet developed by vanderzant et al. (1962) (corn earworm diet, bioserve, frenchtown, nj, usa). rearing temperature was 29±1° c for both the host and parasitoid, whereas t. nigriceps adults were kept at 25±1 °c. in both cases a 16 h light photoperiodic regime was adopted and the relative humidity was 70±5 %. insect hosts h. virescens last instar larvae were staged according to webb and dahlman (1985) and synchronized as reported by pennacchio et al. (1992). enzymatic test for phenoloxidase (po) activity hemolymph was obtained by puncturing with a needle a proleg of last instar larvae cold anesthetized. all bleedings were done on ice-cold petri dishes and the hemolymph was transferred with a micropipette into icecold eppendorf tubes. hemolymph samples were then processed by low-speed centrifugation (1200 rpm for 3 min at 4 °c), to eliminate hemocytes and tissue debris. the supernatant (plasma) was immediately used or stored at –80 °c. time course analysis of po relative activity of plasma samples from parasitized and nonparasitized h. virescens larvae was carried out spectrophotometrically, by recording the formation of dopachrome from the l-dopa (dihydroxyphenylalanine) (sigma chemicals, st. louis, mo, usa) substrate. changes in absorbance were recorded at 490 nm (µa 490 nm/10 min) at 20 °c by a double-beam jasco v-560 spectrophotometer (jasco int. co., tokyo, japan). all assays were performed by adding 5 µl of plasma to 1 ml of l-dopa buffer (4 mm ldopa dissolved in 10 mm tris-hcl, ph 7.2). the ldopa buffer was used as a blank. to assess the effect of protease (ec 3.4.21.4) treatment on phenoloxidase activation, trypsin (15 iu) (sigma) was added to the substrate. in vitro encapsulation h. virescens hemocytes were obtained from last instar larvae 2 h after parasitization by t. nigriceps and from synchronous nonparasitized controls. insects were surface sterilized by rapid immersion in 70 % ethanol, washed in sterile distilled water, dried on sterile filter paper and bled by proleg amputation. samples of 40-60 µl hemolymph per larva were collected onto ice-cold petri-dishes, lined with 100 % ethanol washed parafilm. whole hemolymph samples were transferred in eppendorf tubes containing an equal volume of anticoagulant buffer mead (98 mm naoh, 145 mm nacl, 17 mm edta, 41 mm citric acid, ph 4.5). hemocytes were pelletted by centrifugation (1200 rpm for 10 min at 4 °c, in a sorvall rmc-14 refrigerated microcentrifuge) and twice washed in grace’s insect medium (sigma) containing 10 % fetal bovine serum (fbs), 1 % glutamine and 1 % antibiotic-antimycotic solution (sigma). the hemocytes were resuspended in 1 ml of the same medium and seeded at a final density of 2 x 105 per well and finally cultured in micro-wells (24-well culture plates, flat bottom, corning incorporated, costar, ny, usa). in vitro encapsulation assays were carried out immediately by adding chromatographic beads (dowex 1x2 mesh 100400) to cultured hemocytes, as described by lavine and strand (2001). hemocyte numbers and adhesion hemocytes were obtained as described in the above section from measured volumes of hemolymph extracted from nonparasitized and parasitized h. virescens larvae. cells were counted by using a burker chamber and subsequently cultured in micro-wells (24well culture plates), at 25 °c. after 16 h from cell seeding, the culture medium was removed and the nonadhering cells were counted. transmission and scanning electron microscopy hemocytes, obtained as described above, were fixed for 30 min in 0.1 m cacodylate buffer, ph 7.2, containing 2 % glutaraldehyde. cells were then washed in the same buffer and postfixed for 20 min with 1 % osmic acid in 0.1 m cacodylate buffer, ph 7.2. after a 62 standard step of serial ethanol dehydratation, cells were pelletted and embedded in an epon-araldite 812 mixture. sections were obtained with a reichert ultracut s ultratome (leica, wien, austria). thin sections were stained by uranyl acetate and lead citrate and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). hemocytes for scanning electron microscopy (sem) were fixed and dehydrated as described above, treated with hexamethildisilazane and mounted on polylysinated slides. samples were then air dried and covered with a 9 nm gold film by flash evaporation of carbon in an emitech k 250 sputter coater (emitech, baltimore, md, usa). specimens were then examined with a sem-feg philips xl-30 microscope (philips, eindhoven, netherlands). immunocytochemistry hemocytes were collected as described above and plated on glass coverslips cultured in micro-wells (24well culture plates). cells were washed with pbs and then fixed for 10 min in pbs buffer, ph 7.6, containing 2 % sucrose and 3 % paraformaldehyde. cells were treated for 10 min at 4 °c with a permeabilizing solution (hepes 20 mm, ph 7.4, 300 mm sucrose, 50 mm nacl, 3 mm mgcl2, 0.5 % triton x-100), then washed with pbs buffer containing 2 % bovine serum albumine (bsa) and finally incubated 1.5 h at 37 °c with tetramethylrhodamine (tritc)-labeled phalloidin (sigma) (diluted 50 µg/ml) in pbs buffer containing 2 % bsa. coverslips were mounted in vectashield mounting medium for fluorescence (vector laboratories, burlingame, ca, usa); slides were examined with a confocal laser microscope (laser 568 nm; mrc 1024, bio-rad laboratories, hemel, hempstead, uk) and images were recorded with a delta vision microscope (deltavision real time system (applied precision, elcomind, italy). results prophenoloxidase(propo)-po cascade t. nigriceps oviposition determined a rapid inhibition of the propo cascade. the po activity was nearly abolished by 15 min after parasitization and then 4 h later gradually resumed, reaching levels recorded in nonparasitized controls (fig. 1). in order to assess if the inhibited po was still potentially functioning, a trypsin treatment of the host plasma samples was carried out. the restored po activity in plasma samples of parasitized larvae, previously found to be inactive, indicated that the host po was not damaged, since the pro-enzyme was converted into its active form by trypsin mediated cleavage (fig. 2). hemocyte number morphology and behaviour the total number of hemocytes in parasitized larvae started to decrease shortly after t. nigriceps oviposition. the lowest number of hemocytes was registered after 4 h and reached the 60 % of the value measured in synchronous nonparasitized controls (fig. 3). after 40 h from parasitization the hemocyte number returned to the control condition (fig. 3). sem observations of hemocytes extracted from nonparasitized host larvae allowed to easily discriminate between granulocytes and plasmatocytes. granulocytes were roundish and showed prominent nuclei while plasmatocytes were larger, with a highly ruffled surface and pseudopodia of varying size (fig. 4). both cell types displayed attachment and spreading behaviours (only 5 % of the total amount of cells did not adhere under in vitro condition). adhering cells showed bundles of actin filaments located close to the cell membrane and in the cytoskeleton of the pseudopodia (figs 5, 6). hemocyte morphology in h. virescens larvae rapidly changed after parasitisation. sem observation showed that granulocytes were evidently damaged, while plasmatocytes were apparently unaltered (figs 7-9). the major morphological alterations in granulocytes consisted of cytoplasmic vacuolization, which became more pronounced over time, membrane blebbing, chromatin condensation and nuclear envelope breakdown (figs 7-14). under in vitro condition after two hours from oviposition, about 33 % of the total cell number was unable to adhere to the substrate (fig. 15). non-adhesive hemocytes showed cytoskeleton disruption, with broken filaments visible close to the cell membrane (fig. 16), where actin oligomers were detected (figs 17, 18). encapsulation assay encapsulation and subsequent melanization of dowex beads rapidly occurred in presence of hemocytes extracted from non parasitized h. virescens larvae (figs 19-22). different types of haemocytes were involved in host defense and both plasmatocytes and granulocytes were recruited to form a developing capsule onto the surface of the dowex beads. when the beads were added to the culture of hemocytes extracted from parasitized h. virescens larvae, the capsule did not develop and just a few granulocytes were visible close to the beads, while plasmatocytes fig. 1 after parasitization instead of the expected durable activation of propo system, an inhibition of melanization and a reduction in the enzymatic activity was observed from 15 min to 4 h. data represent mean ± sd, *p< 0,01. 63 fig. 2 tryptic enzyme treatment restores propo activity (trypsin is usually used to check propo integrity). data represent mean ± sd, *p< 0,01. fig. 3 the number of hemocytes decreases in hemolymph of parasitized h. virescens. data represent mean ± sd, *p< 0,01. fig. 4 nonparasitized h. virescens: sem observations of collected hemocytes show different cell types: granulocytes (arrowheads) and plasmatocytes (arrows). figs 5, 6 nonparasitized h. virescens: in hemocytes, actin filaments, detected with phalloidin (arrowheads), are grouped in bundles under the membrane and in the pseudopodia. 64 figs 7-14 parasitized h. virescens: sem observations of collected hemocytes (figs 7-9) show that the granulocyte subpopulation is particularly damaged while plasmatocytes appear morphologically unaltered (arrows). several ultrastructural features typical of apoptotic cell death are visible in the granulocytes: surface blebbing, chromatin aggregation, broken nuclear envelope, altered cytoplasm with swollen vacuoles. granulocytes examined by tem show numerous and large vacuoles in the cytoplasm (figs 10, 14). 65 discussion the “host regulation” by insect parasitoids, as defined by vinson and iwantsch (1980), is the final result of the evolution of host-parasitoid relationship towards host control for the benefit of the parasitoid progeny. parasitoid has to colonize and use the host, that provides food and shelter for the progeny of parasitoid (vinson et al., 2001). the tactics for circumventing the host defense and for redirecting its physiology, growth and reproduction, to support the development of the parasitoid juvenile stages, are key-factors in successful parasitism. the study of the molecular details of the physiological mechanisms underlying host-parasitoid interactions in insects has generated a fairly large amount of information (vinson et al., 2001; schmidt et al., 2001; beckage and gelman, 2004), in particular relatively to the major categories of host regulation factors produced by parasitic hymenoptera, such as polydnaviruses (kroemer and webb, 2004; webb and strand, 2005) and venom (weaver et al., 2001; asgari et al., 2003; zhang et al., 2004a, 2004b) suppressing the host immune responses. the suppression seems to be largely associated with inhibition of serine protease cascades (brehelin et al., 1975; beckage et al., 1990; beck et al., 2000) and hemocyte disruption and/or death (strand and pech, 1995; schimdt et al., 2001). this article is focused on the in vivo and in vitro study of survival strategies adopted by t. nigriceps during the early time of parasitization (i.e. the most vulnerable period of development). t. nigriceps avoids the host immune reaction by a combination of both passive and active mechanisms. the outer fibrous layer of the egg seems to be the first protecting barrier (davies and vinson, 1986), which is effectively complemented by a very precocious and transient suppression of the po activity. this transient block could be interpreted as a mechanism to disrupt the early steps activated by the recognition of an invading organism. the observed reactivation of the enzyme in parasitized plasma samples by trypsin treatment indicates that the po retains its functionality. we can differently interpret this result: a parasitoid-derived molecule could bind the po, protecting the cleavage site, thus inhibiting its activation or a parasitoid factor involved in host regulation likely hit the protease cascade rather than the enzyme itself. this may inhibit both the melanization response and the possible production of signal molecules that would in turn activate the cellular immune reaction. in fact, it is reasonable to speculate that the propo-activating system (propo-as) plays a key-role in the regulation of these important mechanisms, as suggested for crustacea and lepidoptera by soderhall and cerenius (1998). the source of the host regulatory factors involved in the po inactivation is still unknown, but based on preliminary experimental data and on the fact that this alteration is triggered within minutes after parasitization, we can predict that venom and/or ovarian secretes in the calyx fluid may play an important role. as evidenced by several authors (lavine and beckage, 1995; doucet and cusson, 1996; hu et al., 2003), many parasitoids are able to suppress host immune defenses by altering the number and/or the behaviour of the circulating hemocytes. there are a number of studies available on this type of alteration, which, for example, can be induced by parasitoidassociated pdvs, as in choristoneura fumiferanatrasonema rostrale (doucet and cusson, 1996) and pseudoplusia includens-microplitis demolitor (strand and pech, 1995) associations, or by the combined action of pdvs and ovarian proteins, as in heliothis virescens-campoletis sonorensis (luckhart and webb, 1996), or by the venom alone, such as in lacanobia oleracea-pimpla hypochondriaca (richards and parkinson, 2000). in all these model systems, the numerical variation of hemocytes is always paired with an altered morphology and/or functionality of the cells. in h. virescens larvae parasitized by t. nigriceps, the total number of circulating hemocytes transiently decreases, with a minimum peak registered within few hours, followed by a slow recovery towards values normally registered in synchronous nonparasitized controls, which were attained by 40 h after parasitoid oviposition. during this interval, the hemocytes show different structural damages, evident actin cytoskeleton disruption and lost of adhesion properties, with general morphological changes, which suggest the occurrence of apoptosis. these hemocyte alterations seem to be selectively induced in granulocytes, while plasmatocytes appear to be morphologically unaltered. however, the apparently unaltered plasmatocytes do not start any encapsulation process of dowex beads in vitro. it remains to be studied if this is a consequence of granulocytes degeneration, which may prevents the plasmatocyte recruitment they regulate during encapsulation (lavine and strand, 2002), or if a more subtle functional alteration of plasmatocytes occurs. all these changes in hemocyte structure and function could be induced by venom and calyx fluid right after oviposition, and, after few hours, reinforced by the expression of tnbv genes. fig. 15 parasitized h. virescens: hemocytes loose their adhesion capability and the minimum was observed at two hour mark. data represent mean ± sd, *p< 0,01. 66 figs 16-18 parasitized h. virescens: in hemocyte actin filaments are disassembled and are visible, under transmission electron microscope, at the periphery of the cell (arrows). phalloidin evidences the oligomers of actin (red spots). figs 19, 20 hemocytes of nonparasitized h. virescens: both plasmatocytes and granulocytes are recruited onto the surface of beads (arrowheads) forming a capsule. figs. 21, 22 hemocytes of parasitized h. virescens: few granulocytes are visible near the beads and plasmatocytes, parallel disposed, are unable to adhere to dowex beads (arrowheads). 67 fig. 23 summary of different events transiently disabling host immune defenses. the thin lines indicate the interval in which a particular alteration occurs; the broad lines are darker where the inhibition is at the highest level. a few genes expressed in host hemocytes have been isolated and, then, are considered to be putatively involved in the host immune disguise (varricchio et al., 1999; pennacchio et al., 2001; falabella et al., 2003; malva et al., 2004; provost et al., 2004). at the present, only for the viral gene tnbv1, it has been demonstrated that an apoptosis-like degeneration is induced in insect cells (lapointe et al., 2005). detailed functional analyses are required to establish the role of other tnbv genes in the induction of the multifaceted immune syndrome recorded in parasitized h. virescens. however, we can predict that these genes, along with female secretion injected at the oviposition, coordinately induce a set of partially overlapping mechanisms, disabling cellular and humoral responses (fig. 23). acknowledgements we thank luisa guidali for her invaluable technical assistance. this work has been supported by a miur grant (cofin 2002-2004). references asgari s, zhang g, zareie r, schmidt o. a serin proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph. insect biochem. mol. biol. 33: 1017-1024, 2003. beck m, theopold u, schmidt o. evidence of serine protease inhibitor activity in the ovarian calyx fluid of the endoparasitoid venturia canescens. j. insect physiol. 46: 1275-1283, 2000. beckage ne, gelman db. wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control. annu. rev. entomol. 49: 299 330, 2004. beckage ne, metcalf js, nesbit dj, schleifer kw, zetlan sr, de buron i. host hemolymph monophenoloxidase activity in parasitized manduca sexta larvae and evidence for inhibition by wasp polydnavirus. insect biochem. 20: 285294, 1990. bogdan c, rollinghoff m, diefenbach a. reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. curr. opin. immunol. 12: 64-76, 2000. boman hg, faye i, gudmundsson gh, lee jy, lidholm da. cell-free immunity in cecropia. a model system for antibacterial proteins. eur. j. biochem. 201: 23-31, 1991. brehelin m, drif l, baud l, boemare n. activation of prophenoloxidase in insect haemolymph: cooperation between humoral and cellular factors in locusta migratoria. insect biochem. 19: 301-307, 1975. cociancich s, bulet p, hetru c, hoffmann ja. the inducible antibacterial peptides of insects. parasitol. today 10: 132139, 1994. davies dh, vinson sb. passive evasion by eggs of braconid parasitoid cardiochiles nigriceps of encapsulation in vitro by haemocytes of host heliothis virescens. possible role for fibrous layer in immunity. j. insect physiol. 32: 10031010, 1986. doucet d, cusson m. role of the calyx fluid in alterations of immunity in choristoneura fumiferana larvae parasitized by tranosema rostrale. comp. biochem. physiol. 114a: 311-317, 1996. falabella p, varricchio p, gigliotti s, tranfaglia a, pennacchio f, malva c. toxoneuron nigriceps polydnavirus encodes a putative aspartyl protease highly expressed in parasitized host larvae. insect mol. biol. 12: 9 -17, 2003. gillespie jp, kanost mr, trenczek t. biological mediators of insect immunity. ann. rev. entomol. 42: 611643, 1997. hoffmann ja. innate immunity of insects. curr. opin. immunol. 7: 4-10, 1995. hoffmann ja, hetru c, reichhart jm. the humoral antibacterial response in drosophila. febs lett. 325: 6366, 1993. hu j, zhu x, fu w. passive evasion of encapsulation in macrocentrus cingulum brischke (hymenoptera: braconidae), a polyembrionic parasitoid of ostrinia furnacalis guenée (lepidoptera: pyralidae). j. insect physiol. 49: 367-375, 2003. hultmark d. immune reactions in drosophila and other insects: a model for innate immunity. trends genet. 9: 178 -183, 1993. kroemer ja, webb ba. polydnavirus genes and genomes: emerging gene families and new insights into polydnavirus replication. annu. rev. entomol. 49: 431-456, 2004. lapointe r, wilson r, vilaplana l, o'reilly dr, falabella p, pennacchio f. expression of a toxoneuron nigriceps polydnavirus (tnbv) encoded protein, causes apoptosislike programmed cell death in lepidopteran insect cells. j. gen. virol. 2005. in press. lavine md, beckage ne. polydnaviruses: potent mediators of insect immune disfunction. parasitol. today 11: 368-378, 1995. lavine md, strand mr. surface characteristics of foreign 68 targets that elicit an encapsulation response by the moth pseudoplusia includens. j. insect physiol. 47: 965-974, 2001. lavine md, strand mr. insect hemocytes and their role in immunity. insect biochem mol. biol. 32: 1295-1309, 2002. lowenberger ca. form, function and phylogenetic relationships of mosquito immune peptides. adv. exp. med. biol. 484: 113 -129, 2001. luckhart s, webb ba. interaction of a wasp ovarian protein and polydnavirus in host immune suppression. dev. comp. immunol. 20: 1-21, 1996. malva c, varricchio p, falabella p, la scaleia r, graziani f. physiological and molecular interaction in the hostparasitoid system heliothis virescens-toxoneuron nigriceps: current status and future perspectives. insect biochem. mol. biol. 34: 177-183, 2004. pennacchio f, malva c, vinson sb. regulation of host endocrine system by the endophagous braconid cardiochiles nigriceps and its polydnavirus. in: edwards jp, weaver rj (eds), endocrine interactions of insect parasites and pathogens, bios sci. publishers, oxford, pp 123-132, 2001. provost b, varricchio p, arana e, espagne e, falabella p, huguet e, la scaleia r, cattolico l, poirie m, malva c, olszewski ja, pennacchio f, drezen jm. bracoviruses contain a large multigene family coding for protein tyrosine phosphatases. j. virol. 78: 13090-130103, 2004. richards eh, parkinson nm. venom from the endoparasitic wasp pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, lacanobia oleracea. j. invertebr. pathol. 76: 33-42, 2000. schmidt o, theopold u, strand mr. innate immunity and its evasion and suppression by hymenopteran endoparasitoids. bioessays 23: 344 -351, 2001. söderhäll k, cerenius l. role of the prophenoloxidaseactivating system in invertebrate immunity. curr. opin. immunol. 10: 23 -28, 1998. strand mr, pech ll. immunological compatibility in parasitoidhost relationships. annu. rev. entomol. 40: 31-56, 1995. vanderzant es, richardson cd, fort sw jr. rearing of the bollworm on artificial diet. j. econ. entomol. 55:140, 1962. vass e, nappi aj. developmental and immunological aspects of drosophila-parasitoid relationships. j. parasitol. 86: 1259-1270, 2001. varricchio p, falabella p, sordetti r, graziani f, malva c. cardiochiles nigriceps polydnavirus: molecular characterization and gene expression in parasitized heliothis virescens larvae. insect biochem. mol. biol. 29: 1087-1096, 1999. vinson sb, guillot fs, hays db. rearing of cardiochiles nigriceps in the laboratory, with heliothis virescens as hosts. ann. entomol. soc. amer. 66: 1170-1172, 1973 vinson sb, iwantsch gf. host regulation by insect parasitoids. quart. rev. biol. 53: 143-165, 1980. vinson sb, pennacchio f, consoli fl. the parasitoid-host endocrine interaction from a nutritional perspective. in: edwards jp, weaver rj (eds), endocrine interactions of insect parasites and pathogens, bios sci. publishers, oxford, pp 187-205, 2001. weaver rj, marris gc, bell ha, edwards jp. identity and mode of action of the host endocrine disrupters from the venom of parasitoid wasps. in: edwards jp, weaver rj (eds), endocrine interactions of insect parasites and pathogens, bios sci. publishers, oxford, pp 33-58, 2001. webb ba, dahlman dl. developmental pathology of heliothis virescens larvae parasitized by microplitis croceipes: parasite-mediated host developmental arrest. arch. insect biochem. physiol. 2: 131-139, 1985. webb ba, strand mr. the biology and genomics of polydnaviruses. in: gilbert li, k iatrou k, gill ss (eds), comprehensive molecular insect science, vol. 6, elsevier, san diego, pp 323-360, 2005. zhang g, schmidt o, asgari s. novel venom peptide from an endoparasitoid wasp is required for expression of poydnavirus genes in host hemocytes. j. biol. chem. 279: 41580-41585, 2004a. zhang g, lu z-q, jiang h, asgari s. negative regulation of prophenoloxidase (propo) activation by a clip-domain serine proteinase homolog (sph) from endoparasitoid venom. insect biochem. mol. biol. 34: 477483, 2004b. isj 4: 38-xx, 2007 isj 4: 45-50, 2007 issn 1824-307x minireview the amphioxus immune system m pestarino, d oliveri, m parodi, s candiani dipartimento di biologia, università di genova, genova, italy accepted march 19, 2007 abstract the cephalochordate amphioxus is the closest living invertebrate relative of the vertebrates and therefore it can provide useful insights on the evolution of the adaptive immune system. in fact some components of the immune system are present in amphioxus but many other features are lacking. a proto-mhc region has been identified by chromosome walking and the presence of t cell receptor has been demonstrated despite the absence of true lymphocytes. moreover a further important step on the study of the amphioxus immune system is represented by the recent availability of the genome of branchiostoma floridae that will allow to better understand how many genes code for immunological molecules involved in adaptive immune system pathways of amphioxus. key words: evolution; protochordates; amphioxus; innate immunity; adaptive immunity __________________________________________________________________________________________ introduction protochordates, consisting of two subphyla (tunicates or urochordates and cephalochordates) sharing a common ancestry with the vertebrates, are useful model animals for the comprehension of the vertebrate phylogeny (swalla et al., 2000, schubert et al., 2006). in the last decades, several molecular data support the hypothesis that the cephalochordates are the sister group of vertebrates (holland et al., 2004), and therefore the cephalochordate amphioxus, also commonly known as lancelet, is particularly useful in order to study the evolution of the immune system and to identify the related genes. in fact, the recent availability of the genome assembly release v.1.0 (started march 2006 and freely available to the scientific community from january 2007) by the joint genome institute (us department of energy) facilitates the comparative studies of genomes of vertebrate and invertebrate species. on the other hand, the emergence of adaptive immunity has been placed at the jawed vertebrate stage but numerous recent comparative immunological findings suggest the presence in amphioxus not only of innate immunity but also of an ancestral adaptive immune system ___________________________________________________________________________ corresponding author: mario pestarino dipartimento di biologia università di genova viale benedetto xv, 5 16132 genova, italy e-mail: pesta@unige.it (sato et al., 2003; danchin et al., 2004; dong et al., 2005). even if the structure of the amphioxus vascular system has been extensively studied, free blood cells have not been clearly identified. only rhodes and coworkers (1982) described by electron microscope the presence in the perivisceral coelom of free cells able to phagocyte and similar to specialized leukocytes as described in ascidians. moreover, the blood vessels lack in a continuous endothelial layer, the blood is colourless and the vascular system is closed and consists of dorsal and ventral vessels from which vascular channels reach pharynx, intestine and gonads. multiple contractile and peristaltic pumps are present in the subintestinal vessel but few anatomical and histological data support their homology to the vertebrate heart even if amphioxus is the closest model for the vertebrate vascular plan (simõescosta et al., 2005). recently, holland and coworkers (2003) cloned in the amphioxus branchiostoma floridae the gene amphink2-tin, similar in sequence to vertebrate nk2 genes and the tinman gene of drosophila, both involved in cardiogenesis (simões-costa et al., 2005). amphink2-tin is firstly expressed in muscle cell precursors at level of the first five or six somites and afterwards in a ventral row of visceral peritoneal cells, containing non-striated myofibrils, and from which the wall of the contractile subintestinal vessel derives. therefore such data support the presence in amphioxus of a rudimentary cardiac system. 45 mailto:pesta@unige.it fig. 1 schematic representation of the evolution of the nine mhc paralogous region (labelled by an arrow and a roman number from i to ix) (modified from abi-rached et al. 2002 and vienne et al., 2003). for two gene families, duplications specific to the amphioxus lineage are evidenced by an asterisk (1: neuraminidase-like 2 and 3: neuraminidase-like 1; 6: udpgt-like 1 and 7: udpgt-like 2. all the other genes of amphioxus has at least an orthologue as shown for the amphioxus frequenin gene (20). two new predicted genes of amphioxus are orthologous to the human nadph (11) and mgc14327 (12). the yellow boxes show the anchor genes (5: rxra; 9: bat1/ddx39; 14: brd2,3,4,t; 15: c3,c4,c5; 16: cacna1a,b,e; 19: notch1,2,3,4; 24: psmb7; 26: psmb8; 27: prx1,2,3,4). the major histocompatibility complex (mhc) in humans mhc consists of a large genetic region containing more than 100 genes involved in graft rejection and clustered in a major chromosomal region named major histocompatibility complex. graft rejection is due to the recognition of mhc class i peptides that are recognized by t cells from the host. moreover, because also non-mhc genes are involved in the immune system, it has been possible to define a central region between the mhc class ii genes and the mhc class i genes, named mhc class iii region, which includes genes coding for the complement system and some members of the tumor necrosis factor (tnf) family (flajnik and du pasquier, 2004). moreover, further genes involved in mhc presentation have been found within the socalled mhc class i and class ii subregions (flajnik and du pasquier, 2004; danchin et al., 2004). as known, the adaptive immune response starts with the presentation of mhc-bound peptides to the tcell receptors. mhc class i and class ii proteins, like other transmembrane proteins, must be properly folded to be competent for exit from the endoplasmic reticulum. the assembly of the various constituents requires a mechanism that involves er-resident chaperones and the exit of mhc class i and class ii is mediated by housekeeping chaperones and dedicated proteins (cresswell, 1994; antoniou et al., 2003; paulsson and wang, 2003). an example of dedicated proteins in mhc class ii presentation are cd74 and the cathepsins. cd74 is a dedicated chaperone found only in the bony vertebrates (dijkstra et al., 2003) and no homologous of this gene has been found in nonvertebrate groups. the housekeeping chaperones have been co-opted by the neo-mhc molecules after the jawed/jawless vertebrate split, and some of the dedicated proteins have been co-opted directly after duplication (danchin et al., 2004). in fact, the emergence of adaptive immunity has been located for a long time at the appearance of gnathostome vertebrates, mainly because the major components 46 of the mammalian adaptive immune system such as mhc, t cell receptors (tcr), and immunoglobulin (ig) molecules, appear for the first time in the cartilaginous fish (cannon et al., 2004). recently, an ancestral adaptive immune system characterized by the presence of lymphocyte-like cells, has been described in lamprey (mayer et al., 2002; uinookool et al., 2002) and variable lymphocyte receptors (vlr) were also identified in hagfish (pancer et al., 2005). the adaptive immune system (ais) and peptide mhc presentation (fig. 1) probably appeared before the emergence of the last common ancestor of the gnatosthomes and after the jawless/jawed vertebrate split, but a possible proto-mhc region has been described in amphioxus (abi-rached et al., 2002), whereas according to danchin and coworkers (2004) the common ancestors of chordates did not possess an ais. it is known that some of the genes involved in the mhc system were co-opted after duplication (abi-rached et al., 2002). because amphioxus has a key position in chordate evolution, studies were performed in b. floridae in order to test the two rounds of en bloc duplication hypothesis for the mhc and its paralogous regions located on human chromosomes 1, 9, and 19 (abi-rached et al., 2002). nine highly conserved anchor genes located in the mhc were used as probes, and their orthologs were cloned from a b. floridae genomic library. ten cosmid clones containing the anchor genes were identified and sequenced, and 22 genes were detected in their surrounding genomic regions. the distribution of human and amphioxus orthologs in their respective genomes and the relationship between these distributions support the en bloc duplication events hypothesis and the phylogenetic analysis showed that all duplication events in these regions occurred after the divergence of cephalochordata and craniata and before the gnathostomata radiation. recently four new cosmids have been sequenced in the amphioxus mhc-like region, and their phylogenomic analysis supports the previously obtained results (vienne et al., 2003). furthermore, the physical linkage of these cosmids has been tested by two-colour fluorescent in situ hybridization (fish) to amphioxus metaphase chromosomes (castro and holland, 2002). in particular, six cosmids (corresponding to 27 genes) mapped to a single amphioxus chromosome, only the cosmid containing the orthologue amphic3 of the complement genes maps to a different chromosome of the 19 chromosome pairs in amphioxus. the obtained experimental data allow to reconstruct the protomhc in the ancestral chordate. such proto-mhc corresponds to the extant mammal class ii and class iii genomic regions and this correspondence shows that mhc class i genes were translocated recently in the mammal evolution and have colonized new genomic portions (flajnik and kasahara, 2001). it has been also hypothesized that the mhc genomic region is part of an ancient syntenic group present in the ancestor of protoand deuterostomes (trachtulec and forejt, 1999). even though the description of the conserved synteny was not supported by phylogenetic and statistical analysis (hughes and pontarotti, 2000), it has been possible to demonstrate that an mhc-like region was certainly present in the common ancestor of protoand deuterostomes (danchin et al., 2003). the immunoglobulin superfamily a multigene family containing ig-like variable regions, v region-containing chitin-binding protein (vcbp) and an immunoglobulin superfamily (igsf) gene homologous to cd47, have been identified in the intestine of amphioxus (cannon et al., 2002, sato et al., 2003). these studies suggest that some ancestral molecules involved in the adaptive immunity existed in protochordates (fig. 2). a fragment of est with significant similarity to a vertebrate tcr sequence was found in a cdna library of adult branchiostoma lanceolatum (sato et al., 2003). in particular an orf encoding a 351 aa long peptide has been found and the putative protein is named brla-vdb for “branchiostoma lanceolatum v-domain bearing”. brla-vdb consists of a n terminal domain starting with a putative leader peptide and followed by a sequence resembling the v domain of the cortical thymocytes of xenopus (ctx) protein. furthermore, the c terminal domain contains five hydrophobic segments separated by short hydrophilic stretches and therefore it may be considered a protein that crosses the plasma membrane five times. these findings support the hypothesis that v domain resembling those found in t cell receptors evolved in invertebrates before the emergence of the adaptive immune system and it may have been involved in non immunological functions (sato et al., 2003). some igsf members bearing the v or v-c structures have been found in amphioxus (cannon et al., 2002, sato et al., 2003). two kinds of igsf members have been described in an intestine cdna library of branchiostoma belcheri. the first one is a vcbp having approximately 70 % identities in amino acid sequence with the vcbp4 of b. floridae that encodes 338 amino acid residues. vcbp comprises a signal peptide, one v-type domain, one c-type domain, a transmembrane region, and a cytoplasmic region, and therefore named as v and c domain-bearing protein (vcp). as known in mammals, expression of recombination activating genes (rag) that are involved in the v (d) j recombination is regulated by the rag1 gene activator (rga) in mammals. recently, the sequence of a cdna clone from an amphioxus cdna library was found to be homologous to that of rag from mouse stromal cells (cannon et al., 2002). the full-length cdna sequence comprises 1119 bp and encodes a putative protein of 210 amino acid residues. characterization of the amino acid sequence revealed that two mtn3 domains and seven span transmembrane domains are present in this protein, indicating a potential role as a plasma membrane protein. a high expression level of rga, detected in gonads, gastrula embryos and adult stages of amphioxus suggests that the signal pathway required for the expression of rag could occur in cephalochordates (dong et al., 2005). therefore a 47 fig. 2 dendrogram showing bilaterian metazoan phylogeny. the possible origin time of the adaptive immune system is shown as well as the two en bloc genomic duplications: the first before the emergence of vertebrates and the second before the appearance of gnathostomes. during the evolutionary history of chordates, amphioxus acquires some of the immune molecules and mechanisms involved in pre-adaptive immunity. tcr, t cell receptor; igsf, immunoglobulin super family; vcbp, v region-containing chitin-binding protein receptors; gilt, ifn-γinduced lysosomal thiol; mhc synteny, major histocompatibility complex synteny (as described in the text). primitive adaptive immunity may have existed in amphioxus although the complete machinery of v(d)j rearrangement may be not formed. recently, a nucleotide sequence similar to ifnγ-induced lysosomal thiol (gilt) reductase have been found in amphioxus (yu et al., 2005). gilt is expressed constitutively in antigen presenting cells (apcs) and facilitates processing and presentation of ag peptide (zheng and chen, 2006). the amphigilt contains the conserved active site cxxc, and nine cysteins downstream of the active site just like the counterparts in human and mouse. the structure of amphi-gilt is vertebrate-like suggesting a closer function to its counterparts in vertebrates. furthermore, gamma-interferon (ifnγ)-inducible lysosomal thiol reductase (gilt) is involved not only in the internalization and delivering to lysosomes of exogenous antigens (watts, 1997), but also in negative regulation of t cell activation (barjaktarevic et al., 2006) and neutralization of extracellular pathogen (lackman and cresswell, 2006). gilt has been identified in several species of vertebrates and invertebrates (maric et al., 2001; woods et al., 2005; zheng and chen, 2006). recently, amphigilt has been demonstrated to have a conserved active domain probably involved in the innate immune responses in amphioxus (liu et al., 2007). innate immunity and humoral immune responses macrophage migration inhibitory factor (mif), a cytokine involved in host defenses and autoimmune diseases, has been found in b. belcheri in which unusually the mif gene is present in multi-copy per haploid genome (du et al., 2004). in particular two mif homologues, called bbt-mif-i and bbt-mif-ii, have been sequenced in amphioxus. mif exerts a crucial role in innate immunity and therefore it can be considered a key molecular marker of evolution of immune system, even if in amphioxus it has more broad function than in vertebrates (du et al., 2006). humoral fluids of b. belcheri contain lysozyme, microbial agglutinin and hemagglutinins before and after challenge with escherichia coli (pang et al., 2006), but also the coelomic fluid contains phenoloxidase, lectin and complement component c3. therefore it is possible to argue that amphioxus has a simple humoral immune defense system. as 48 known in vertebrates mannose-binding lectinassociated serine protease (masp) are involved in complement activation through the lectin pathway, in amphioxus a masp gene has been cloned (endo et al., 2003) and its structure is similar to the human masp1/3 (dahl et al., 2001). furthermore, the evolutionary history of the masp gene family seems to be parallel to that of their substrates such as c3 and c4. in fact, both c3 and masp homologue genes have been found in amphioxus (fujita, 2002), as well as a gene encoding a protein containing the membrane attack complex (mac)/perforin module, homologue to vertebrate c6 called amphic6 has been identified (suzuki et al., 2002). such evidences suggest that a primordial complement system is probably emerged after the cephalochordates. in conclusion several findings on the amphioxus immune system incline us to suggest that innate and adaptive immunity appeared early during the chordate phylogeny. in amphioxus it is possible to find genes coding for molecules typical of the vertebrate immune system therefore a protovertebrate immune system is present in amphioxus. nevertheless, further studies are needed in order to define the molecular and functional features of the amphioxus immune system as well as to ascertain how many blood cell types are present in amphioxus. acknowledgments the authors wish to thank the anonymous reviewers for useful comments and suggestions that have greatly improved the quality of the article. references abi-rached l, gilles a, shiina t, pontarotti p, inoko h. evidence of en bloc duplication in vertebrate genomes. nat. genetics 31: 100–105, 2002. antoniou an, powis sj, elliott t. assembly and export of mhc class i peptide ligands. curr. opin. immunol. 15: 75–81, 2003. barjaktarevic i, rahman a, radoja s, bogunovic b, vollmer a, vukamanovic s, et al. inhibitory role of ifn-gamma-inducible lysosomal thiol reductase in t cell activation. j. immunol. 177: 4369-4375, 2006. cannon jp, haire rn, litman gw. identification of diversified genes that contain immunoglobulinlike variable regions in a protochordate. nat. immunol. 3: 1200-1207, 2002. cannon jp, haire rn, rast jp, litman gw. the phylogenetic origins of the antigen-binding receptors and somatic diversification mechanisms. immunol. rev. 200: 12–22, 2004. castro lf, furlong rf, holland pwh. an antecedent of the mhc-linked genomic region in amphioxus. immunogenetics 55: 782-784, 2004. cresswell p. assembly, transport, and function of mhc class ii molecules. annu. rev. immunol. 12: 259–293, 1994. dahl mr, thiel s, matsushita m, fujita t, willis ac, christensen t, et al. masp-3 and its with distinct complexes of the mannang-binding lectin complement activation pathway. immunity 15: 127-132, 2001. danchin eg, abi-rached l, gilles a, pontarotti p. conservation of the mhc-like region throughout evolution. immunogenetics 55: 141-148, 2003. danchin e, vitiello v, vienne a, richard o, gouret p, mcdermott mf, et al. the major histocompatibility complex origin. immunol. rev. 198: 216-232, 2004. dijkstra jm, kiryu i, kollner b, yoshiura y, ototake m. mhc class ii invariant chain homologues in rainbow trout (oncorhynchus mykiss). fish shellfish immunol. 15: 91–105, 2003. dong m, fu y, yu c, su j, huang s, wu x, et al. identification and characterisation of a homolog of an activation gene for the recombination activating gene 1 (rag 1) in amphioxus. fish shellfish immunol 19: 165-174, 2005. du j, xie x, chen h, yang w, dong m, su j, et al. macrophage migration inhibitory factor (mif) in chinese amphioxus as a molecular marker of immune evolution during the transition of invertebrate/vertebrate. dev. comp. immunol. 28: 961-71, 2004. du j, yu y, tu h, chen h, xie x, mou c, et al. new insights on macrophage migration inhibitory factor: based on molecular and functional analysis of its homologue of chinese amphioxus. mol. immunol. 43: 2083-20888, 2006. du pasquier l, zucchetti i, de santis r. immunoglobulin superfamily receptors in protochordates: before rag time. immunol. rev. 198: 233-243, 2004. endo y, nonaka m, saiga h, kakinuma y, matsushita a, takahashi m, et al. origin of mannose-binding lectin-associated serine protease (masp)-1 and masp-3 involved in the lectin complement pathway traced back to the invertebrate, amphioxus. j. immunol. 170: 4701-4707, 2003. flajnik mf, du pasquier l. evolution of innate and adaptive immunity: can we draw a line? trends immunol. 25: 640-644, 2004. flajnik mf, kasahara m. comparative genomics of the mhc: glimpses into the evolution of the adaptive immune system. immunity 15: 351362, 2001. fujita t. evolution of the lectin-complement pathway and its role in innate immunity. nat. rev. immunol. 2: 346-347, 2002. holland nd, venkatesh tv, holland lz, jacobs dk, bodmer r. amphink2-tin, an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart. dev. biol. 255: 128-137, 2003 holland lz, laudet v, schubert m. the chordate amphioxus: an emerging model organism for developmental biology. cell. mol. life sci. 61: 2290-2308, 2004. hughes al, pontarotti p. gene duplication and mhc origins. immunogenetics 51: 982-983, 2000. lackman rl, cresswell p. exposure of the promonocytic cell line thp-1 to escherichia coli induces ifn-gamma-inducible lysosomal thiol reductase expression by inflammatory cytokines. j. immunol. 177: 4833-4840, 2006. 49 liu n, zhang s, liu z, gaowa s, wang y. characterization and expression of gammainterferon-inducible lysosomal thiol reductase (gilt) gene in amphioxus branchiostoma belcheri with implications for gilt in innate immune response. mol immunol 44: 26312637, 2007. maric m, arunachalam b, phan ut, chen d, garrett ws, cannon ks, alfonso c, karlsson l, flavell ra, cresswell p. defective antigen processing in gilt-free mice. science 294: 1361-1365, 2001. mayer we, uinook-ool t, tichy h, gartland la, klein j, cooper md. 2002. isolation and characterization of lymphocyte-like cells from a lamprey. proc. natl. acad. sci. usa 99: 14350– 14355, 2002. pancer z, saha nr, kasamatsu j, suzuki t, amemiya ct, kasahara m, cooper md. variable lymphocyte receptors in hagfish. proc. natl. acad. sci. usa 102: 9224-9229, 2005. pang q, zhang s, liu x, wu d. humoral immune responses of amphioxus branchiostoma belcheri to challenge with escherichia coli. fish shellfish immunol. 21: 139-45, 2006. paulsson k, wang p. chaperones and folding of mhc class i molecules in the endoplasmic reticulum. biochim. biophys. acta 1641: 1–12, 2003. klein j. natural history of the major histocompatibility complex. wiley interscience, new york, usa, 1986. rhodes cp, ratcliffe na, rowley af. presence of coelomocytes in the primitive chordate amphioxus (branchiostoma lanceolatum). science 217: 263-265, 2003. sato a, mayer we, klein j. a molecule bearing an immunoglobulin-like v region of the ctx subfamily in amphioxus. immunogenetics 55: 423-427, 2003. schubert m, escriva h, xavier-neto j, laudet v. amphioxus and tunicates as evolutionary model systems. trends ecol. evol. 21: 269-277, 2006. simǒes-costa ms, vasconcelos m, sampaio ac, cravo rm, linhares vl, hochgreb t, et al. the evolutionary origin of cardiac chambers. dev biol. 277: 1-15, 2005. suzuki mm, satoh n, nonaka m. c6-like and c3like molecules from the cephalochordate, amphioxus, suggest a cytolytic complement system in invertebrates. j. mol. evol. 54: 671679, 2002. swalla bj, cameron cb, corley ls, garey jr. urochordates are monophyletic within the dueterostomes. syst. biol. 49: 52-64, 2000. trachtulec z, forejt j. transcription and rna processing of mammalian genes in saccharomyces cerevisiae. nucleic acid res. 27: 526-531, 1999. uinook-ool t, mayer we, sato a, dongak r, cooper md, klein j. lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes. proc. natl. acad. sci. usa 99: 14356–14361, 2002. vienne a, shiina t, abi-rached l, danchin e, vitiello v, cartaulta f, et al. evolution of the proto-mhc ancestral region: more evidence for the plesiomorphic organisation of human chromosome 9q34 region. immunogenetics 55: 429-436, 2003. watts c. capture and processing of exogenous antigens for presentation on mhc molecules. annu. rev. immunol. 15: 821-850, 1997. woods ig, wilson c, friedlander b, chang p, reyes dk, nix r, et al. the zebrafish gene map defines ancestral vertebrate chromosomes. genome res. 15: 1307-1314, 2005. yu c, dong m, wu x, li s, huang s, su j, et al. genes "waiting" for recruitment by the adaptive immune system: the insights from amphioxus. j. immunol. 174: 3493-500, 2005. zheng w, chen x. cloning and expression analysis of interferon-γ-inducible lysosomal thiol reductase gene in large yellow croaker (pseudosciaena crocea). mol. immunol. 43: 2135-2141, 2006. 50 << /ascii85encodepages false /allowtransparency false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice exogenous il-8 induces phagocyte activation in the compound ascidian botryllus schlosseri isj 3: 18-24, 2006 issn 1824-307x research report exogenous il-8 induces phagocyte activation in the compound ascidian botryllus schlosseri a menin, l ballarin department of biology, university of padova, padova, italy accepted february 28, 2006 ______________________________________________________________________________ abstract we studied the responses of botryllus schlosseri phagocyte to human recombinant il-8 (hril-8) at three different concentrations (10, 25 and 50 ng/ml). both spreading ability and phagocytosis were significantly enhanced by the exogenous chemokine at 25 and 50 ng/ml in the culture medium and the effects are coupled to modifications of the actin cytoskeleton. the addition of the signal transduction inhibitors suramin, calphostin c and h-89 to the incubation medium, inhibits the above-reported effects and suggests that exogenous il-8 acts via protein kinase (pk) c and pka pathways through its binding to a g protein-coupled receptor. key words: botryllus schlosseri; immunocytes; il-8 _____________________________________________________________________________________________________________________ introduction interleukin-8 (il-8, cxcl8) is an inducible chemokine released during infections and inflammation in response to bacteria toxin or inflammatory cytokines like interleukin-1 (il-1) and tumour necrosis factor alpha (tnf-α) (rollins, 1997; baggiolini, 2001; casilli et al., 2005; esche et al., 2005). it causes rapid changes in the morphology of leucocytes, such as neutrophils, t-lymphocytes and natural killer cells (nk), as the consequence of a reorganisation of the actin cytoskeleton. in addition, il-8 induces the up-regulation and activation of integrins on leucocytes required for their adhesion to both endothelial cells, prior to extravasation, and extracellular matrix, during their way to inflammation sites (luster, 1998; imhof and aurrand-lions, 2004; casilli et al., 2005). as a chemokine, il-8 exerts its action by interacting with seven transmembrane, g protein-coupled receptors which, upon ligand binding, activate a heterotrimeric g-protein which dissociates into the gtp-bound α and the βγ subunits. the latter, ______________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padova via u. bassi 58/b, 35100 padova, italy email: loriano.ballarin@unipd.it in turn activates phospholipase c (plc) leading to the production of inositol triphosphate (ip3) and diacylglycerol (dag). in the case of il-8, the βγ subunit of the gprotein also recruits and activates phosphatidylinositol-3-kinase (pi3k) which has a central role in inflammation as it allows the recruitment and activation of phagocytes and nk cells (smith et al., 1992; hirsh et al., 2000; naccache et al., 2000; fuhler et al., 2005 maghazachi, 2005). as far as invertebrates are concerned, although the existence of molecules sharing homology with vertebrate cytokines is still a matter of debate (beschin et al., 2001, 2004), the presence of endogenous immunoregulatory cytokines has been indirectly suggested in various phyla (beck and habicht, 1991; hughes et al., 1990, 1991; ouwemissi-oukem-boyer et al., 1994; granath et al., 1994; cooper et al., 1995; ottaviani et al., 1995, 1996; franchini et al., 1996; beck, 1998). according to beschin et al. (2001, 2004), these molecules share with vertebrate cytokines some lectin domains which can explain the observed effects of mammalian cytokines on invertebrate immunocytes (kletsas et al., 1998; ottaviani et al., 2000, 2004). in tunicates, the presence of endogenous chemotactic molecules, is known from many studies. they are represented by either homologues of the 18 mammalian complement c3, as in styela plicata (raftos et al., 2002), halocynthia roretzi (nonaka et al., 1999), pyura stolonifera (raftos et al., 2003) and ciona intestinalis (pinto et al., 2003), or different molecules cross-reacting with anti-il-1 antibodies as in styela clava (raftos et al., 1998). in the colonial ascidian botryllus schlosseri, we have recently demonstrated that activated immunocytes release cytokines (i.e. immunomodulatory molecules, in the broad sense of the term) able to enhance yeast phagocytosis (menin et al., 2006). the above molecules are recognised by antibodies raised against mammalian il-1α and tnfα and are mainly produced by cytotoxic morula cells upon the recognition of non-self molecules (ballarin et al., 2001). these cells are known to be involved in the non-fusion reaction which occurs when genetically incompatible colonies contact each other and shares many features, such as chemotaxis, extravasation, degranulation and cytotoxicity, with vertebrate inflammation where chemokines play a key role (ballarin et al., 1995, 1998; rinkevich et al., 1998; cima et al., 2004). recently, we undertook a new research aimed to identify and characterise chemokines in our model. as a preliminary approach, we studied the effects of human recombinant il-8 (hril-8) on the activity of b. schlosseri immunocytes. results indicate that ascidian immunocytes can sense the presence of exogenous il-8 and consequently modify their activity. materials and methods animals colonies of botryllus schlosseri from the lagoon of venice were used. they were kept in aerated aquaria, attached to glass slides and fed with liquifry marine (liquifry co., dorking, england) and algae (dunaliella sp.). blood plasma preparation blood was collected with a glass micropipette after puncturing, with a fine tungsten needle, the tunic marginal vessels of colonies previously blotted dry. blood was centrifuged at 780 x g and the supernatant was referred as blood plasma (bp). the terms “autologous” and “heterologous” refer to bp from the same colony used for hemocyte collection or from a different, non-fusible colony, respectively. hemocyte collection and culture blood was collected, as described above, from colonies previously rinsed in 0.38 % na-citrate in filtered sea water (fsw), ph 7.5, as anti-clotting agent. it was then centrifuged at 780 x g for 10 min and pellets were finally resuspended in fsw to give a concentration of 5 x 106 cells/ml. sixty µl of hemocyte suspension were placed in the centre of culture chambers prepared as described elsewhere (ballarin et al., 1994) and left to adhere to coverslips for 30 min at room temperature. light microscopy of hemocytes after the adhesion of hemocytes to the coverslips, cells were exposed for 60 min to fsw containing hril8 (peprotech ec; 10, 25 and 50 ng/ml) in the presence or in the absence of 0.7 mm suramin, an antagonist of g protein (huang et al., 1990; ottaviani et al., 2000); fsw alone was used in controls. cells were then fixed for 30 min at 4 °c in 1 % glutaraldehyde in fsw containing 1 % sucrose, rinsed in phosphate-buffered saline (pbs: 1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4, 0.065 m na2hpo4, ph 7.2) and stained with 10 % giemsa solution for 5 min. the morphology of hemocytes was observed under a leitz dialux 22 light microscope at a magnification of 1250 and the cell-spreading index, i.e. percentage of hemocytes with amoeboid shape, was finally calculated after counting at least 300 cells per coverslip. in another series of experiments, calphostin c, a specific inhibitor of protein kinase c (pkc; tamaoki, 1991) and h-89, a specific inhibitor of protein kinase a (pka; chijiwa et al., 1990) were added to the incubation media, in the presence or in the absence of hril-8, at the sublethal concentration of 0.1 and 1 µm, respectively. these concentrations were already used in experiments with invertebrate immunocytes (ottaviani et al., 2000). in addition, computer-assisted image analysis (casting image nt) was performed on fixed hemocyte monolayers, previously treated as described above, to evaluate the phagocyte shape factor, defined as in ottaviani et al. (1997). lower shapes factors indicate larger perimeters with respect to the areas and, therefore, an increased amoeboid shape. immunocytochemical assays for cytoskeleton hemocyte monolayers on coverslips, treated as described above, were fixed for 30 min at 4 °c in 4 % paraformaldehyde (serva electrophoresis gmbh, heidelberg, germany) and 1 % sucrose in isotonic salt solution (iso: 20 mm tris, 0.5 m nacl, ph 7.5; edds, 1985), washed in pbs plus 1 % sucrose and permeabilised with 0.1% triton x-100 (merck kgaa, darmstadt, germany) in pbs for 5 min. for the specific detection of f-actin, the monolayers were then incubated for 30 min at 25 °c in fitc-labelled phalloidin (sigma-aldrich co, st louis, mo, usa), 1 μg/ml in pbs. lastly, the coverslips were rinsed in 0.1 m carbonate buffer, ph 9.5, to amplify the fluorescent signal, mounted with vectashield (vector laboratories inc., burlingame, ca, usa) on glass slides, and observed at a magnification of 1250 under a leitz dialux 22 light and fluorescence microscope equipped with i2/3 filter block for fitc excitation. phagocytosis assay after adhesion, hemocytes were incubated with 60 µl of a suspension of yeast cell (yeast:hemocyte ratio = 10:1) in fsw containing hril-8 at the same concentrations indicated above, in the presence or 19 fig. 1 fixed b. schlosseri phagocytes. a, c-e) hyaline amoebocytes; b) univacuolated macrophage-like cell. a-c) giemsa’s stain; d-e) treatment with fluorescent falloidin to reveal f-actin. scale bar = 3 µm. absence of 0.7 mm suramin; fsw alone was used in controls. cultures were kept upside down for 60 min at room temperature. hemocyte monolayers were then washed by dipping the coverslips repeatedly in a large volume (100 ml) of fsw, fixed in a solution of 1 % glutaraldehyde and 1 % sucrose in fsw for 30 min at 4 °c and stained with 10 % giemsa for 5 min. fig. 2 a) cell-spreading index of botryllus hemocytes left to adhere for 60 min in fsw containing hril-8 in the presence or in the absence of 0.7 mm suramin; fsw alone was used in controls; b) phagocytic index of botryllus hemocytes exposed to yeast-containing fsw and hril-8 in the presence or in the absence of 0.7 mm suramin; fsw alone was used in controls. significant differences with respect to controls are marked by asterisks. * = p < 0.05. the coverslips were then mounted on glass slides with the aqueous medium “acquovitrex” (carlo erba reagenti spa, milan, italy) and at least 300 hemocytes were observed under the light microscope, in ten optical fields at a magnification of 1250, to determine the phagocytic index, i.e. the percentage of hemocytes with ingested yeast cells. in another series of experiments, calphostin c and h-89 were added to the incubation media, in the presence or in the absence of hril-8, at the sublethal concentration of 0.1 and 1 µm, respectively, as reported above. statistical analysis experiments were replicated at least three times; data are expressed as mean ± sd. at least 300 cells, in 10 optical fields at a magnification of 1250, were counted for each experiment aiming to determine the frequencies of amoeboid or yeast-containing cells; frequencies were subjected to angular transformation. the shape factor was calculated on 20 hyaline amoebocytes for each treatment. means were compared with the duncan’s test. results phagocyte spreading b. schlosseri phagocytes are represented by hyaline amoebocytes and macrophage-like cells. the former are motile cells able to spread on the substrate (fig. 1a) and actively engulf foreign particles. as a consequence of the ingestion, they change their morphology to globular, vacuolated macrophage-like cells (fig. 1b). in our controls, we had about 15 % of hemocytes with spreading morphology (fig. 2). similar results were obtained with the protein kinase inhibitors (fig. 3a). in the presence of hril-8 at the concentrations of 25 and 50 ng/ml, a significant (p < 0.05) increase in the cell spreading index was observed (fig. 1c) which was abolished by the presence of the g protein inhibitor suramin (fig. 2a). no significant effects were observed in the presence of 10 ng/ml hril-8 or suramin alone (data not shown). the shape factor of hyaline amoebocytes resulted significantly (p < 0.05) decreased by hril-8 at 25 and 50 ng/ml (table 1). no significant variation in the shape factor was observed in the presence of the protein kinase inhibitors. 20 control spreading phagocytes had a typical cytoskeletal organisation, with actin filaments organized in bundles of stress fibres without a preferential orientation. the same was true for pseudopodia which radiated from most of the cell contour and resulted strongly fluorescent (fig. 1d). in the presence of hril-8, phagocytes appeared remarkably spread with stress fibres oriented according to a well defined major axis and their cytoplasmic projections, rich in fluorescent actin, emerging from the apical endings (fig. 1e). phagocytosis about 15 % of hemocytes were able to ingest yeast cells. this fraction increased significantly (p < 0.05) in presence of hril-8 at the concentrations of 25 and 50 ng/ml, while the chemokine effect was suppressed by suramin (fig. 2b). in the absence of the chemokine, both calphostin c and h-89 significantly (p < 0.05) inhibit phagocytosis. the protein kinase inhibitors were able to significantly (p < 0.05) reduce the hril-8-stimulated phagocytosis when the chemokine was used at the concentration of 10 ng/ml. the inhibitory effect of h-89 only was still detectable at 25 ng/ml, while at the fig. 3 effects of 0.1 μm calphostin c (red) and 1 μm h-89 (yellow) on cell spreading index (a) and phagocytosis (b) in the presence or in the absence of hril-8. asterisks mark significant differences with respect to controls (incubation in yeast-containing fsw). * = p < 0.05. table 1 shape factors of hyaline amoebocytes in various experimental conditions ______________________________________________________________________________ treatment shape factor ______________________________________________________________________________ fsw (control) 0.21 ± 0.10 fsw + hril-8 10 ng/ml 0.19 ± 0.06 fsw + hril-8 25 ng/ml 0.14 ± 0.07 * fsw + hril-8 50 ng/ml 0.12 ± 0.04 *** ______________________________________________________________________________ asterisks mark significant differences with respect to controls. *: p < 0.05; ***: p < 0.001 concentration of 50 ng/ml the phagocytic index was not influenced by none of the inhibitors used (fig. 3b). discussion circulating immunocytes represent an important feature of coelomate metazoans as they represent the main component of the immune system. once sensed a non-self molecular pattern, immunocytes must be able to leak from the circulation into the tissues and migrate towards the source of the stimulus in order to kill and/or engulf it in the course of an inflammatory reaction. cytokines are immunoregulatory soluble molecules which modulate the activity of immunocytes (rubinstein et al., 1998; borish and steinke, 2003), whereas chemokines represent chemotactic cytokines, released by activated immunocytes and damaged tissues, which mediate immunocytes recruitment to the site of inflammation. several chemokines were identified in vertebrates, grouped in four families on the basis of their primary sequence (esche et al., 2005). the situation is less clear in invertebrates, even if the presence of chemotactic factors was reported (alvarez et al., 1995; nonaka et al., 1999; raftos et al., 2002; pinto et al., 2003; raftos et al., 2003; ottaviani et al., 2004). il-8, one of the most extensively studied vertebrate chemokine, is a key mediator in polymorphonuclear leucocyte, t lymphocyte and nk cell recruitment and activation (baggiolini, 2001; casilli et al., 2005; esche et al., 2005). it induces changes in the organisation of the actin cytoskeleton and expression of adhesion molecules on leucocytes required for their migration to the inflammation sites (luster, 1998; imhof and aurrand-lions, 2004; casilli et al., 2005). invertebrate immunocytes can change their functionality in the presence of exogenous mammalian cytokines (hughes et al., 1990; beck and habicht, 1991; granath et al., 1994, 2001; ottaviani et al., 1995; barcia et al., 1999). this study demonstrates that b. schlosseri immunocytes can respond to exogenous hril-8 with an increase in both phagocyte spreading, as indicated by the higher haemocyte cell spreading index and the reduced amoebocyte shape factor, and phagocytic index. 21 in vertebrates il-8-induced leucocyte modifications are mediated by both changes in the organisation of the actin cytoskeleton and upregulation of integrin expression (luster, 1998; imhof and aurrand-lions, 2004; casilli et al., 2005), the latter required for adhesion to substrata during migration and for the establishment of close contacts with foreign particles in the course of phagocytosis (matricon-gondran and letocart, 1999). we have already demonstrated the importance of cytoskeletal modifications and integrin expression in phagocyte spreading and phagocytosis in botryllus (ballarin et al., 2002). our results confirm previous data and indicate that exogenous il-8 can induce a cytoskeletal reorganisation which is related to the reported increase in phagocyte spreading and phagocytosis. il-8-induced modifications in immunocytes activity were also reported by ottaviani et al. (2000) in the bivalve mollusc mytilus galloprovincialis, suggesting that responsiveness to exogenous il-8 can be common among invertebrate immunocytes. in vertebrates, it is known that il-8 exerts its activity through its interaction with g protein-coupled receptors with seven α-helix transmembrane domains (esche et al., 2005). although no sequences sharing homology with vertebrate chemokine receptor genes have been identified in invertebrates so far (de vries et al., 2006), the presence of g protein-coupled receptors is firmly established and orthologues of the vertebrate receptors have been identified in both radiata and bilateria (brody and cravchik, 2000; bouchard et al., 2003; keating et al., 2003; kawada et al., 2004); in the compound ascidian ciona intestinalis a g protein-coupled receptor involved in intercellular signalling was recently described (elphick et al., 2003). the inhibition of the il-8-induced increase in cell spreading and phagocytosis by suramin, which is known to disrupt receptor-g protein coupling (chung and kermode, 2005), in botryllus (this report) and m. galloprovincialis (ottaviani et al., 2000), suggests that the chemokine interacts with some g protein-coupled receptor in invertebrate immunocytes, unrelated to vertebrate cytokine receptors, which can be the natural target of endogenous chemotactic molecules. vertebrate g protein-coupled receptors mainly act through two well known signal transduction pathways: the cyclic amp and the phosphoinositide pathways (gomberts et al., 2002). in the first case, the adenylate cyclase is activated with the consequent production of cyclic amp which, in turn, activates pka. in the phosphoinositide pathway, a plc is activated upon ligand-receptor interaction which results in the production of ip3 and dag, the former mobilising ca2+ from intracellular stores, the latter activating pkc. as suggested by experiments with calphostin c and h-89, both the transduction pathways are involved in il-8-induced botryllus cell spreading and phagocytosis. the observed decrease of the phagocytic index in the presence of the protein kinase inhibitors in the absence of the exogenous chemokine or in presence of il-8 at the ineffective concentration of 10 ng/ml indicates that both pka and pkc are routinely required for phagocytosis. in addition, in the presence of 25 ng/ml of il-8, h-89, but not calphostin c, reduces the phagocytic index to levels lower than the controls suggesting that the pka pathway is more important than that mediated by pkc in botryllus phagocytosis. similar effect were reported for il-8induced changes in cell shape in bivalve molluscs (ottaviani et al., 2000). further studies are now required to better characterise invertebrate g protein-coupled receptors, their natural ligands and the signal transduction pathways involved in immunocyte activation. acknowledgements authors wish to thank mr. m. del favero for technical help. this work was supported by the italian miur (prin, 2004). references alvarez mr, friedl fe, ruiz roman f. in vivo chemoactivation of oyster hemocytes induced by bacterial secretion products. j. invertebr. pathol. 6: 287292, 1995. baggiolini m. chemokines in pathology and medicine. j. intern. med. 250: 91-104, 2001. ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri. dev. comp. immunol. 22: 479-492, 1998. ballarin l, scanferla m, cima f, sabbadin a. phagocyte spreading and phagocytosis in the compound ascidian botryllus schlosseri: evidence for an integrin-like, rgddependent recognition mechanism. dev. comp. immunol. 26: 39-48, 2002. barcia r, cao a, arbeteta j, ramos-martinez ji. the il-2 receptor in hemocytes of the sea mussel mytilus galloprovincialis lmk. life 48: 419-423, 1999. beck g, habicht gs. primitive cytokines: harbingers of vertebrate defense. immunol. today 12: 180-183, 1991. beck g. macrokines: invertebrate cytokine-like molecules? front. biosci., d559-d569, 1998. beschin a, bilej m, torreele e, de baetselier p. on the existence of cytokines in invertebrates. cell. mol. life sci. 58: 801–814, 2001. beschin a, bilej m, magez s, lucas r, de baetselier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on a similar lectin-like activity. prog. mol. subcell. biol. 34: 145-163, 2004. borish lc, steinke jw. cytokines and chemokines. j. allergy clin. immunol. 111: s460-s475, 2003. bouchard c, ribeiro p, dubé f, anctil m. a new g proteincoupled receptor from a primitive metazoan shows homology with vertebrate aminergic receptors and displays constitutive activity in mammalian cells. j. neurochem. 86: 1149-1161, 2003. brody t, cravchik a. drosophila melanogaster g proteincoupled receptors. j. cell biol. 150: f83-f88, 2000. casilli f, bianchini a, gloaguen i, biordi l, alesse e, festuccia c et al. inhibition of interleukin-8 (cxcl8/il8) responses by repertaxin, a new inhibitor of the 22 chemokine receptors cxcr1 and cxcr2. biochem. pharmacol. 69: 385-394, 2005. chijiwa t, mishima a, hagiwara m, sano m, hayashi k, inoue t et al. inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic amp-dependent protein kinase, n-[2-(p-bromocinnamylamino)ethyl]-5 isoquinolinesulfonamide (h-89), of pc12d pheochromocytoma cells. j. biol. chem. 265: 52675272, 1990. chung w-c, kermode jc. suramin disrupts receptor-g protein coupling by blocking association of g protein α and βγ subunits. j. pharmacol. exp. ther. 313: 191-198, 2005. cima f, sabbadin a, ballarin l. cellular aspects of allorecognition in the compound ascidian botryllus schlosseri. dev. comp. immunol. 28: 881-889, 2004. cooper el, franchini a, ottaviani e. earthworm coelomocytes possess immunoreactive cytokines and pomc-derived peptides. anim. biol. 4: 25-29, 1995. de vries me, kelvin aa, xu l, ran l, robinson j, kelvin dj. defining the origin and evolution of the chemokine/chemokine receptor system. j. immunol. 176: 401-415, 2006. edds kt. morphological and cytochemical transformation in sea urchin coelomocytes. in: cohen wd (ed), blood cells of marine invertebrates: experimental systems in cells, vol 4, liss ar, new york, 53-74, 1985. elphick mr, satou y, satoh n. the invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate ciona intestinalis. gene 302: 95-101, 2003. esche c, stellato c, beck la. chemokines: key players in innate and adaptive immunity. j. invest. dermatol. 125: 615-628, 2005. franchini a, miyan ja, ottaviani e. induction of acthand tnf-α-like molecules in the hemocytes of calliphora vomitoria (insecta, diptera). tissue cell 28: 587-592, 1996. fuhler gm, knol gj, drayer al, vellenga e. impaired interleukin-8 and groα-induced phosphorylation of extracellular signl-regulated kinase result in decreased migration of neutrophils from patients with myelodysplasia. j. leukoc. biol. 77: 257-266, 2005. gomperts bd, tatham per, kramer im. signal transduction. academic press, san diego, ca, 2002. granath wo, connors va, tarleton rl. interleukin 1 activity in haemolymph from strains of the snail biomphalaria glabrata varying in susceptibility to the human blood fluke schistosoma mansoni: presence, differential expression, and biological function. cytokine 6: 21-27, 1994. granath wo, connors va, raines ae. effects of exogenous interleukin-1β on primary sporocysts of schistosoma mansoni (trematoda) incubatd with plasma and hemocytes from schistosome-susceptible and resistant biomphalaria glabrata (gastropoda). invertebr. biol. 120: 365-371, 2001. huang r-r, dehaven rn, cheung ah, diehl re, dixon re, strader cd. identification of allosteric antagonists of receptor-guanine nucleoside-binding protein interactions. mol. pharmacol. 37: 304-310, 1990. hirsch e, katanaev vl, garlanda c, azzolino o, pirola l, silengo l, et al. central role for g protein-coupled phosphoinositide 3-kinase γ in inflammation. science 287: 1049-1053, 2000. hughes tk, smith em, chin r, cadet p, sinisterra j, leung mk et al. interactions of immunoreactive monokines (interleukin-1 and tumor necrosis factor) in the bivalve mollusc mytilus edulis. proc. natl. acad. sci. usa 87: 4426-4429, 1990. hughes tk, smith em, stefano gb. detection of immunoreactive interleukin-6 in invertebrate hemolymph and nervous tissue. prog. neurol. endocrin. immunol. 4: 234-239, 1991. imhof ba, aurrand-lions m. adhesion mechanisms regulating the migration of monocytes. nat. rev. immunol. 4: 432-444, 2004. kawada t, kanda a, minakata h, matsushima o, satake h. identification of a novel receptor for an invertebrate oxytocin/vasopressin superfamily peptide: molecular and functional evolution of the oxytocin/vasopressin superfamily. biochem. j. 382: 231-237, 2004. keating cd, kriek n, daniels m, ashcroft nr, hopper na, siney ej, et al. whole-genome analysis of 60 g proteincoupled receptors in caenorhabditis elegans by gene knockout with rnai. curr. biol. 13: 1715-1720, 2003. kletsas d, sassi d, franchini a, ottaviani e. pdgf and tgf-β induce cell shape changes in invertebrate immunocytes via specific cell surface receptors. eur. j. cell biol. 75: 362-36, 1988. luster ad. chemokines, chemotactic cytokines that mediate inflammation. new engl. j. med. 338: 436-445, 1998. maghazachi aa. insights into seven and single transmembrane-spanning domain receptors and their signaling pathways in human natural killer cells. pharmacol. rev. 57: 339-357, 2005. matricon-gondran m, letocart m. internal defenses of the snail biomphalaria glabrata. j. invertebr. pathol. 74: 235-247, 1999. menin a, del favero m, cima f, ballarin l. release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian. mar. biol. 2006 (in press). naccache ph, levasseur s, lachance g, chakravarti s, bourgoin sg, mccoll sr. stimulation of human neutrophils by chemotactic factors is associated with the activation of phosphatidylinositol 3-kinase γ. j. biol. chem. 275: 23636-23641, 2000. nonaka m, azumi k, ji x, namikawa-yamada c, sasaki m, saiga h et al. opsonic complement component c3 in the solitary ascidian, halocynthia roretzi. j. immunol. 162: 387-391, 1999. ottaviani e, franchini a, cassanelli s, genedani s. cytokines and invertebrate immune responses. biol. cell 85: 87-91, 1995. ottaviani e, sassi d, kletsas d. pdgfand tgf-β-induced changes in cell shape of invertebrate immunocytes: effect of calcium entry blockers. eur. j. cell biol. 74: 336-341, 1997. ottaviani e, franchini a, malagoli d, genedani s. immunomodulation by recombinant human interleukin-8 and its signal transduction pathways in invertebrate hemocytes. cell. mol. life sci. 57: 506-513, 2000. ottaviani e, franchini a, malagoli d. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. ouwe-missi-oukem-boyer o, porchet e, capron a, dissous c. characterization of immunoreactive tnfα molecules in the gastropod biomphalaria glabrata. dev. comp. immunol. 18: 211-218, 1994. pinto mr, chinnici cm, kimura y, melillo d, marino r, spruce la, et al. cic3-1a-mediated chemotaxis in the deuterostome invertebrate ciona intestinalis (urochordata). j. immunol. 171: 5521-5528, 2003. raftos da, stillman dl, cooper el. chemotactic response of tunicate (urochordata: ascidiacea) hemocytes in vitro. j. invertebr. pathol. 72: 44-49, 1998. raftos da, nair s, robbins j, newton r, peters r. a complement component c3-like protein from the 23 tunicate, styela plicata. dev. comp. immunol. 26: 307312, 2002. rubinstein m, dinarello ca, oppenheim jj, hertzog p. recent advances in cytokines, cytokine receptors and signal transduction. cytokine growth factor rev. 9: 175-181, 1998. raftos da, robbins j, newton r, nair s. a complement component c3-like peptide stimulates chemotaxis by hemocytes from an invertebrate chordate, the tunicate pyura stolonifera. comp. biochem. physiol. 134a: 377386, 2003. smith rj, sam lm, leach kl, justen jm. postreceptor events associated with human neutrophil activation by interleukin-8. j. leukoc. biol. 52: 17-26, 1992. rinkevich b, tartakover s, gershon h. contribution of morula cells to allogeneic responses in the colonial urochordate botryllus schlosseri. mar. biol. 131: 227236, 1998. tamaoki t. use and specificity of staurosporine, ucn-01, and calphostin c as protein kinase inhibitors. methods enzymol. 201: 340-347, 1991. rollins bj. chemokines. blood 90: 909-928, 1997. 24 a menin, l ballarin department of biology, university of padova, padova, italy animals immunocytochemical assays for cytoskeleton phagocytosis assay phagocyte spreading phagocytosis references review isj 4: 127-136, 2007 issn 1824-307x review new immune systems: pathogen-specific host defence, life history strategies and hypervariable immune-response genes of invertebrates l bowden, nm dheilly, da raftos, sv nair department of biological sciences, macquarie university, north ryde, new south wales, 2109, australia accepted december 14, 2007 abstract our understanding of invertebrate immune systems is undergoing a paradigm shift. until recently, the host defence responses of invertebrates were thought to rely on limited molecular diversity that could not tailor reactions toward specific microbes. this view is now being challenged. highly discriminatory defence responses, and hypervariable gene systems with the potential to drive them, have been identified in a number of invertebrate groups. these systems seem to be quite distinct, suggesting that pathogen-specific responses might have evolved on numerous occasions. here, we review evidence that inducible, disease-specific immunity might be commonplace in the animal kingdom. key words: adaptive immunity; host defence; immunization; invertebrate immunology; hypervariability introduction the continuing growth of invertebrate aquaculture worldwide, and its seemingly inherent problems with catastrophic disease outbreaks, is driving a renewed demand for research into invertebrate immune systems. much of this research revolves around two unresolved questions can invertebrate immune systems be fine-tuned to fight specific infections, and, can that fine-tuning provide protective immunity to re-infection? for the first time, powerful new molecular technologies are parallelling classical immunization experiments to provide evidence for disease-specific immunity among invertebrates. however, these two experimental approaches have rarely been unified to elucidate the mechanisms underpinning pathogen-specific defense. providing this synthesis will continue to drive the paradigm shift in invertebrate immunology. it could lay the foundation for the use of immunization as a powerful new method for disease control in invertebrate aquaculture, and provide fundamental insights into the adaptive selection pressures that drive the evolution of immune systems. in this article , we explore evidence for pathogen-specific immunity ___________________________________________________________________________ corresponding author: david a raftos department of biological sciences, macquarie university, north ryde, new south wales, 2109, australia e-mail: draftos@rna.bio.mq.edu.au among invertebrates, and the role that hypervariable gene families play in those responses. traditional views of invertebrate immune systems until recently, highly variable immune-response molecules were thought to be confined to the jawed vertebrates (gnathostomes) (raftos and raison, 1992; raftos, 1993; litman et al., 2005). adaptive immune responses among gnathostomes revolve around hypervariable antibodies and t-cell receptors (tcrs), which act as pathogen-specific recognition proteins. the enormous diversity of antigen-specific antibodies and tcrs is generated using enzymes encoded by somatic recombination activating (rag) genes. production of each unique antibody or tcr isotype is confined to individual lymphocyte clones, which are responsible for providing lasting immunity against specific pathogens via the process of clonal selection. since burnet (1959) put forward his theory of clonal selection, it has been assumed that invertebrates lacked highly specific immune responses based on pathogen-specific immunorecognition (raftos, 1993). this presumption was based, almost exclusively, on the failure to find hypervariable antibodies or tcrs amongst invertebrates (raftos and raison, 1992). remarkably, there is only a limited history of appropriately controlled immunization experiments 127 mailto:draftos@rna.bio.mq.edu.au being used to test whether antibody/tcrindependent pathogen-specific immune responses occur among invertebrates. the purported lack of pathogen-specific immunity among invertebrates has often been explained by the differential use of rand k selection strategies by invertebrates and vertebrates (klein, 1989, 1989; rinkevich, 1999). the terms, r and k, come from ecological algebra defined by verhulst's equation describing population dynamics (verhulst, 1838). dn/dt = rn (1-n/k), where "r" is the growth rate of a population, "k" is the carrying capacity of the environment and "n" is population size. the r/k-selection hypothesis has been used to distinguish invertebrates most often as r-selected, indicating that they are smaller, have higher fecundity, shorter lifespans and less stable populations than most vertebrates (pianka, 1970). this is seen as a high risk reproductive strategy that is more susceptible to chance events. it explains why organisms that are r-selected display higher fecundity. vertebrates, on the other hand, were seen to have lower reproductive outputs, longer lifespans and generally smaller, more stable populations that could be described as k-selected. the r/k-selection argument with respect to the evolution of pathogen-specific immune systems is simple invertebrates don't live as long as vertebrates, and they don't invest as much in each of their offspring. therefore, they do not require sophisticated immune responses that rely on pathogen-specific immunorecognition to maintain population stability. this is a simplistic argument that has always been flawed (stearns, 1977; getz, 1993). in terms of life history strategies, it is difficult to imagine that some form of adaptive immunity would not have evolved among invertebrates. based on the r/k selection paradigm, it has been taken for granted that vertebrates, with their presumed long life cycles and relatively lower reproductive outputs, had a greater evolutionary "need" for adaptive immunity than invertebrates (klein, 1989; medzhitov and janeway, 1997). however, r/k-selection does not neatly distinguish vertebrates from invertebrates (stearns, 1977; getz, 1993). both rand k-selection strategies can be observed in both taxa, including important stem groups. some invertebrates, such as bivalves, gastropods, echinoids and decapods, can live for many decades (up to 100 years in some cases), and clearly express features of k-selected strategies, including large body size and relatively low effective fecundity (powell and cummings, 1985). conversely, some gnathostomes, including fish, in which adaptive immunity appears to have first evolved, have short lifespans and high fecundity. for instance, coral fish (eviota sigillata) reach sexual maturity within weeks of birth and have a maximum lifespan of 59 days (depczynski and bellwood, 2005). so, using the r/k model to determine whether a broad taxonomic group "requires" highly specific responses to particular pathogens has never been well supported by the available evidence. factors other than r/k life history strategies are also likely to provide significant selection pressures for the evolution of pathogen-specific immune systems. many invertebrates occupy niches that place them in close contact with a broad range of pathogens. others establish high density populations that are prone to epizootic disease outbreaks (stow et al., 2007). many flies and other arthropods feed and/or breed in faeces or decomposing plant material, whilst some other insects, such as ants, bees and termites, live in highly social colonies that facilitate the transmission of virulent pathogens and parasites. eusociality, or the production of sterile workers to care for reproductive members of the population, is perhaps the most extreme example of a life history strategy that might necessitate pathogenspecific immunity. eusociality often involves asexual reproduction that limits genetic variability within populations, combined with high population densities in confined environments (hives or nests). according to van valen's (1973) red queen theory, which suggests that genetic diversity within populations is required to counter rapidly evolving pathogens, the genetic and social strictures of eusociality should make eusocial populations more susceptible to epizootic disease. as a result, there should be a strong link between eusociality and enhanced defensive capacity. this prediction fits well with recent data indicating that, among social insects, immunological capacity increases proportionately with the degree of sociality. increased eusociality and genetic relatedness among bees is strongly correlated with an increase in the production of antimicrobial compounds (stow et al., 2007). some eusocial species also show strong evidence for pathogen specific immunity (see below). new evidence for pathogen-specific immunity among invertebrates even though the r/k selection argument has been used since burnet's time to predict that invertebrates lack pathogen-specific recognition of the type provided by vertebrate antibodies and tcrs (klein, 1989), new data question this assumption. recent work indicates that arthropods and molluscs can generate fine-tuned responses against specific pathogens. these studies have used classical immunization experiments to test for pathogen specificity; usually by measuring host survival after inoculation and by comparing responses between primary and secondary inoculations using different pathogens (fig. 1). in these experiments, more rapid and powerful secondary immune responses against the original, inoculating pathogen are presumed to indicate that the host has developed an induced response targeting that pathogen (little et al., 2005). pathogen specificity is further implied if enhanced responses do not extend to microbes other than the one used in the initial, priming inoculation. this approach is not new. in the past it has been used to show that drosophila melanogaster can distinguish between broad groups of pathogens, such as fungi and bacteria (lemaitre et al., 1997). these broad responses are often mediated by the 128 fig. 1 a generic protocol for immunization experiments. host organisms are inoculated with one of two different pathogens (circles and squares). some time later, they are re-challenged either with the same pathogen used for the initial inoculation, or with the other pathogen. the intensity of host responses to the secondary challenge is then measured, often in terms of host mortality over time or the clearance rate of the pathogen (modified from little et al., 2005). toll and imd/relish signalling pathways and probably differentiate between highly conserved molecular structures on pathogens, such as bacterial lipopolysaccharides (lps) and fungal βglucans (leclerc and reichhart, 2004; beutler et al., 2006). janeway (1989, 1992) has designated these types of highly conserved target molecules as "pathogen-associated molecular patterns (pamps)". pamps define broad groups of pathogens for detection by generic "pattern recognition" receptors (prrs), such as toll-like receptors and lectins. pattern recognition, which lacks fine-scale differentiation between closely related microbes, is common throughout the animal kingdom and was thought to be the only molecular recognition paradigm available to invertebrates (medzhitov and janeway, 1997, 2002; janeway and medzhitov, 2002). however, more recent immunization experiments have shown that d. melanogaster is capable of much finer scale discrimination between microbes. pham et al. (2007) found that immune responses by d. melonogaster against streptococcus pneumoniae could be enhanced by inoculation with heat-killed s. pneumoniae. primed responses against s. pneumoniae did not provide protection against other pathogens, even closely related bacterial species. and prior exposure to a variety of other bacteria did not elicit protection against s. pneumoniae. specific protection could also be stimulated by injecting beauveria bassiana, a natural pathogen of d. melanogaster. however, inoculation with salmonella typhimurium, listeria monocytogenes and mycobacterium marinum, did not induce pathogen-specific protective responses. the enhanced responses to s. pneumoniae and b. bassiana in drosophila clearly have features in common with gnathostome adaptive immunity, yet they appear to be mediated by the same toll-like pathways and phagocytic responses that are associated with simple pattern recognition (pham et al., 2007). even more compelling evidence for pathogenspecific immunity among insects comes from studies of bumblebees (bombus terrestris). sadd and schmid-hempel (2006) immunized bees with a defined isolate of the gram-negative bacterium, pseudomonas fluorescens, and two closely-related gram-positive bacteria (paenibacillus alvei and paenibacillus larvae). bees were re-challenged either 8 or 22 days after the initial, priming injection, and their relative survival was recorded. the data showed that b. terrestris were capable of mounting highly specific immune responses that could differentiate even between the congeneric bacteria (sadd and schmid-hempel, 2006). such targeted responses only became evident when bees were left for 22 days before being re-challenged. pathogen-specific responses were masked by less specific reactions when bees were re-challenged within 8 days of the initial inoculation. these examples of pathogen-specific immunity in drosophila and b. terrestris are among a number of highly targeted immune responses so far identified among invertebrates. in 2006, kurtz cited five studies of either insects, crustaceans or molluscs, in which there is strong evidence for the induction of pathogen specific immunity by inoculation with trematodes, bacteria, tapeworms or trypanosomes (webster and woodhouse, 1998; schmid-hempel et al., 1999; carius et al., 2001; little et al., 2003; kurtz and franz, 2003). among these studies, kurtz and franz (2003) showed that infections of the copepod crustacean, 129 macrocyclops albidus, with the tapeworm, schistocephalus solidus, were far less severe if the hosts had been primed with siblings of the worms used for the subsequent infections. this acquired protection was not evident if the tapeworms used in the initial and subsequent challenges were genetically distinct. similar immunization experiments in the prawn, penaeus monodon, identified discriminatory responses to virulent white spot syndrome virus (wssv) (witteveldt et al., 2004). injecting the wssv envelope protein, vp28, provided substantial protection against white spot infection, whereas a closely related antigen, vp19, did not. in a another series of immunization experiments, it was shown that induced, specific protection against pathogens in the water flea, daphnia magna, can be transmitted from mother to offspring (little et al., 2005). the interpretation of these data is extremely controversial (hauton and smith, 2007). it does suggest that some protostomes can mount defensive responses that are fine-tuned toward some invading pathogens (schmid-hempel et al., 1999; kurtz and franz, 2003; schmid-hempel and ebert, 2003; little and kraaijeveld, 2004; kurtz, 2005; little et al., 2005; kurtz and armitage, 2006). however, there is still insufficient evidence to tell whether these systems are widespread throughout the metazoa. it is also unclear if they can generate targeted protection against many different pathogens, or whether they have been selected only to provide protection against pathogens that are the most relevant to the host species. hypervariable immune-response gene families of invertebrates at roughly the same time that traditional immunization experiments were revealing the existence of pathogen-specific responses in some invertebrates, new molecular technologies were being used to identify hypervariable immune response molecules that might underpin pathogenspecificity (flajnik, 2004; flajnik and du pasquier, 2004; loker et al., 2004; litman et al., 2005; du pasquier, 2006). it is now evident that a range of highly variable immune-response gene families are expressed among invertebrates and jawless vertebrates. some of these families have already been found to play key roles in immunological functions, such as self/non-self recognition and differentiation between broadly different pathogen types (i.e. fungi vs. bacteria). from the existing data it is clear that the gene families encoding highly variable immune-response proteins have arisen independently on numerous occasions in higher plants and in animals (flajnik, 2004; flajnik and du pasquier, 2004). none of the families that have been identified to date are closely related to each other, even though many incorporate immunoglobulin-superfamily (igsf) domains (flajnik, 2004; flajnik and du pasquier, 2004; litman et al., 2005). this suggests that there has been strong selection pressure within individual taxa to develop mechanisms that promote survival in the presence of rapidly evolving pathogens. a number of the highly variable immuneresponse families identified among invertebrates and "lower" vertebrates are described in more detail below. variable lymphocyte receptors in agnathans the two extant groups of agnathans (jawless fish; hagfish and lampreys) do not express highly diverse antibodies or tcr (raftos and raison, 1992). however, expressed sequence tag (est) analyses of lamprey lymphocytes revealed immuneresponse genes that encode highly variable lymphocyte receptors, or vlrs. these molecules contain numerous leucine-rich-repeat (lrr) modules, and may act as cell surface receptors implicated in immunological defence (flajnik, 2004; pancer et al., 2004; litman et al., 2005). vlrs have non-variable glycosyl-phosphatidyl-inositol (gpi) lipid anchors for attachment to the plasma membranes lymphocytes. these gpi anchors may become detached to free humoral forms of vlr molecules (litman et al., 2005). the high level of diversity displayed among vlrs arises from variation in their nucleotide sequences and in the number of lrr modules that they incorporate (pancer et al., 2004). there is some evidence that different lamprey lymphocytes express different and unique vlrs, implying a clonal expression system akin to that of gnathostome antibodies (flajnik, 2004). v region-containing chitin binding proteins in cephalochordates cannon et al. (2002) identified five families of highly variable immune-response molecules in the protochordate, amphioxus (branchiostoma floridae), that incorporate two n-terminal igsf v-region domains and one chitin-binding domain at the carboxy-terminus. these molecules have been designated v region-containing chitin binding proteins (vcbps). their v regions show high levels of diversification and might function as specific immune-recognition domains akin to the adaptive immunological receptors of gnathostomes. the five distinct families of vcbp transcripts identified by cannon et al. (2002) are highly diversified, showing only 27 % 41 % sequence identity among families. the chitin-binding region incorporates a diagnostic sequence of spaced cysteine residues that is characteristic of chitin-binding proteins in other species, and recombinant vcbps can bind chitin. in situ hybridisation revealed that vcbps are expressed exclusively in the intestine. given the filter feeding life history of amphioxus, this distribution fits well with a potential role in the control of pathogens in the gut. cannon et al. (2002) concluded that vcbps may have a dual function, combining pattern recognition by the chitin-binding domain with diversified specific immunorecognition afforded by the highly variable igsf v-regions. 185/333 proteins in sea urchins the 185/333 gene family from the purple sea urchin, strongylocentrotus purpuratus, has high levels 130 fig. 2 185/333 proteins are expressed primarily on the surface of one sea urchin (s. purpuratus) coelomocyte type, small phagocytes. in this image, 185/333 staining is shown in green and nuclei are counterstained red. note the expression of 185/333 on the extensive filopodia of small phagocytes. f, filopodia; n, nuclei. of both cdna and genomic dna sequence diversity (nair et al., 2005; terwilliger et al., 2006, 2007; buckley and smith, 2007). about 50-60 distinct genomic 185/333 loci have been estimated and almost 700 unique mrna transcripts have been sequenced to date (terwilliger et al., 2007; k buckley, george washington university, personal communication). there is also evidence for 185/333 gene sequences and gene expression in other sea urchin species (m roth, macquarie university, personal communication). unlike the highly variable gene families of other invertebrates, sequence comparisons have failed to reveal any similarities between 185/333 genes and those from other organisms (nair et al., 2005). however, based on their expression patterns, 185/333 proteins appear to be involved in immune responses. titres of both 185/333 mrna transcripts and proteins increase after immunological challenge (brockton et al., 2007; nm dheilly, unpublished data). the frequency of 185/333 mrnas increases 70-fold during bacterial infections, and 185/333 transcripts can comprise more than 60 % of the mrna specifically induced by immune responses (nair et al., 2005). there is also some evidence that different pamps elicit the expression of different 185/333 variants (nm dheilly, unpublished data; terwilliger et al., 2007). proteomics and transcriptome analysis have shown that 185/333 molecules have high levels of molecular diversity within and between sea urchins. individual sea urchins can express in excess of 200 distinct 185/333 proteins, and each animal has a distinctive suite of the proteins that differs from all other individuals (nm dheilly, unpublished data; terwilliger et al., 2007). 185/333 proteins are localized to the surface of a distinct class of coelomocytes, the frequency of which is substantially enhanced in coelomic fluid after immunological challenge (fig. 2) (brockton et al., 2007). the observed molecular weights of 185/333 proteins are much higher than those predicted from mrnas, suggesting that 185/333 proteins form strong associations with other molecules, or with each other (brockton et al., 2007; nm dheilly, unpublished data ). the variability of 185/333 genes can be explained by combinatorial diversity, single nucleotide polymorphisms (snps) and small insertions and deletions (indels). 185/333 mrna transcripts are comprised of 25 different blocks of sequence, or “elements”, that are either present or absent in many different combinations (nair et al., 2005; terwilliger et al., 2006; buckley and smith, 2007). snps and indels also occur frequently in all 185/333 transcripts, magnifying their diversity. nucleotide substitutions between 185/333 variants are often non-conservative, resulting in amino acid changes. this suggests that 185/333 diversity has been driven by positive selection of the type associated with pathogen-driven adaptation (nair et al., 2005; terwilliger et al., 2006). the processes that generate 185/333 diversity remain unclear, but they could include genomic or somatic recombination, alternative splicing, rna editing and post translational modifications (buckley and smith, 2007). the biological activities of 185/333 proteins have not yet been defined and their lack of homology with other molecules makes it difficult to predict structure/function relationships. however, evidence from sequence analyses is shedding more light on the structure of these proteins. 185/333 mrnas encode polypeptides with a hydrophobic leader, separate glycineand histidinerich regions, numerous n-linked and o-linked glycosylation sites, acidic sequence patches, and an arginine-glycineaspartic acid (rgd) motif (terwilliger et al., 2006). predicted polypeptides do not contain cysteines, transmembrane regions, gpi linkage sites or identifiable domains. the only regions with at least some similarity to other molecules are the rgd motif and one of the histidine-rich domains, which is comparable to histatins, a group of mammalian salivary proteins with powerful antifungal activities (xu et al., 1990). more detailed analysis of 185/333 sequences reveals many short amino acid sequence repeats that are found at numerous positions in the predicted proteins (nair et al., 2005; buckley and smith, 2007) (fig. 3). distinctive types of repeats are found in different regions of predicted polypeptides, most notably in the n-terminal glycinerich region and a c-terminal region that incorporates numerous potential n-glycosylation sites. in many cases, different types of short repeats are clustered together, and these clusters are duplicated throughout the polypeptide. distinct combinatorial patterns of repeats define eight distinct 185/333 sub-families, which phylogenetic analysis suggests arose by progressive recombination events (nm dheilly, unpublished data; k buckley, george washington university, personal communication). freps in molluscs highly variable fibrinogen-related proteins (freps) in the snail, biomphalaria glabrata, and in at least five other genera of gastropods, consist of 131 fig. 3 a) the pattern of amino acid repeats evident in a model 185/333 protein from the sea urchin, s. purpuratus. each colored shape represents a unique amino acid repeat. boxed areas show where different clusters of repeats are duplicated throughout the predicted polypeptide. distinct repeat clusters are found in the glycine-rich and n-glycosylated regions of the polypeptide. b) amino acid repeat patterns found in 8 distinct 185/333 sub-families. each family is distinguished by a unique combinatorial pattern of sequence repeats. either two or three domains. one is a c-terminal fibrinogen domain, while the other one or two domains are members of the igsf (fig. 4a) (zhang et al., 2004). freps are reported to have lectin-like (carbohydrate-binding) activity and their expression increases in response to challenge with the trematode parasites, echinostoma paraensei and schistosoma mansoni (adema et al., 1997). one strain of b. glabrata that is resistant to s. mansoni increases frep expression by up to 57-fold after exposure to the parasite, whilst frep expression in s. mansoni-susceptible snails is unaltered by immunological challenge (hertel et al., 2005). freps can precipitate antigens that are secreted by s. mansoni and can bind to the surface of e. paraensei. all of these observations suggest that freps are involved in host/parasite interactions and may act as highly diversified recognition and/or effector proteins (loker et al., 2004; hertel et al., 2005). substantial diversity is evident in the igsf domain(s) of freps. expressed sequence tag (est) analysis of transcripts enriched for the freps of s. mansoni-resistant snails by suppression subtractive hybridisation (ssh) identified 88 unique ssh-ests among the 112 clones sequenced (nowak et al., 2004). sequence diversity incorporates a number of distinct frep subfamilies, and additional variability is evident in the form of snps within each of these sub-families. diversity seems to be generated through a combination of alternative exon splicing, gene conversion and somatic hypermutation, meaning that different individuals express different suites of freps (fig. 4b) (zhang and loker, 2003; zhang et al., 2004). frep genes appear have diversified faster at the genomic level than other genes, and all of the variants identified to date seem to be derived from just nine ancestral or "mother" sequences (zhang et al., 2004). dscams in insects the down’s syndrome cell adhesion (dscam) molecules of d. melanogaster and other insects are members of the igsf. dscam genes comprise clusters of ig-like exons with high levels of sequence diversity that are flanked by relatively conserved regions (fig. 5) (schmucker et al., 2000). alternative splicing recombines exons by mutually exclusive excision, potentially generating more than 38,000 distinct extracellular domains. individual hemocytes can generate between 14-50 different isoforms of dscam and there are a total of about 19,000 hemocyte-specific isoforms (watson et al., 2005). hemocytes from mutant flies with impaired dscam expression, and hemocytes in which dscam expression had been specifically knocked down using interference rna (rnai), have significantly lowered phagocytic activities than hemocytes from normal flies. antibodies that specifically bind to the extracellular ig domains of dscams can also inhibit phagocytosis and dscam isoforms can bind onto pathogen surfaces (watson et al., 2005). this suggests 132 fig. 4 a) schematic polypeptide structure of the frep3 sub-family showing different domains (n-terminus on left). sp, signal peptide; igsf1 and 2, immunoglobulin superfamily domains; scr, small connecting region; icr, interceding region; fbg, fibrinogen domain. b) comparison of freps expressed by two different individuals of the same snail species (b. glabrata). the region of igsf1 shown by the black bar in a. was amplified from transcripts isolated from the two snails and sequenced. the numbers shown in the figure are the number of clones that were unique to each snail, and the number shared by the two snails (modified from zhang et al., 2004). that dscams are involved in the phagocytic uptake of pathogens, possibly acting as pathogen-specific recognition proteins (watson et al., 2005). interestingly, dscams also act as axon guidance receptors during neuronal development, with different dscam isoforms being expressed in the brain compared to hemocytes (watson et al., 2005). what role do highly variable gene families play in pathogen-specific immunity? the data on 185/333 genes, vcbps, freps and dscams imply that all of these gene families contribute to novel forms of inducible, pathogenspecific immune systems (flajnik, 2004; flajnik and du pasquier, 2004; litman et al., 2005). however, there is little direct evidence to support this conclusion. the molecular approaches that have been used to identify these genes have often lacked accompanying information about their physiological functions. in hindsight, resolving the biological activities of hypervariable proteins, particularly their contribution to pathogen-specific immune responses, is proving to be problematic and time consuming. in most cases, we still do not understand the biological relevance of these gene systems, or whether they help to combat infection. traditional immunization experiments have suffered from the opposite problem compared to molecular biological approaches. whilst immunization studies have been very useful in demonstrating the existence of acquired, pathogenspecific reactions, they have not provided information about the molecular mechanisms underlying induced responses (little and kraaijeveld, 2004; little et al., 2005). a new goal of invertebrate immunology will be to address this gap between molecular immunology and immunization experiments (hauton and smith, 2007). new work needs to combine classical immunization protocols with comprehensive molecular analyses to simultaneously identify pathogen-specific immune responses and the genes that control them. to date there are only a few convincing studies that have implemented this approach. dong et al. (2006) showed that particular dscam splice-variants in the mosquito, anopheles gambiae, are associated with resistance to malarial (plasmodium) and bacterial infections. rnai silencing of dscam expression significantly reduced survival rates from bacterial infections and increased plasmodium ooycst development in the midgut of mosquitoes. most significantly, the a. gambiae dscam (agdscam) variants produced in response to inoculation with particular bacteria, (escherichia coli and pseudomonas veronii) could bind onto these microbes with higher affinity than the agdscams produced in response to challenge with grampositive bacteria (staphylococcus aureus) or nonchallenged cell lines. silencing specific forms of agdscams associated with responses to different bacteria made mosquitoes more sensitive to infections by the target bacterial species, but not 133 fig. 5 gene organization of dscams from the mosquito anopheles gambiae showing clusters of highly variable igsf domains and constant domains. the numbers below the figure show the number of ig exons per cluster (modified from dong et al., 2006). others. these data imply a strong association between dscams and pathogen-specific immunity in insects (dong et al., 2006). similarly, robalino et al. (2005) have demonstrated a clear link between pathogenspecific systems in shrimp and protective immunity to wssv. they found that protection against wssv in litopenaeus vannamei could be elicited by injecting double-stranded rna (dsrna) (robalino et al., 2005). even though randomly generated dsrnas had some effect, much higher pathogenspecific protection against white spot disease was induced when dsrnas based on wssv sequences were used, indicating that shrimp can use pathogenspecific rnai systems to generate highly targeted protection against viral diseases. conclusions studies that combine immunization experiments with manipulative molecular analyses represent a new emphasis for invertebrate immunology. the data presented in this article suggest two things that pathogen-specific, inducible immune systems might be common among invertebrates, and that these systems have evolved independently on numerous occasions. this implies that many more pathogen-specific immune systems, and their associated hypervariable recognition molecules, await discovery. acknowledgments our work on immune-response proteins in sea urchins is funded by an australian research council discovery grant (dp0556486 with lc smith). l. courtney smith, k buckley, d terwilliger and v brockton from the george washington university in washington, dc, have been responsible for most of the work on sea urchin 185/333 molecules. our collaboration with them has helped to develop many of the ideas expressed in this article. we also thank courtney smith for helping to edit this article. the photomicrograph shown in fig. 4 was taken by da raftos in courtney smith's laboratory. our studies of disease resistance in molluscs are supported by an australian research council linkage grant in conjunction with the nsw department of primary industries (lp0453461). references adema cm, hertel la, miller rd, loker es. a family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection. proc. natl. acad. sci. usa 94: 8691-8696, 1997. beutler b, jiang z, georgel p, crozat k, croker b, rutschmann s, et al. genetic analysis of host resistance: toll-like receptor signalling and immunity at large. annu. rev. immunol. 24: 353-389, 2006. brockton v, henson jh, raftos da, majeske aj, kim yo, smith lc. localization and expression of 185/333 proteins encoded by a diverse gene family in coelomocytes from the purple sea urchin, strongylocentrotus purpuratus (stimpson). j. cell sci. [in press], 2007. buckley km, smith lc. extraordinary diversity among members of the large gene family, 185/333, from the purple sea urchin, strongylocentrotus purpuratus. bmc mol. biol. 8: 68, 2007. burnet fm. the clonal selection theory of acquired immunity. cambridge university press, cambridge, 1959. cannon jp, haire rn, litman gw. identification of diversified genes that contain immunoglobulinlike variable regions in a protochordate. nature immunol. 3: 1200-1207, 2002. carius hj, little tj, ebert d. genetic variation in a host–parasite association: potential for coevolution and frequency-dependent selection. evol. int. j. org. evol. 55: 1136– 1145, 2001. 134 depczynski m, bellwood dr. shortest recorded vertebrate lifespan found in a coral reef fish. curr. biol. 15: 288-289, 2005. dong y, taylor he, dimopoulos g. agdscam, a hypervariable immunoglobulin domaincontaining receptor of the anopheles gambiae innate immune system. plos biol. 4: 11371146, 2006. du pasquier l. germline and somatic diversification of immune recognition elements in metazoa. immunol. lett. 104: 2-17, 2006. flajnik mf. another manifestation of god. nature 430: 157-158, 2004. flajnik mf, du pasquier l. evolution of innate and adaptive immunity: can we draw a line? trends immunol. 25: 640-644, 2004. getz wm. metaphysiological and evolutionary dynamics of populations exploiting constant and interactive resources r-k selection revisited. evol. ecol. 7: 287-305, 1993. hauton c, smith vj. adaptive immunity in invertebrates: a straw house without a mechanistic foundation. bioessays 29: 11381146, 2007. hertel la, adema cm, loker es. differential expression of frep genes in two strains of biomphalaria glabrata following exposure to the digenetic trematodes schistosoma mansoni and echinostoma paraensei. dev. comp. immunol. 29: 295-303, 2005. janeway ca. approaching the asymptote? evolution and revolution in immunology. cold spring, harbor symp. quant. biol. 54: 1-13, 1989. janeway ca. the immune system evolved to discriminate infectious nonself from noninfectious self. immunol. today 13:11-16, 1992. janeway ca, medzhitov r. innate immune recognition. annu. rev. immunol. 20: 197-216, 2002. klein j. are invertebrates capable of anticipatory immune responses? scand. j. immunol. 29: 499-505, 1989. klein j. homology between immune responses in vertebrates and invertebrates: does it exist? scand. j. immunol. 46: 558-564, 1997. kurtz j. specific memory within innate immune systems. trends immuol. 26: 186-191, 2005. kurtz j, franz k. evidence for memory in invertebrate immunity. nature 425: 37-39, 2003. kurtz j, armitage sao. alternative adaptive immunity in invertebrates. trends immunol. 27: 493-496, 2006. leclerc v, reichhart jm. the immune response of drosophila melanogaster. immunol. rev. 198: 59-71, 2004. lemaitre b, reichhart jm, hoffman ja. drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. proc. natl. acad. sci. usa 94: 14614-14619, 1997. litman gw, cannon jp, dishaw lj. reconstructing immune phylogeny: new perspectives. nature 5: 866-879, 2005. little tj, o’connor b, colegrave n, watt k, read af. maternal transfer of strain-specific immunity in an invertebrate. curr. biol. 13: 489-492, 2003. little tj, kraaijeveld ar. ecological and evolutionary implications of immunological priming in invertebrates. trends ecol. evol. 19: 58-60, 2004. little tj, hultmark d, read a. invertebrate immunity and the limits of mechanistic immunology. nature immunol. 6: 651-654, 2005. loker es, adema cm, zhang s, kepler tb. invertebrate immune systems – not homogenous, not simple, not well understood. immunol. rev. 198: 10-24, 2004. medzhitov r, janeway ca. innate immunity: the virtues of a nonclonal system of recognition. cell 91: 295-298, 1997. medzhitov r, janeway ca. decoding the patterns of self and nonself by the innate immune system. science 296: 298-300, 2002. nair sv, del valle h, gross ps, terwilliger dp, smith lc. macroarray analysis of coelomocyte gene expression in the sea urchin. identification of unexpected immune diversity in an invertebrate. physiol. genomics 22: 33-47, 2005. nowak ts, woodards ac, jung y, adema cm, loker es. identification of transcripts generated during the response of resistant biomphalaria glabrata to schistosoma mansoni infection using suppression subtractive hybridization. j. parasitol. 90: 1034-1040, 2004. pancer z, amemlya ct, ehrhardt gr, ceitlin j, gartland l, cooper md. somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey, nature 430: 174-180, 2004. pham ln, dionne ms, shirasu-hiza m, schneider ds. a specific primed immune response in drosophila is dependent on phagocytes. plos pathogens 3: 1-8, 2007. pianka er. on r and k selection. amer. nat. 104: 592-597, 1970. powell en, cummings h. are molluscan maximum life spans determined by long-term cycles in benthic communities. oecologia 67: 177-182, 1985. raftos, da. development of primitive recognition systems in invertebrates. in: developmental immunology. cooper el, nisbet-brown e (eds), oxford university press, new york, 1993. raftos da, raison rl. out of the primordial slime: evolution and the immune system. today's life sci. 14: 16-20, 1992. rinkevich b. invertebrates versus vertebrates innate immunity: in the light of evolution. scand. j. immunol. 50: 456-460, 1999. robalin j, bartlett t, shepard e, prior s, jaramillo g, scura e, et al. double-stranded rna induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of rna interference and innate immunity in the invertebrate antiviral response? j. virol. 79: 13561-13571, 2005. sadd bm, schmid-hempel p. insect immunity shows specificity in protection upon secondary 135 pathogen exposure. curr. biol. 16: 1206-1210, 2006. schmid-hempel p, puhr k, kruger n, reber c, schmid-hempel r. dynamic and genetic consequences of variation in horizontal transmission for a microparasitic infection. evol. int. j. org. evol. 53: 426–434, 1999. schmid-hempel p, ebert d. on the evolutionary ecology of specific immune defence. trends ecol. evol. 18: 27-32, 2003. schmucker d, clemens jc, shu h, worby ca, xiao j, muda m, et al. drosophila dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. cell 101: 671-684, 2000. stearns sc. evolution of life-history traits critique of theory and a review of data. annu. rev. ecol. systematics 8, 145-171: 1977. stow a, briscoe d, gillings m, holley m, smith s, leys m, et al. antimicrobial defences increase with sociality in bees. biol. lett. 3: 422-424, 2007. terwilliger dp, buckley km, mehta d, moorjani pg, smith lc. unexpected diversity displayed in cdnas expressed by the immune cells of the purple sea urchin, strongylocentrotus purpuratus. physiol. genomics 26:134-144, 2006. terwilliger dp, buckley km, brockton v, ritter nj, smith lc. distinctive expression patterns of 185/333 genes in the purple sea urchin, strongylocentrotus purpuratus: an unexpectedly diverse family of transcripts in response to lps, β-1,3-glucan and dsrna. bmc mol. biol. 8: 16, 2007. van valen l. a new evolutionary law. evol. theory 1: 1-30, 1973. verhulst pf. notice sur la loi que la population pursuit dans son accroissement. correspondance mathématique et physique. 10:113-121, 1838. watson fl, puttmann-holgado r, thomas f, lamar dl, hughes m, kondo m, et al. extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 18741878, 2005. webster jp, woolhouse mej. selection and strain specificity of compatibility between snail intermediate hosts and their parasitic schistosomes. evol. int. j. org. evol. 52: 16271634, 1998. witteveldt j, cifuentes cc, vlak jm, van hulten mcw. protection of penaeus monodon against white spot syndrome virus by oral vaccination. j. virol. 78: 2057-2064, 2004. xu t, telser e, troxler fg, oppenheim fg. primary structure and anticandidal activity of the major histatine from parotid secretion of the subhuman primate, macaca fascicularis. j. dental res. 51: 1717-1723, 1990. zhang sm, adema cm, kepler tb, loker es. diversification of ig superfamily genes in an invertebrate. science 305: 251-254, 2004. zhang sm, loker es. the frep gene family in the snail biomphalaria glabrata: additional members, and evidence consistent with alternative splicing and frep retrosequences. dev. comp. immunol. 27: 175-187, 2003. 136 transcriptome analysis of hemocytes and hepatopancreas in red swamp crayfish, procambarus clarkii, challenged with white spot syndrome virus 119 isj 7: 119-131, 2010 issn 1824-307x research report transcriptome analysis of hemocytes and hepatopancreas in red swamp crayfish, procambarus clarkii, challenged with white spot syndrome virus x-z shi, x-c li, s wang, x-f zhao, j-x wang school of life sciences, shandong university, jinan, shandong 250100, china accepted march 22, 2010 abstract red swamp crayfish procambarus clarkii is used for the innate immune defense of crustaceans due to its convenience for laboratory culture and study. to know more about the transcriptome of the crayfish, we constructed and sequenced a cdna library from a mixture of hemocytes and hepatopancreas from white spot syndrome virus (wssv)-infected crayfish. by random sequencing, we obtained 9115 high-quality expressed sequence tags with a mean length of 370 bp, representing 3033 unigenes. most of the unigenes are first reports for the red swamp crayfish. besides the metabolic genes, many genes that may be involved in the innate immune system of the crayfish are also obtained from the library, such as antimicrobial peptides, pattern recognition receptors, proteases and protease inhibitors, signal transduction proteins, apoptosis-, antioxidant-, and rna interference-related proteins. we chose ten immune-related genes to analyze their expression pattern by quantitative real time polymerase chain reaction (qrt-pcr) from the hemocytes of normal and wssv-challenged crayfish. seven of them, including anti-lipopolysaccharide factor, astacidin, crustin 1, h3 histone family 3a, serine/threonine protein kinase, tgf beta-inducible nuclear protein, and tar rna binding protein, were upregulated after wssv injection, but the mrna expression levels of crustin 2, a lectin, and a digestive cysteine protease decreased after wssv infection. our results showed that the transcriptome analysis provides a useful resource for identification of immune related genes and understanding the immune responses of the crayfish. key words: hemocytes; hepatopancreas; expression sequence tags; white spot syndrome virus; red swamp crayfish; procambarus clarkii introduction the crustacean farming industry has been suffering serious problems and enormous economic losses from an outbreak of white spot syndrome virus (wssv) since 1993 (yan et al., 2007). wssv is a serious pathogen and can infect numerous crustaceans including shrimp, crab, and lobster (shi et al., 2005; zeng et al., 2009). the cultivation of red swamp crayfish (procambarus clarkii) has become an important economic activity in china as crayfish is a delicious food. p. clarkii can be infected by white spot syndrome virus (wssv) and is easy to be used as a laboratory model for immunity analysis. the investigation of the immune defense mechanisms of red swamp crayfish could be helpful in the control of the wssv disease of cultivated crustaceans by ___________________________________________________________________________ corresponding author: jin-xing wang school of life sciences shandong university jinan, shandong 250100, china e-mail: jxwang@sdu.edu.cn enhancing the defense activity. crustaceans lack an adaptive immune system and rely totally on the innate defense system to resist pathogen invasion. the innate immune reaction comprises cellular reactions such as phagocytosis, nodule formation and encapsulation performed by hemocytes, and humoral reactions mediated by antimicrobial peptides. crayfish hemocytes play important roles in the initiation of several immune responses and production of antimicrobial peptides (jiravanichpaisal et al., 2007). the hemocytes and hepatopancreas are crucial for the immune system in crustaceans as it is the main production site for immune recognition molecules, initiates the humoral reaction, and takes part in the cellular reaction by some specialized cells and phagocytes (gross et al., 2001). many immune-related genes have been found in hemocytes of wssv-challenged crayfish (jiravanichpaisal et al., 2007; zeng et al., 2009). expressed sequence tag analysis is widely used to identify novel and differentially expressed genes 120 (leu et al., 2007; jiang et al., 2008). liu (liu et al., 2006) firstly used suppression subtractive technique on crayfish to study transcripts after wssv infection. by suppression subtractive hybridization analysis, zeng and lu (zeng et al., 2009) found thirty-three differently expressed genes in wssv-infected hemocytes of crayfish. the transcriptome information for the hepatopancreas is still limited, so we constructed and sequenced a cdna library from wssv-challenged hemocytes and hepatopancreas in red swamp crayfish. the aim of this study is to identify and annotate more immune-related genes in the two immune organs and also provide some information to uncover the mechanisms of wssv pathogenesis in crustaceans. in this study, we obtained 3033 unigenes; most of them are novel for red swamp crayfish. many genes in the library were found to be related to the innate immune system in other species, such as pattern recognition receptors, antimicrobial peptides, proteases and protease inhibitors, signal transduction proteins, apoptosis-related proteins, antioxidant proteins, rna silencing-related proteins, molecular chaperones, cuticle proteins, and calcium-binding proteins. materials and methods wssv challenge of crayfish and collection of hepatopancreas and hemocytes red swamp crayfish (procambarus clarkii) (about 10-20 g each) were obtained from a seafood market in jinan, shandong province, china. they were cultured in 500 l tanks in fresh water at room temperature (~20 c) in the laboratory. wssv was extracted from the gills of infected crayfish, and diluted in pbs (phosphate buffered saline, containing 140 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, and 1.8 mm kh2po4). using a 100 l syringe, 3.2×10 7 virus particles were injected into the abdominal segment of each crayfish. the preparation and quantification of viral inocula followed a previously described method (wang et al., 2009b). ninety-six hours after injection, the hemolymph was collected from the ventral sinus of ten crayfish and mixed with 1⁄10 volume of anticoagulant buffer (10 % sodium citrate, ph 7) containing 200 mm phenylthiourea as a melanization inhibitor. the hemolymph was then centrifuged at 800 g for 5 min (4 c) to collect the hemocytes. the hepatopancreas was also collected from the crayfish for rna extraction 96 h after wssv injection. rna extraction and cdna library construction the total rna was extracted from hemocytes and hepatopancreas of 96 h infected crayfish using unizol reagent following the manufacturer’s instructions (biostar, shanghai, china). messenger rna (mrna) was extracted with the polyatract mrna isolation system (promega, usa). the mrnas from hemocytes and hepatopancreas were mixed together and used to construct a cdna library. the creator smart cdna library construction kit (clontech, usa) was used for the cdna library construction following the manufacturer’s instructions. the double strand cdna was digested and ligated with the pdnr-lib vector, and then transformed into competent dh5 cells. individual colonies were randomly selected, and plasmids were extracted for sequencing from the 5'-ends. analysis and classification of the expression sequence tags (ests) high quality ests (more than 100 bp after removal of the vector sequence) were assembled by cap3 (http://seq.cs.iastate.edu./) in order to obtain contig sequences. unigenes (including contigs and singlets) were analyzed by blastx against the national center for biotechnology information (ncbi) protein database, blastn against the ncbi nucleotide database, and blastx against the swissprot protein database. the e-value of significant matches by the blastx and blastn is less than 0.001, and the alignment score is more than 30. the concrete functional information and metabolic profiles of unigenes were obtained using the kyoto encyclopedia for genes and genomes (kegg) (kanehisa et al., 2004). unigenes were assembled into different functional classes with clusters of orthologous groups of proteins (cogs) (http://www.ncbi.nlm.nih.gov/cog/). quantitative real time pcr (qrt-pcr) total rnas were extracted from hemocytes of ten normal and 96 h wssv-challenged crayfish using unizol reagent separately. then rnas were reverse transcribed to the first strand cdnas. the cdnas were diluted 100 fold and used as the template for qrt-pcr. ten genes including anti-lipopolysaccharide factor, astacidin, crustin 1, crustin 2, h3 histone family 3a, serine/threonine protein kinase (srk), tgf beta-inducible nuclear protein, tar rna binding protein (trbp), lectin and digestive cysteine protease from this cdna library were chosen for qrt-pcr analysis. these genes were chosen because they belonged to different groups and were firstly found in crayfish. we wanted to know whether they participate the antivirus innate immune reaction of crayfish. 18s rrna was used as the control. primer sequences used in this study are listed in table 1. qrt-pcr was performed following the manufacturer’s instruction with sybr premix ex taq (takara, japan) using a real-time thermal cycler (bio-rad, usa). the qrt-pcr was performed in 10 μl reactions containing 5 μl of 2×premix extaq, 1 μl of the 1:100 diluted cdna, 2 μl each of the forward and reverse primer (1 m). the amplification conditions were initial denaturation at 95 c for 5 min, 40 cycles of 95 c for 30 s, 60 c for 50 s, followed by a melting curve from 60 to 95 c. pcr products were detected by agarose gel analysis after each pcr reaction to confirm the specific gene amplification. data were analyzed using a comparative method (2 -∆∆ct ). qrt-pcr analysis was repeated three times for each sample. results and discussion general of the cdna library to get more information on the virus immunity-related proteins from crayfish, total rna was extracted from important immune tissues including hemocytes and hepatopancreas at 96 h http://www.ncbi.nlm.nih.gov/cog/ 121 table 1 primers used in the study after wssv challenge, and then a cdna library was constructed and randomly sequenced. as shown in table 2, a total of 10145 clones were sequenced, and among these, 9115 ests were equal to or longer than a cutoff length (100 bp) after removal of vector sequences. they are high quality ests with an average length of 370 bp. in total, 3033 unigenes were acquired including 859 contigs which have more than one est and 2174 singlets which have only one est; the singlet rate is 71.68 . the unigene hemocyanin has 213 ests, which is the largest unigene size (table 3). the novelty of the library is 33.27 . among the unigenes, only a small fraction is previously reported genes and most of them are novel genes in red swamp crayfish. we discovered many new genes from this library, and it also provides some information about the transcriptome of crayfish hemocytes and hepatopancreas. all the unigenes were aligned to the ncbi nr database using blastx, and to the ncbi nt database using blastn as a complementary analysis. moreover, the unigenes were repeatedly aligned against the blastx swissprot database. from the annotation of the alignments, we found that most of the ests shared high similarities with nucleotide sequences mainly from table 2 summary statistical analysis of the cdna library from hemocytes and hepatopancreas total ests: low and high quality ests; low quality ests: after remove empty ests and vector ests, the length of est less than a cutoff length (100bp); high quality ests: after remove empty ests and vector ests, the length of est more than a cutoff length (100bp); unigenes: contigs and singlets; contigs: the number of ests is equal to or bigger than 2; singlets: the number of ests is 1; novelty: unigenes/assembled ests; redundancy: 1-novelty gene primer sequence(5 -3 ) astacidin astacidin-f atgcgtcttctccatctcc astacidin-r ttacttgcctggacggta anti lipopolysaccharide factor alf-f ccgcctccttcaccccaca alf-r tccacctcaccgttccgcc crustin1 crustin1-f tattcctcgctgcacaaaca crustin1-r cacatagcacctccctctca crustin2 crustin2-f gggaagaaaagcacaatggt crustin2-r ggtatggaggtcgagacagg digestivecysteine protease dcysp-f aagtatgttgacgcagaggagg dcysp-r aaattggttcattgccaggttg h3 histone family 3a h3-f gtcaccatcatgcccaaggata h3-r gccagcactgcgaagtcaattc lectin lectin-f gttattgacgactccacctt lectin-r gtcttcccattgacccactt serine/threonine protein kinase spk-f tgctatgtgaagctcggctct spk-r: gcgatctgatgctcctcctct tgf beta-inducible nuclear protein tgfinp-f gcctgggtgctggtatcttgg tgfinp-r gttcgttctgtggcattgtgt tar rna binding protein trbp-f aaaatgtatcgtcaaccaccac trbp-r caccctctatctgcaacaagtc 18srna 18srna-f accgattgaatgatttagtgag 18srna-r tacggaaaccttgttacgac description number total ests 10145 low quality ests 1030 high quality ests 9115 unigenes 3033 contigs 859 singlets 2174 novelty (%) 33.27 redundancy (%) 66.73 122 fig. 1 the number of annotated unigenes with different e-values. crayfish (pacifastacus leniusculus), narrow-fingered crayfish (pontastacus leptodactylus), broad-fingered crayfish (astacus fluviatilis), and california spiny lobster (panulirus interruptus). about 68 of the unigenes have best hits and annotations against available databases, and the other 961 unigenes that have no hits may be undiscovered or unknown function genes in the database. a number of genes had identity scores of a given e value (1.00e 10). from the e-value distribution profile of all the annotated unigenes shown in fig. 1, we could see that about 90 of the annotated unigenes had an e-value 1.00e 10, so the annotation results of the blast searches in three databases are believable. the most highly expressed gene is hemocyanin 2, and its contig contains 213 ests. in arthropods, hemocyanin is a multifunctional protein which can transport oxygen in the hemolymph, shows phenoloxidase activity in its proteolytically cleaved n-terminal, and the c-terminal part sequence serves as antimicrobial peptides (lee et al., 2004). the hemocyanin of horseshoe crab could be directly activated by microbial protease and was enhanced by pathogen associated molecular patterns. the activated hemocyanin can generated highly reactive oxygen intermediates that kills microbes (jiang et al., 2007). therefore, hemocyanin plays important roles in the immune system of arthropods. here we obtained 213 ests of hemocyanin in our cdna library, and nine kinds of hemocyanin can be divided by alignment and phylogenetic analysis. this suggested that the hemocyanin might have function in antiviral immune response. megalin is also a highly expressed gene which contains 152 ests. megalin is a low-density lipoprotein receptor-related protein that is a cell surface endocytic receptor and could bind extracellular ligands for degradation (li et al., 2001). several proteases such as cathepsin l, trypsin, and zinc protease are all highly abundant expressed genes and have more than 100 ests. table 3 analysis of unigenes including contigs and singlets max unigene size：213 *unigene size: the number of ests in a unigene unigene size number of unigenes percent of unigenes(%) 1 2174 71.68 2 358 11.8 3 157 5.18 4–5 116 3.82 6–10 103 3.4 11–20 60 1.98 21–50 40 1.32 51–100 17 0.56 >100 8 0.26 123 genes potentially involved in the defense reactions hemocytes and hepatopancreas are important immune-related cells and organs in arthropods, and 33 differentially expressed genes have been discovered in hemocytes of wssv-challenged crayfish by suppression subtractive hybridization (ssh) and cdna microarrays (zeng et al., 2009). many upregulated genes in the ssh library such as serine protease inhibitor, tubulin, zinc finger protein, synaptosome-associated protein, fatty acid binding protein, superoxide dismutase precursor, arginine kinase, and heat shock protein were also found in our cdna library from hemocytes and hepatopancreas of wssv-challenged red swamp crayfish. among the newly discovered genes of our cdna library, there are many genes potentially involved in the defense reaction of crayfish (table 4). they could be assembled into seven groups such as antimicrobial peptides, pattern recognition receptors, proteases and protease inhibitors, signal transduction proteins, apoptosis-related proteins, antioxidant proteins, and others which could not be classified into groups. antimicrobial peptides in this cdna library, five kinds of antimicrobial peptides have been found, including anti-lipopolysaccharide factor (alf), astacidin, lysozymes, single wap domain-containing protein, and crustins. alfs are potential antimicrobial peptides which could bind to the lipopolysaccharide (lps) from the cell wall of gram-negative bacteria and inhibit the lps-mediated coagulation cascade in arthropod (rosa et al., 2008). alf could interfere with wssv replication in crayfish pacifastacus leniusculus and protect shrimp from wssv infection (liu et al., 2006). alf was upregulated in hemocytes 96 h after wssv challenge in qrt-pcr analysis (fig. 2a), and it may be related to the anti-wssv reaction in crayfish p. clarkii. astacidin 2 is a small proline/arginine-rich antibacterial peptide that shows strong antimicrobial activity against gram-positive and gram-negative bacteria, and has been purified from the hemolymph of freshwater crayfish p. leniusculus (jiravanichpaisal et al., 2007). it was upregulated in hemocytes 96 h after wssv challenge (fig. 2b). lysozymes are well known antimicrobial proteins that show lytic activity to a range of gram-positive and gram-negative bacteria (de-la-re-vega et al., 2006). both the single wap domain-containing protein (swd) and two crustins belong to the wap-containing proteins were also found in the crayfish. swd was upregulated in the wssv-challenged shrimp (amparyup et al., 2008; du et al., 2010). crustins are a large antimicrobial peptide family. they can be divided into three groups according to structural characters (smith et al., 2008). from an antimicrobial experiment in vitro, we could see that crustins mainly showed strong bactericidal activity against gram-positive bacteria and gram-negative bacteria so far (amparyup et al., 2008; smith et al., 2008). also in vivo experiment showed that after crustin was depleted, the mortality increased significantly after shrimp were infected by v. penaeicida (shockey et al., 2009). crustin 2 is a kind of carcinin like protein. crustin 1 and crustin 2 belong to different groups. crustin 1 was upregulated and crustin 2 was downregulated in hemocytes 96 h after challenge of wssv (figs 2c, d). pattern recognition receptors (prrs) in invertebrates, pattern recognition receptors can recognize and bind molecules present on the surface of pathogens such as -1,3-glucans, lipopolysaccharides, lipoteichoic acid, and peptidoglycans, which are called pathogen-associated molecular patterns (janeway, 1989). we discovered several kinds of pattern recognition receptors including c-type lectins, -1,3-glucan binding protein, lipopolysaccharide and -1,3-glucan binding protein, and lysm and putative peptidoglycan binding domain-containing protein. c-type lectins have been reported to participate in the innate defense reaction of nonself recognition by recognizing and binding to the molecules on the surfaces of cell walls and then inducing pathogen phagocytosis and agglutination (sun et al., 2007; ma et al., 2008). reports on chinese shrimp showed that c-type lectin served not only as a pattern recognition receptor but also an effector (sun et al., 2008). c-type lectin from the shrimp litopenaeus vannamei also has activity against wssv (zhao et al., 2009). a c-type lectin was downregulated 96 h after wssv injection, which was the same pattern as a c-type lectin, pmav, from penaeus monodon (leu et al., 2007) (fig. 2e). in crustaceans, -1,3-glucan binding protein (bgbp) and lipopolysaccharide and -1,3-glucan binding protein (lgbp) serve as pattern recognition receptors to recognize cell wall components of bacteria and fungi, such as -1,3-glucans and lipopolysaccharide. bgbp binding to -1,3-glucan induces hemocyte degranulation and subsequently activates the prophenoloxidase (propo) system (lin et al., 2008). lgbp from the crayfish p. leniusculus have been reported to participate in the activation of the propo system (lee et al., 2000). proteases and protease inhibitors the hepatopancreas was reportedly the site of synthesis of digestive enzymes in crustaceans. several protease and protease inhibitors were found in the crayfish. the proteases include astacin, zinc protease, zinc metalloproteinase, cathepsin, cysteine protease, carboxypeptidase, and trypsin. the protease inhibitors include three kinds of serine protease inhibitors such as hemocyte-specific kazal-type protease inhibitor, hepatopancreas kazal-type protease inhibitor, and the putative serine protease inhibitor serpin. astacin is a small zinc protease that can digest fibrillar collagen and other proteins (reyda et al., 1999). it was significantly upregulated by bacterial or lps challenge in oyster (roberts et al., 2009). zinc metalloproteinase is a protease family involved in growth factor activation, polypeptide degradation and extracellular protein processing (bond et al., 1995). trypsin is one of the most important proteases and contributes about 6 % of soluble protein in the 124 table 4 genes potentially included in innate immune response systems gene name species most similar to e-value no. of ests antibacterial peptide pl-crustin 1 pacifastacus leniusculus 2e-37 3 pl-crustin 2 pacifastacus leniusculus 3e-13 2 anti-lipopolysaccharide factor pacifastacus leniusculus 1e-19 3 astacidin 2 pacifastacus leniusculus 4e-20 1 lysozyme precursor crassostrea gigas 2e-06 2 single wap domain-containing protein marsupenaeus japonicus 1e-09 1 pattern recognition receptor c-type lectins fenneropenaeus chinensis 2e-06 72 -1,3-glucan binding protein penaeus monodon 1e-11 5 lipopolysaccharide and -1,3-glucan binding protein pacifastacus leniusculus 7e-56 1 proteases/protease inhibitors astacin astacus fluviatilis 5e-96 1 zinc proteases astacus astacus 4e-25 123 trypsin pacifastacus leniusculus 1e-12 124 cysteine protease homarus americanus 4e-11 60 cathepsin c marsupenaeus japonicus 7e-44 1 cathepsin d apriona germari 8e-34 1 cathepsin l nephrops norvegicus 2e-16 141 carboxypeptidase a1 scophthalmus maximus 4e-06 1 carboxypeptidase b astacus fluviatilis 7e-20 7 carboxypeptidase a5 precursor mus musculus 1e-06 8 serine carboxypeptidase precursor mus musculus 3e-10 3 semigranular hemocyte specific kazal-type protease inhibitor pacifastacus leniusculus 3e-13 1 hepatopancreas kazal-type protease inhibitor penaeus monodon 1e-07 17 zinc metalloproteinase caenorhabditis elegans 2e-07 1 putative serine protease inhibitor serpin pacifastacus leniusculus 4e-21 1 signal transduction proteins ras-related protein rab-1a lymnaea stagnalis 2e-40 3 ras-related protein rab-18 gallus gallus 2e-63 1 ras-related protein rab-5b pongo pygmaeus 2e-49 1 ras-related protein rab-7a rattus norvegicus 3e-34 1 ras-related gtp-binding protein c mus musculus 1e-10 1 ras-like gtp-binding protein rho1 isoform 1 apis mellifera 2e-25 2 125 rho family small gtp binding protein cdc42 nasonia vitripennis 6e-69 1 vacuolar atpase g subunit-like protein maconellicoccus hirsutu 2e-19 1 nucleolar gtp-binding protein 1 homo sapiens 2e-56 1 gtp-binding nuclear protein ran (gtpase ran) xenopus tropicalis 2e-45 3 ran-binding protein 16 mus musculus 7e-51 1 protein kinase n2 tribolium castaneum 2e-38 1 receptor for activated protein kinase c-like protein lepeophtheirus salmonis 9e-73 1 toll-like receptor anopheles gambiae 1e-06 2 relish litopenaeus vannamei 2e-25 1 insulin like growth factor binding protein mus musculus 2e-10 1 casein kinase i isoform alpha xenopus laevis 2e-06 1 mitogen-activated protein-binding protein-interacting protein nasonia vitripennis 5e-29 1 serine/threonine-protein phosphatase pp2a drosophila melanogaster 1e-25 1 arginine kinase homarus gammarus 3e-46 6 serine/threonine-protein kinase n2 homo sapiens 2e-38 1 apoptosis senescence-associated protein pisum sativum 1e-19 2 zinc finger protein xenopus laevis 2e-11 3 antioxidation thioredoxin-like protein maconellicoccus hirsutus 4e-33 1 thioredoxin domain containing 11 isoform 1 apis mellifera 1e-31 1 thioredoxin reductase 1 rattus norvegicus 9e-08 1 superoxide dismutase pontastacus leptodactylus 6e-18 1 glutathione peroxidase 5 mus musculus 1e-09 23 glutathione peroxidase 1 canis familiaris 4e-21 14 ferritin pacifastacus leniusculus 1e-09 11 glutathione s-transferase mu 2 mus musculus 1e-32 6 glutathione-s-transferase-like protein galleria mellonella 4e-40 1 glutathione s-transferase 1-1 (gst class-theta) tribolium castaneum 1e-36 1 glutamine synthetase panulirus argus 1e-42 1 microsomal glutathione s-transferase 3 mus musculus 1e-24 1 putative thiosulfate sulfurtransferase fmp31 saccharomyces cerevisiae 2e-13 1 omega class glutathione s-transferase crassostrea gigas 1e-27 1 farnesoic acid o-methyltransferase marsupenaeus japonicus 4e-08 6 o-methyltransferase fenneropenaeus chinensis 5e-25 1 acyl-coenzyme a oxidase 3 strongylocentrotus purpuratus 9e-07 1e-12 2 126 digestive gland. trypsins as important digestive proteases mainly take part in food digestion, hydrolysis, and activation of zymogens (muhlia-almazan et al., 2008). three kinds of cathepsins have been discovered in the crayfish, including cathepsin c, cathepsin d, and cathepsin l. cathepsin c is a multifunctional protease and is essential for the activation of other enzymes (turk et al., 2001). cathepsin d is an aspartic protease involved in several physiological functions such as protein degradation, apoptosis and autophagy (zaidi et al., 2008). cathepsin l could digest food in the gastrointestinal juice of crustaceans (hu et al., 2007). a digestive cysteine protease was downregulated 96 h after wssv injection (fig. 2f). carboxypeptidases are hydrolases that cleave in the c-terminus of proteins. carboxypeptidases a and b are digestive carboxypeptidases and serve in the degradation of proteins in the digestive tract. serine protease inhibitors of arthropods include the kazal, kunitz, -macroglobulin, and serpin families. they play important roles in prophenoloxidase and cytokine activation, blood coagulation, and pathogen digestion. three kinds of serine protease inhibitors including hemocyte-specific kazal-type protease inhibitor, hepatopancreas kazal-type protease inhibitor, and the putative serine protease inhibitor serpin have been isolated from this library. kazal-type protease inhibitors have been found in the hemocytes of shrimp, and were suggested to be involved in the host defense against wssv challenge (jarasrassamee et al., 2005). serpin from hemocytes cdna library of chinese shrimp has been shown to fluctuate after wssv and bacteria challenges (liu et al., 2009). proteins in signal transduction pathway ras-related protein rab, ran-binding protein, and ras-like gtp-binding protein rho were found in the cdna library; they all belong to the ras-related protein superfamily. the ras family of small gtpases plays roles in a series of cellular processes such as phagocytosis, vesicle transportation, and development. rab gtpase from japanese shrimp participates in the defense response to virus and might function as an intracellular virus recognition protein in virus-infected shrimp (wu et al., 2008). moreover ran from japanese shrimp is involved in antiviral defense immunity and could regulate hemocytic phagocytosis by interacting with myosin (liu et al., 2009). the studies of rho gtpase have mainly focused on phagocytosis and actin dynamics control (pan et al., 2005). toll is a receptor of the toll signal pathway responsible for antifungal and anti-gram-positive bacterial response in drosophila. toll like receptor from invertebrates could not bind pathogens directly, but it was activated by binding to spaetzle (hoffmann, 2003). it was also found in the crayfish cdna library. relish is a downstream nfb transcription factor in the imd signal pathway. the translocation of cleaved relish to the nucleus could activate the signal pathway (hoffmann, 2003). this means that the toll and imd pathways might existed in the crayfish, and the pathways might have functions in antiviral responses. insulin-like growth factor binding protein (igfbp) was also found in the crayfish. it is a member of the others lysm and putative peptidoglycan binding domain containing protein xenopus tropicalis 4e-09 1 megalin danio rerio 1e-16 152 hemocyanin 2 pacifastacus leniusculus 2e-15 213 atp-dependent rna helicase mus musculus 1e-23 6 paz and piwi domain protein/ piwi-like protein paramecium tetraurelia 5e-19 1 cuticle protein 6 blaberus craniifer 2e-11 1 crustacean calcium-binding protein 23 orconectes limosus 2e-46 1 heat shock 70 kda protein blastocladiella emersonii 4e-37 3 heat shock protein 27 drosophila melanogaster 2e-06 1 tgf beta-inducible nuclear protein brugia malayi 8e-07 1 h3 histone family 3a salmo salar 3e-29 1 tar rna binding protein fenneropenaeus chinensis 1e-09 1 chitinase fenneropenaeus chinensis 9e-14 43 127 insulin-like growth factor pathway. the insulin-like growth factor pathway takes part in growth and development, metabolic homeostasis, fecundity and stress resistance, and also lifespan in multicellular organisms (broughton et al., 2009). serine/threonine-protein kinases and phosphatases play roles in signal transduction of anoxia in the crayfish orconectes virilis (cowan et al., 2001). serine/threonine-protein kinase n2 and serine/threonine-protein phosphatase pp2a were isolated from our cdna library. casein kinase1 is a member of the serine/threonine protein kinase family, and it has seven isoforms in mammals and vertebrates. casein kinase1 could enhance receptor interacting protein-mediated nfb activation (wang et al., 2008). the mrna expression level of serine/threonine-protein kinase n2 from this cdna library increased 96 h after wssv injection (fig. 2g), and it may be involved in the antiviral innate immune reaction of crayfish. the transforming growth factor (tgf) signal pathway plays important roles in diverse biological processes. in caenorhabditis elegans, the tgf pathway participates in immune responses (schulenburg et al., 2004). tgf beta-inducible nuclear protein could be induced by stimulation of tgf, so it may be involved in the defense reaction. tgf beta-inducible nuclear protein was upregulated in hemocytes of crayfish after wssv challenge, so it may participate in the anti-virus immune response (fig. 2h). apoptosis-related proteins a senescence-associated protein was also found from this cdna library. the senescence-associated protein takes part in the early embryonic development of silkworm bombyx mori (hong et al., 2006). several zinc finger proteins were also found in the library, just as in the ssh library of hemocytes from wssv-challenged crayfish. one zinc finger protein could bind to viral mrna and prevent its accumulation in the cytoplasm (garcia et al., 2007). thus the zinc finger proteins of our cdna library may be involved in the anti-virus immune defense reaction of crayfish. antioxidant proteins reactive oxygen species, the products of normal aerobic metabolism, can cause oxidative damage to organisms. antioxidant proteins are needed to eliminate the reactive oxygen species and regulate the redox homeostasis. to protect against damage and regulate redox homeostasis, molecules in the glutaredoxin and thioredoxin systems are employed. glutaredoxins and thioredoxins are conserved proteins involved in many cellular processes including repair of oxidatively damaged proteins and protein refolding and regulation (grant, 2001). the thioredoxin system contains thioredoxin and thioredoxin reductase, and thioredoxin is catalyzed directly by thioredoxin reductase and electrons from nicotinamide adenine dinucleotide (nadh) (aispuro-hernandez et al., 2008). by the random sequencing of the cdna library, thioredoxin-like proteins and thioredoxin reductase were isolated from the wssv-challenged crayfish. thioredoxin was characterized in pacific white shrimp (l. vannamei) and the antioxidant activity of the recombinant protein was tested by reducing insulin disulfides using the trolox equivalent antioxidant capacity assay. the results showed that thioredoxin is an important antioxidant (aispuro-hernandez et al., 2008). glutathione peroxidase is a component of the glutaredoxin system. glutathione s-transferases belong to a multigene family that plays important roles in detoxification of xenobiotic compounds (rosa de lima et al., 2002). a selenium-dependent glutathione peroxidase and two glutathione s-transferases have been cloned from chinese shrimp and were involved in detoxification defense reactions (ren et al., 2009). a glutamine synthetase was also found in this library. superoxide dismutase is a kind of antioxidant enzyme. it was also found in the ssh library of wssv-challenged crayfish and upregulated after virus injection (zeng et al., 2009). ferritin is an iron storage protein that participates in iron metabolism and detoxification. ferritins from shrimp have been reported to be involved in the anti-virus defense reaction (zhang et al., 2006). others lysm and putative peptidoglycan binding domain-containing protein (pbp) has not been found in crustaceans so far, so this pbp may be the first one. it may also be involved in the innate immune reaction in crayfish. we found paz and piwi domain protein and atp-dependent rna helicase, which are involved in rna-mediated silencing and the defense response against viruses (cerutti et al., 2006). in humans, the trans-activation response (tar) rna-binding protein plays a role in passing small interfering rna to the rna-induced silencing complex and functions in rna interference(wang et al., 2009a). a tar rna binding protein was found in the cdna library; it was upregulated after wssv challenge in the hemocytes of crayfish (fig. 2i), so it may take part in the antiviral immune reaction. chitinases are members of the glycoside hydrolase family. it has been reported that in the mollusc crassostrea gigas, two chitinases are stimulated in response to lps challenge, which suggested that they are involved in the immune defense reaction of oyster (badariotti et al., 2007). chitinase was upregulated in hepatopancreas of wssv-resistant shrimp (pan et al., 2005). cuticle protein and crustacean calcium-binding protein were also found by an est approach in the wssv-infected shrimp p. mondon. calcium-binding proteins act as cytosolic ca 2 buffers and were significantly downregulated in wssv-injected shrimp (leu et al., 2007). a calcium-binding peptide from the exoskeleton of crayfish functions as a regulator of exoskeleton calcification (inoue et al., 2004). cuticle proteins were upregulated as a result of wssv infection, as the wssv mainly infects the cuticular epidermis in shrimp (leu et al., 2007). heat shock proteins are members of the chaperone family and take part in protein folding. in shrimp, heat shock protein 70 expression was influenced by wssv infection, and the association 128 fig. 2 qrt-pcr analysis of ten genes in total rna extracted from red swamp crayfish hemocytes 96 h after wssv challenge. the qrt-pcr data of the expression level of these genes in response to bacterial and viral challenge were calculated by 2 -∆∆ct . bars stand for the mean±sd. of three independent pcr amplifications and quantifications. the data obtained were subjected to the statistical analysis followed by an unpaired sample t-test. asterisks indicate significant differences (*p<0.05) when comparing to that in the healthy hemocytes (0 h). astacidin (genbank accession no. gq301199); crustin 1 (genbank accession no. gq301201); crustin 2 (genbank accession no. gq301202); anti-lipopolysaccharide factor (alf) hm005306; digestive cysteine proteinase hm005305; h3 histone family protein hm005304;serine/threonine protein kinase(ser) hm005303; tgf beta-inducible nuclear protein hm005302; tar rna binding protein (trbp)hm005301; lectin hm005300. 129 between the major envelope protein vp28 of wssv and heat shock protein 70 is direct and atp-dependent (xu et al., 2009). heat shock protein 70 was upregulated after wssv infection and involved in the anti-virus response in crayfish (zeng et al., 2009). another heat shock protein of this cdna library showed similarity to heat shock protein 27 from drosophila melanogaster, and it had anti-apoptotic activity by inhibition of cytochrome c and tnf-mediated cell death (arya et al., 2007). histones are basic protein components of chromatin. as early as 1942, some reports showed that histone possessed antibacterial properties (miller et al., 1942). recent work demonstrate that the histones and histone-derived fragments have antimicrobial activities in diverse range of organisms from shrimp to human (kawasaki and iwamuro, 2008). the h3 family contains two replacement histone genes, h3 histone family 3a and 3b. here we found a h3 histone family 3a. it was upregulated after wssv infection (fig. 2j). it was reported that histone h2b could mediate anti-virus immune defense reactions, so the upregulation of h3 histone 3a may also be involved in the antiviral defense reaction of crayfish (kobiyama et al., 2010). acknowledgements the authors thank all other laboratory members for some data analysis and helpful discussions. this work was supported by grants from the national high technology research and development program of china (863 program) (no. 2007aa09z425, 2006cb101806), the national natural science foundation of china (no. 30770282). references amparyup p, donpudsa s, tassanakajon a. shrimp single wap domain (swd)-containing protein exhibits proteinase inhibitory and antimicrobial activities. dev. comp. immunol. 32: 1497-509, 2008. amparyup p, kondo h, hirono i, aoki t, tassanakajon a. molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp penaeus monodon. mol. immunol. 45: 1085-1093, 2008. aispuro-hernandez e, garcia-orozco kd, muhlia-almazan a, del-toro-sanchez l, robles-sanchez rm, hernandez j, et al. shrimp thioredoxin is a potent antioxidant protein. comp. biochem. physiol. 148c: 94-99, 2008. arya r, mallik m, lakhotia sc. heat shock genes integrating cell survival and death. j. biosci. 32: 595-610, 2007. badariotti f, lelong c, dubos mp, favrel p. characterization of chitinase-like proteins (cg-clp1 and cg-clp2) involved in immune defence of the mollusc crassostrea gigas. febs j. 274: 3646-3654, 2007. bond js, beynon rj. the astacin family of metalloendopeptidases. protein sci. 4: 1247-1261, 1995. broughton s, partridge l. insulin/igf-like signalling, the central nervous system and aging. biochem. j. 418: 1-12, 2009. burge ej, madigan dj, burnett le, burnett kg. lysozyme gene expression by hemocytes of pacific white shrimp, litopenaeus vannamei, after injection with vibrio. fish shellfish immunol. 22: 327-339, 2007. cerutti h, casas-mollano ja. on the origin and functions of rna-mediated silencing: from protists to man. curr. genet. 50: 81-99, 2006. cowan kj, storey kb. protein kinase and phosphatase responses to anoxia in crayfish, orconectes virilis: purification and characterization of camp-dependent protein kinase. comp. biochem. physiol. 130b: 565-577, 2001. de-la-re-vega e, garcia-galaz a, diaz-cinco me, sotelo-mundo rr. white shrimp (litopenaeus vannamei) recombinant lysozyme has antibacterial activity against gram negative bacteria: vibrio alginolyticus, vibrio parahemolyticus and vibrio cholerae. fish shellfish immunol. 20: 405-408, 2006. du zq, li xc, wang zh, zhao xf, wang jx. a single wap domain (swd)-containing protein with antipathogenic relevance in red swamp crayfish, procambarus clarkii. fish shellfish immunol. 28: 134-142, 2010. garcia cc, damonte eb. zn finger containing proteins as targets for the control of viral infections. infect. disord. drug targets 7: 204-212, 2007. gross ps, bartlett tc, browdy cl, chapman rw, warr gw. immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the pacific white shrimp, litopenaeus vannamei, and the atlantic white shrimp, l. setiferus. dev. comp. immunol. 25: 565-577, 2001. hoffmann ja. the immune response of drosophila. nature 426: 33-38, 2003. hong sm, nho sk, kim ns, lee js, kang sw. gene expression profiling in the silkworm, bombyx mori, during early embryonic development. zoolog. sci. 23: 517-528, 2006. hu kj, leung pc. food digestion by cathepsin l and digestion-related rapid cell differentiation in shrimp hepatopancreas. comp. biochem. physiol. 146b: 69-80, 2007. inoue h, ohira t, ozaki n, nagasawa h. a novel calcium-binding peptide from the cuticle of the crayfish, procambarus clarkii. biochem. biophys. res. commun. 318: 649-654, 2004. janeway ca, jr. approaching the asymptote? evolution and revolution in immunology. cold spring harb. symp. quant. biol. 54 pt 1: 1-13, 1989. jarasrassamee b, supungul p, panyim s, klinbunga s, rimphanichayakit v, tassanakajon a. recombinant expression and characterization of five-domain kazal-type serine proteinase inhibitor of black tiger shrimp (penaeus monodon). mar. biotechnol. (ny) 7: 46-52, 2005. jiang n, tian ns, ho b, ding jl. respiratory protein-generated reactive oxygen species as an antimicrobial strategy. nat. immunol. 8: 1114-1122, 2007. http://www.ncbi.nlm.nih.gov/pubmed?term=%22amparyup%20p%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22donpudsa%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22tassanakajon%20a%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); 130 jiravanichpaisal p, lee sy, kim ya, andren t, soderhall i. antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish pacifastacus leniusculus: characterization and expression pattern. dev. comp. immunol. 31: 441-455, 2007. kanehisa m, goto s, kawashima s, okuno y, hattori m. the kegg resource for deciphering the genome. nucleic acids res. 32: d277-280, 2004. kobiyama k, takeshita f, jounai n, sakaue-sawano a, miyawaki a, ishii kj, et al. extrachromosomal histone h2b mediates innate antiviral immune responses induced by intracellular double-stranded dna. j. virol. 84: 822-832, 2010. lee sy, lee bl, soderhall k. processing of crayfish hemocyanin subunits into phenoloxidase. biochem. biophys. res. commun. 322: 490-496, 2004. lee sy, wang r, soderhall k. a lipopolysaccharide and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish pacifastacus leniusculus. purification, characterization, and cdna cloning. j. biol. chem. 275: 1337-1343, 2000. leu jh, chang cc, wu jl, hsu cw, hirono i, aoki t, et al. comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected penaeus monodon. bmc genomics 8: 120, 2007. li y, cam j, bu g. low-density lipoprotein receptor family: endocytosis and signal transduction. mol. neurobiol. 23: 53-67, 2001. lin yc, vaseeharan b, chen jc. identification and phylogenetic analysis on lipopolysaccharide and beta-1,3-glucan binding protein (lgbp) of kuruma shrimp marsupenaeus japonicus. dev. comp. immunol. 32: 1260-1269, 2008. liu h, jiravanichpaisal p, soderhall i, cerenius l, soderhall k. antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish pacifastacus leniusculus. j. virol. 80: 10365-10371, 2006. liu w, han f, zhang x. ran gtpase regulates hemocytic phagocytosis of shrimp by interaction with myosin. j. proteome res. 8: 1198-1206, 2009. liu y, li f, wang b, dong b, zhang x, xiang j. a serpin from chinese shrimp fenneropenaeus chinensis is responsive to bacteria and wssv challenge. fish shellfish immunol. 26: 345-351, 2009. ma th, benzie ja, he jg, chan sm. pmlt, a c-type lectin specific to hepatopancreas is involved in the innate defense of the shrimp penaeus monodon. j. invertebr. pathol. 99: 332-341, 2008. miller b, abrams r, dorfman a, klein m. antibacterial properties of protamines and histone. science 96: 428-430,1942. muhlia-almazan a, sanchez-paz a, garcia-carreno fl. invertebrate trypsins: a review. j. comp. physiol. [b] 178: 655-672, 2008. pan d, he n, yang z, liu h, xu x. differential gene expression profile in hepatopancreas of wssv-resistant shrimp (penaeus japonicus) by suppression subtractive hybridization. dev. comp. immunol. 29: 103-112, 2005. ren q, sun rr, zhao xf, wang jx. a selenium-dependent glutathione peroxidase (se-gpx) and two glutathione s-transferases (gsts) from chinese shrimp (fenneropenaeus chinensis). comp. biochem. physiol. 149c: 613-623, 2009. reyda s, jacob e, zwilling r, stocker w. cdna cloning, bacterial expression, in vitro renaturation and affinity purification of the zinc endopeptidase astacin. biochem. j. 344 pt 3: 851-857, 1999. roberts s, goetz g, white s, goetz f. analysis of genes isolated from plated hemocytes of the pacific oyster, crassostreas gigas. mar. biotechnol. (ny) 11: 24-44, 2009. rosa de lima mf, sanchez ferreira ca, joaquim de freitas dr, valenzuela jg, masuda a. cloning and partial characterization of a boophilus microplus (acari: ixodidae) glutathione s-transferase. insect biochem. mol. biol. 32: 747-754, 2002. rosa rd, stoco ph, barracco ma. cloning and characterisation of cdna sequences encoding for anti-lipopolysaccharide factors (alfs) in brazilian palaemonid and penaeid shrimps. fish shellfish immunol. 25: 693-696, 2008. schulenburg h, kurz cl, ewbank jj. evolution of the innate immune system: the worm perspective. immunol. rev. 198: 36-58, 2004. shi z, wang h, zhang j, xie y, li l, chen x, et al. response of crayfish, procambarus clarkii, haemocytes infected by white spot syndrome virus. j. fish dis. 28: 151-156, 2005. shockey je, o'leary na, de la vega e, browdy cl, baatz je, gross ps. the role of crustins in litopenaeus vannamei in response to infection with shrimp pathogens: an in vivo approach. dev. comp. immunol. 33: 668-673, 2009. smith vj, fernandes jm, kemp gd, hauton c. crustins: enigmatic wap domain-containing antibacterial proteins from crustaceans. dev. comp. immunol. 32: 758-772, 2008. sun j, wang l, wang b, guo z, liu m, jiang k, et al. purification and characterisation of a natural lectin from the serum of the shrimp litopenaeus vannamei. fish shellfish immunol. 23: 292-299, 2007. sun yd, fu ld, jia yp, du xj, wang q, wang yh, et al. a hepatopancreas-specific c-type lectin from the chinese shrimp fenneropenaeus chinensis exhibits antimicrobial activity. mol. immunol. 45: 348-361, 2008. turk d, janjic v, stern i, podobnik m, lamba d, dahl sw, et al. structure of human dipeptidyl peptidase i (cathepsin c): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases. embo j. 20: 6570-6582, 2001. wang s, liu n, chen aj, zhao xf, wang jx. trbp homolog interacts with eukaryotic initiation factor 6 (eif6) in fenneropenaeus chinensis. j. immunol. 182: 5250-5258, 2009a. wang s, zhao xf, wang jx. molecular cloning and characterization of the translationally controlled 131 tumor protein from fenneropenaeus chinensis. mol. biol. rep. 36: 1683-1693, 2009b. wang y, sun x, wu j, xu be, gu c, wang h, et al. casein kinase 1alpha interacts with rip1 and regulates nf-kappab activation. biochemistry 47: 441-448, 2008. wu w, zong r, xu j, zhang x. antiviral phagocytosis is regulated by a novel rab-dependent complex in shrimp penaeus japonicus. j. proteome res. 7: 424-431, 2008. xu h, yan f, deng x, zou t, ma x, zhang x, et al. the interaction of white spot syndrome virus envelope protein vp28 with shrimp hsc70 is specific and atp-dependent. fish shellfish immunol. 26: 414-421, 2009. yan dc, dong sl, huang j, zhang js. white spot syndrome virus (wssv) transmission from rotifer inoculum to crayfish. j. invertebr. pathol. 94: 144-148, 2007. zaidi n, maurer a, nieke s, kalbacher h. cathepsin d: a cellular roadmap. biochem. biophys. res. commun. 376: 5-9, 2008. zeng y, lu cp. identification of differentially expressed genes in haemocytes of the crayfish (procambarus clarkii) infected with white spot syndrome virus by suppression subtractive hybridization and cdna microarrays. fish shellfish immunol. 26: 646-650, 2009. zhang j, li f, wang z, zhang x, zhou q, xiang j. cloning, expression and identification of ferritin from chinese shrimp, fenneropenaeus chinensis. j. biotechnol. 125: 173-184, 2006. zhao zy, yin zx, xu xp, weng sp, rao xy, dai zx, et al. a novel c-type lectin from the shrimp litopenaeus vannamei possesses anti-white spot syndrome virus activity. j. virol. 83: 347-356, 2009. conservation of cytokine-mediated responses in innate immunity: a flow cytometric study investigating the effects of human proinflammatory cytokines on phagocytosis in the earthworm eisenia hortensis isj 5: 124-134, 2008 issn 1824-307x research report conservation of cytokine-mediated responses in innate immunity: a flow cytometric study investigating the effects of human proinflammatory cytokines on phagocytosis in the earthworm eisenia hortensis sl fuller-espie1, l goodfield1, k hill1, k grant2, n derogatis3 1 science department, cabrini college, radnor, pennsylvania, usa 2 college of graduate studies, thomas jefferson university, philadelphia, pennsylvania, usa 3 the children’s hospital of philadelphia, department of pathology and laboratory medicine, philadelphia, pennsylvania, usa accepted september 12, 2008 abstract this study was aimed at determining the influence of human proinflammatory cytokines on innate immune responses in the earthworm eisenia hortensis. preincubation of earthworm coelomocytes in vitro with either interleukin-1 beta (il-1 beta), granulocyte-macrophage colony stimulating factor (gmcsf), interleukin-2 (il-2), or tumor necrosis factor-alpha (tnf-alpha) followed by subsequent bacterial challenge was carried out to investigate whether human proinflammatory cytokines would induce a state of enhanced responsiveness in phagocytic cells derived from the coelomic cavity of e. hortensis. the effect on phagocytosis by large coelomocytes (hyaline amebocytes) was evaluated using flow cytometry where the uptake of escherichia coli expressing green fluorescence protein in the presence or absence of pretreatment with proinflammatory cytokines was measured. our results show that proinflammatory cytokines enhanced phagocytosis to a statistically significant (p ≤ 0.05) degree in 1018 % of earthworms tested for il-1 beta, 20 % for gm-csf, 20-27 % for il-2, and 27-30 % for tnfalpha, depending on the cytokine concentration used. our results favor the suggestion that receptorcoding genes have been conserved through evolution between vertebrates and invertebrates. key words: eisenia hortensis; proinflammatory cytokines; phagocytosis; innate immunity; flow cytometry; hyaline amebocytes introduction invertebrates possessing a coelomic cavity utilize innate immune responses consisting of highly effective cellular and humoral components (roch, 1996; cooper et al., 2002). for example, the coelomic fluid in earthworms consists of lytic and antimicrobial components such as eiseniapore (lange et al., 1997; lange et al., 1999), the cytokine-like cytolytic protein known as coelomic cytolytic factor 1 (ccf-1), and the highly homologous proteins fetidin and lysenin (bruhn et al., 2006; procházková et al., 2006). ccf-1 is functionally analogous with the mammalian cytokine tumor necrosis factor (bilej et al., 1995). when earthworms ___________________________________________________________________________ corresponding author: sheryl l fuller-espie science department cabrini college, 610 king of prussia road radnor, pennsylvania 19087-3698, usa email: sfuller-espie@cabrini.edu were injected intracoelomically with the endotoxin lipopolysaccharide (lps) both humoral and cellular levels of ccf-1 expression increased significantly (bilej et al., 1998), an effect also observed with tnf when mammals were injected with lps (carswell et al., 1975). despite functional analogy between ccf1 and tnf, it is interesting to note the absence of gene homology between the two (beschin et al., 2004). it has been suggested that ccf-1 is representative of a primitive inflammatory cytokinelike factor which participates in humoral and cellular inflammatory responses including the activation of prophenoloxidase activity (bilej et al., 1998; beschin et al., 1999). cytokine-like factors have also been reported in other invertebrates. prendergast et al. (1983), working with the starfish, asterias forbesi, isolated sea star factor (ssf) and demonstrated its ability to function across species by stimulating chemotaxis and macrophage activation in mammals. 124 mailto:sfuller-espie@cabrini.edu 0 10 20 30 40 50 60 fsc-h 0 10 20 30 40 50 60 s s c -h r1 r2 r3 a. 0 10 20 30 40 50 60 fsc-h 0 10 20 30 40 50 60 s s c -h r1 r2 r3 b. fig. 1 typical scatter profile of earthworm coelomocytes using flow cytometry. a two parameter dot plot measuring forward scatter (fsc) versus side scatter (ssc) of a typical earthworm coelomocyte population after extrusion and incubation overnight in sdmem medium is shown. this data shows the results obtained from ew14. three distinct populations are indicated; region 1 (r1) represents the eleocytes, region 2 (r2) represents the large coelomocytes (lc), and region 3 (r3) represents the small coelomocytes (sc). dot plots are shown without (a) and with (b) contour marks to emphasize densities of subpopulations. the fact that earthworms possess cytokine-like factors such as ccf-1 has prompted much attention on the biochemical activities of these cytokine-like factors, their evolution and conservation, and crossspecies activity using mammalian-derived cytokines. the studies of renzelli-cain et al. (1995) demostrated that mammalian interleukin-1 alpha (il-1 alpha) enhanced phagocytic activity of opioid-treated amebocytes of the earthworm lumbricus terrestris, and also influenced their aggregation and conformation. interleukin-8 (il-8) has been shown to mediate alterations in cell shape and increase chemotaxis and phagocytic activity in the mussel mytilus galloprovincialis (ottaviani et al., 2000). in the freshwater snails planorbarius corneus and viviparus ater, preincubation with il-1 and tnf-alpha caused a decrease in the release of biogenic amines when subsequently exposed to corticotrophin-releasing factor, a proto-type stress response inducer (ottaviani et al., 1995). in addition, the natural killer (nk)-like activity of p. corneus hemocytes was preserved in vitro when incubated with interleukin-2 (il-2) (franceschi et al., 1991). franchini et al. (2000; 2006) have also demonstrated that the giant leopard slug limax maximus had accelerated tissue repair when platelet-derived growth factor-ab (pdgf-ab) and transforming growth factor-beta (tgf-beta) were applied to wounds. investigation of the cellular immune responses of earthworms has resulted in the categorization of coelomocytes (leukocytes) which reside in the coelomic cavity into three major subpopulations, hyaline amebocytes (large coelomocytes), granular amebocytes (small coelomocytes), and chloragocytes (eleocytes). the coelomocytes are easily harvested from experimentally-induced earthworms following extrusion of coelomic fluid from the dorsal pores of the body wall, and can be manipulated in vitro to study their immune functions. the large coelomocytes (lc) are the major phagocytic cells, the small coelomocytes (sc) constitute the population exhibiting nk-like activity, and the eleocytes contain chloragosomes and do not participate in either phagocytic or nk-like activities (cooper, 1996; cossarizza et al., 1996; adamowicz et al., 2001; engelmann et al., 2002; engelmann et al., 2005). owing to the physical differences between coelomocyes including their granularity and size, flow cytometry permits lc, sc and eleocytes to be distinguished based on forward light scatter (fsc) and side light scatter (ssc) properties (cossarizza et al., 1996; engelmann et al., 2004; cossarizza et al., 2005; patel et al., 2007). in addition, flow cytometry can be used to selectively analyze immune functions of the subpopulations by drawing regions around and gating only those cells desired for investigative purposes. although light microscopy has been used by researchers to study phagocytosis in earthworms (adamowicz et al., 2001; kalaç et al., 2002), this approach lends itself to subjectivity, and places restrictions on the number of cells that can be included in quantitative assays based on time constraints. flow cytometry, in contrast, is an objective quantitative methodology which is capable of analyzing several thousands of cells per second, and limiting analysis to predetermined subpopulations. the selected examples cited above illustrate that mammalian cytokines can trigger immune responses in invertebrate models. we sought to investigate the effects of four proinflammatory cytokines, namely il-1 beta, gm-csf, il-2 and tnf-alpha, on the phagocytic activity of 125 coelomocytes isolated from the earthworm eisenia hortensis. our study used flow cytometry to follow the uptake of escherichia coli expressing green fluorescent protein after gating on coelomocytes that satisfied the fsc and ssc parameters characteristic of lc. our results show that preincubation of lc with the proinflammatory cytokines il-1 beta, gm-csf, il-2 and tnf-alpha induced a stimulatory effect on in vitro phagocytosis in 10-30 % of the earthworms tested in this study. materials and methods reagents general laboratory reagents and plasticware were purchased from fisher scientific unless otherwise noted. cell culture all cell culture reagents and phosphate-buffered saline (pbs) were purchased from invitrogen unless otherwise noted. dulbecco’s modified eagle medium (dmem) was supplemented with 10 % fetal calf serum, 100 μg/ml ampicillin (shelton scientific), 10 μg/ml kanamycin (shelton scientific), 10 μg/ml tetracycline, 5 μg/ml chloramphenicol (fluka biochemika), 1x penicillin/streptomycin/amphotericin b, 1x nonessential amino acids and 1x l-glutamine (gibco) to comprise super dmem (sdmem). earthworm husbandry eisenia hortensis (european nightcrawlers) were purchased from vermitechnology unlimited, orange lake, florida, usa, who import e. hortensis from star food, holland, scherpenzeelseweg 95, 3772me barneveld, the netherlands. species identity was determined by the united states department of agriculture, usda permit #52262 (vermitechnology, personal communication). shortterm colonies were maintained at room temperature (rt) in the dark on autoclaved shredded paper moistened with water and single grain rice cereal or rice with bananas cereal (gerber) until use. shredded paper was changed twice weekly. extrusion of coelomocytes prior to experimentation, earthworms were chosen based on their color and activity; earthworms with healthy deep coloration, lacking yellow appearance, and with high activity were placed overnight on moist paper towels saturated with 2.5 μg/ml fungizone (fisher scientific) to reduce the level of fecal material and other surface contaminants. to collect coelomocytes from an earthworm, earthworms were extruded according to engelmann et al. (2004) with minor modifications. briefly, earthworms were placed in a 100 mm petri dish containing 3 ml ice cold extrusion buffer (71.2 mm nacl, 5 % v/v ethanol, 50.4 mm guaiacol-glycerylether, 5 mm egta, ph 7.3). the coelomocytes were then transferred to 1 ml accumax (innovative cell technology) for a 5 min incubation period at rt. then the cells were washed with 5 ml lubricus balanced salt solution (lbss, 71.5 mm nacl, 0.3 mm nah2po4, 4.2 mm nahco3,4.8 mm kcl, 0.4 mm kh2po4, 1.1 mm mgso4 x 7 h2o, ph 7.3), prior to centrifugation at 150xg for 5 min at 4 oc. coelomocytes were resuspended in 1 ml sdmem and enumerated using a hemocytometer. only large coelomocytes (lc) and small coelomocytes (sc) were included in the cell count; eleocytes were not counted but did factor into a quality score. samples with large numbers of eleocytes compared to lc and sc were not used in phagocytosis assays. cytokines all cytokines were reconstituted and stored according to vendors’ recommendations. recombinant human il-1 beta was purchased from r & d systems (201-lb). recombinant human gm-csf, recombinant human il-2, and recombinant human tnf-alpha were purchased from prospec-tany technogene (cyt-221a, cyt-209 and cyt-223, respectively). in all cases purity was greater than 97 % and endotoxin levels were less than 1.0 eu/μg. il-1 beta was used at 20 ng/ml and 40 ng/ml; gmcsf was used at 2 ng/ml and 4 ng/ml; il-2 was used at 12.5 ng/ml and 25 ng/ml; and tnf-alpha was used at 2.5 ng/ml and 5 ng/ml. bacteria escherichia coli hb101 transformed with pglo (biorad) and expressing green fluorescent protein (gfp) were grown on tryptic soy agar containing 100 μg/ml ampicillin and 0.2 % arabinose at 32 °c for 24 h. cells were chemically fixed using 4 % paraformaldehyde for 1 h at rt, and then washed three times with pbs. centrifugation was carried out at 3273xg for 5 min at 4 oc, cells were resuspended in pbs, enumerated using a hemocytometer, and stored in the dark at 4 oc until used. these cells hereafter are referred to as e. coli-gfp. phagocytosis assay phagocytosis assays were carried out in sdmem. coelomocytes [20,000 per well for earthworm (ew) 1 and ew2; 50,000 per well for all others] were pretreated with individual cytokines at concentrations indicated above for 20 h, 5 % co2, at 20 oc in 96-well, v-bottom plates in 100 μl. depending on coelomocyte yield and scope of each experiment, the number of replicates differed between assays affecting the degrees of freedom in the statistical analyses. experiments were set up in duplicates for ew5 and ew15, in triplicates for ew1, ew4, ew6, ew8-14, and ew16, and quadruplicates for ew2, ew3, and ew5. the co2 incubator was placed in a 4 oc walkin cold room in order to obtain these conditions. following cytokine pretreatment, e. coli-gfp was added to each well at a multiplicity of infection of 1000 bacteria: 1 coelomocyte in 200 μl final volume per well. to control for non-specific binding of e. coli to the external surface of coelomocytes, 5 μm cytochalasin b was added to control wells 30 min before the addition of e.coli-gfp. incubation times for e. coli-gfp uptake ranged from 1-4 h at 30 oc. following e. coli-gfp uptake, trypan blue was used at a final concentration of 0.02 % for 30 min at room temperature in the dark, for quenching purposes to reduce background fluorescence (mosiman et al.,1997). the cells were centrifuged at 150xg for 5 min at 4 oc, washed once with pbs, and then resuspended in facs flow buffer (bd bioscience) for flow cytometry analysis. 126 cytokine (concentration) number of earthworms treated with cytokine number of responding earthworms (p ≤ 0.05) (ew identity number) statistically significant response rate to cytokine (p ≤ 0.05) il-1 beta (40ng/ml) 28 5 (ew 4,5,8,12,15) 18 % il-1 beta (20ng/ml) 20 2 (ew 3,5) 10 % gm-csf (4ng/ml) 10 2 (ew 4,5) 20 % gm-csf (2ng/ml) 50 10 (ew 1,2,5,6,8,9,10,11,14,16) 20 % il-2 (25ng/ml) 22 6 (ew 4,5,9,11,13,16) 27 % il-2 (12.5ng/ml) 10 2 (ew 4,5) 20 % tnf-alpha (5ng/ml) 22 6 (ew 4,5,9,11,13,14) 27 % tnf-alpha (2.5ng/ml) 10 3 (ew 5,6,7) 30 % table 1 summary of total number of earthworms used in this study and percent responders. the four cytokines and the concentrations used are indicated. the total number of earthworms pretreated with the cytokines, the number responding with p ≤ 0.05 to each cytokine, the identity of the earthworm responders, and the percentage of statistically significant responses are shown. flow cytometry fluorescence was measured using a facscalibur flow cytometer and cell quest software (bd biosciences) with the following specifications for all experiments described in this study: forward light scatter (fsc) e00-linear, side light scatter (ssc) 332v-linear, green fluorescent protein fluorescence (fl-1) 312v-log. listmode data was acquired with cell quest software, and analysis of data was carried out using winlist 5.0 (verity software house, inc.). only coelomocytes with the appropriate granularity and size corresponding to the large coelomocyte population were gated for analysis in two-dimensional dot plots of fsc vs fl-1. percent specific phagocytosis was determined by subtracting the autofluorescent background of the controls (coelomocytes incubated with cytochalasin b and e. coli-gfp) from the sample fluorescence measured by the fl-1detector. all histograms and dot plots were created using winlist 5.0. see figs 1-2 for representation of data acquisition and analysis. statistical analysis all graphs and data analysis were created and processed using microsoft excel 2007. only statistically relevant results with p ≤ 0.05 (as defined by student’s t-test) comparing untreated versus cytokine-treated samples are shown in figs 3-6. results enhanced phagocytosis was detected when coelomocytes were preincubated with the proinflammatory cytokines il-1 beta, gm-csf, il-2 and tnf-alpha fig. 1 illustrates a typical profile of earthworm coelomocytes obtained when measuring forward light 127 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 a. lr 96.57;ur3.43 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 b. lr 92.77;ur 7.23 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 c. lr 55.29;ur 44.71 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 d. lr 37.72;ur 62.28 fig. 2 characteristic profile of large coelomocytes (lc) before and after phagocytosis and proinflammatory cytokine treatment. two dimensional dot plots measuring forward scatter (fsc) on the x-axis versus green fluorescence (fl-1) on the y-axis were generated after gating on the r2 subpopulation (lc) as indicated in fig. 1. this data shows the results obtained for ew11. lr = % gated lc in lower right quadrant; ur = % gated lc in upper right quadrant. a. lc illustrating the level of autofluorescent background. b. lc in the presence of e. coligfp plus cytochalasin b, an antibiotic which interferes with microfilament activity and inhibits phagocytosis. c. lc incubated with e. coli-gfp. d. lc preincubated with il-2 (25 ng/ml) prior to the addition of e. coli-gfp (p = 0.016). e. coli-gfp control was not detected using the fsc, ssc and fl-1 settings employed (data not shown). scatter (fsc) versus side light scatter (ssc) properties. coelomocytes from ew14 are depicted. three characteristic populations were routinely observed and three regions, r1, r2, and r3, were drawn around the subpopulations corresponding to the eleocytes, large coelomocytes (lc) and small coelomocytes (sc), respectively. a dot plot represents the total population acquired (fig. 1a) while a contour plot shows the relative densities of each of the three subpopulations (fig. 1b). percent specific phagocytosis was determined for each earthworm treated with il-1 beta, gm-csf, il-2 and tnf-alpha. fig. 2 represents a typical profile of responding earthworm coelomocytes measuring forward scatter (fsc) versus gfp fluorescence (fl-1) before and after treatment with il-2 (25 ng/ml) (p = 0.016). coelomocytes from ew11 are depicted. fsc versus fl-1 dot plots were gated on the r2 subpopulation (lc) as illustrated in fig. 1. each dot plot was divided into four quadrants, upper left (ul) (4), upper right (ur) (5), lower left (ll) (6), and lower right (lr) (7). since analyses included r2-gated cells, events fall only in the ur and the lr quadrants. the lr quadrant corresponded to coelomocytes which did not phagocytose e. coli-gfp (i.e. nonfluorescent), and the ur corresponded to those coelomocytes which did, exhibited by the higher relative fluorescence intensity (fl-1) compared to controls. fig. 2a shows the large coelomocytes of ew11 incubated in the absence of e. coli-gfp illustrating the level of background autofluorescence observed. nonfluorescent cells comprised 96.57 % of the total gated lc population (lr), while autofluorescent cells comprised 3.43 % (ur). the lc had relatively low levels of autofluorescence in all samples analyzed, while the eleocytes had significantly higher levels, but the eleocytes were excluded from the analysis. fig. 2b shows the lc of ew11 incubated with e. coli-gfp together with cytochalasin b, an antibiotic which interferes with microfilament activity and thereby inhibits phagocytosis (axline et al., 1974). this control was important to exclude the possibility of nonspecific 128 earthworm il-1 β 40ng/ml il-1β 20ng/ml gm-csf 4ng/ml gm-csf 2ng/ml il-2 25ng/ml il-2 12.5ng/ml tnf-α 5ng/ml tnf-α 2.5ng/ml ew1 t t/r ew2 t t/r ew3 t/r t ew4 t/r t t/r t t/r t/r t/r t ew5 t/r t/r t/r t/r t/r t/r t/r t/r ew6 t t t t/r t t t t/r ew7 t t t t t t t t/r ew8 t/r t/r t t ew9 t t/r t/r t/r ew10 t/r ew11 t t/r t/r t/r ew12 t/r t t t ew13 t t t/r t/r ew14 t t/r t t/r ew15 t/r t t t ew16 t t/r t/r t table 2 summary of earthworms used in this study exhibiting a statistically significant response to at least one of the cytokines used in pretreatment. only worms exhibiting statistically significant responses to at least one of the cytokine treatments are shown. for each earthworm shown, the cytokines used are indicated. t = earthworm treated with cytokine at indicated concentration; t/r = earthworm treated with cytokine at indicated concentration and exhibiting a statistically significant response (p ≤ 0.05). binding of e. coli-gfp to the cell surface of the large coelomocytes and provided background values which were subtracted from the experimental values to obtain percent specific phagocytosis. in this example nonfluorescent cells comprised 92.77 % of the population (lr), while fluorescent cells made up 7.23 % (ur). percent specific phagocytosis was calculated by first subtracting the fluorescent background of the averaged cytochalasin b control samples (i.e., 7.23 %) from the fluorescence observed 129 fig. 3 effects of il-1 beta on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 20 ng/ml (black) or (b) 40 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. with e. coli-gfp only (fig. 2c, ur quadrant), or treated with e. coli-gfp plus cytokine (fig. 2d, ur quadrant). the % specific phagocytosis in this example for e. coli-gfp is 44.71 % 7.23 % = 37.48 %, and the % specific phagocytosis for e. coli-gfp plus il-2 is 62.28 % 7.23 % = 55.05 %. then statistical analyses were performed to determine if the differences between the averaged untreated and cytokine-treated samples were statistically significant. using these criteria analyses were carried out for assays investigating the effects of il1 beta, gm-csf, il-2 and tnf-alpha. this study analyzed the response of the coelomocytes extruded from 50 earthworms to il-1 beta, gm-csf, il-2 and tnf-alpha in 9 separate assays. cytokines were used individually in all cases, and not in combination. coelomocytes from 28 of these earthworms were treated with all four of these cytokines at one or both of the concentrations chosen for the assays. coelomocytes from the remaining earthworms were treated with gm-csf only, or gm-csf and il-1 beta. table 1 illustrates the number of earthworms tested for each cytokine and the corresponding concentrations. sixteen of the 50 earthworms exhibited a statistically significant (p ≤ 0.05) response to at least one of the four cytokines used and only those earthworms were included in our final analyses. table 2 lists the 16 responding earthworms, indicates which cytokines were used and the concentrations employed, and whether or not enhancement of phagocytosis was statistically significant. a figs 3-6 show the results for only those earthworms which exhibited statistically significant responses (p ≤ 0.05) to the cytokines employed. results for earthworms with p > 0.05 are not shown. when il-1 beta was used at 20 ng/ml, 2 out of 20 (10 %) responded significantly ( fig. 3a). at the higher concentration of 40 ng/ml, 5 out of 28 (18 %) responded significantly (fig. 3b). gm-csf used at 2 ng/ml induced a 20 % response rate with 10 out of 50 earthworms responding significantly (fig. 4a). at 4 ng/ml, 2 out of 10 (20 %) responded significantly. (fig. 4b). il-2 gave similar results with 2 out of 10 (20 %) responding at 12.5 ng/ml (fig. 5a), and 6 out of 22 (27 %) responding at 25 ng/ml (fig. 5b). higher response rates were observed with tnfalpha; 3 out of 10 (30 %) responded to 2.5 ng/ml (fig. 6a), and 6 out of 22 (27 %) responded to 5 ng/ml (fig. 6b). in summary, these results show an overall increase in phagocytosis in 10 30 % of earthworms subjected to proinflammatory cytokine treatment compared to untreated controls. b a b fig. 4 effects of gm-csf on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 2 ng/ml (black) or (b) 4 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. 130 fig. 5 effects of il-2 on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 12.5 ng/ml (black) or (b) 25 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. discussion innate immune cells utilize pattern recognition receptors (prrs) to recognize chemical entities that are common to different classes of pathogens. prrs bind to pathogen-associated molecular patterns (pamps) and initiate cell-signaling pathways of innate immunity. one type of prr is the evolutionarily conserved toll/toll-like receptor, which has the ability to recognize a broad spectrum of ligands. the toll receptor was originally discovered in drosophila during an investigation of dorsoventral patterning in embryonic development (reviewed in janssens et al., 2003). homologues of insect toll receptors have been identified in mammals and other animals and are known as tolllike receptors (tlrs). two examples of tlr signaling pathways are: 1) tlr2, which binds to peptidoglycan of both gram-positive and gramnegative bacteria, lipoteichoic acid of gram-positive bacteria, and zymosan of yeast; and 2) tlr4, which binds to lps of gram-negative bacteria, and certain viruses (reviewed in leulier et al., 2008). it has been shown that toll receptor and tlr signaling induces phagocytosis and the synthesis of anti-microbial compounds in vertebrates, insects, and the invertebrate caenorhabditis elegans, but it is not yet known if they are needed in order for these mechanisms to occur in earthworms (reviewed in cooper et al., 2006). a our results show that the proinflammatory cytokines, il-1 beta, gm-csf, il-2, and tnf-alpha significantly increased the levels of phagocytosis of lc in 10-18 % of earthworms tested for il-1 beta, 20 % for gm-csf, 20 27 % for il-2, and 27 30 % for tnf-alpha, depending on the cytokine concentration used in the assay. it is interesting to note that of the 28 earthworms that were treated with all four cytokines, 12 (43 %) responded significantly to at least one of the cytokines and in some cases to all four cytokines. specifically, 3 responded to only one cytokine (ew7 to tnf-alpha; ew12 and ew15 to il-1 beta), 5 responded to two cytokines (ew6 and ew14 to gm-csf and tnfalpha; ew8 to il-1 beta and gm-csf; ew13 to il-2 and tnf-alpha; and ew16 to gm-csf and il-2), 2 responded the three cytokines (ew9 and ew11 to gm-csf, il-2 and tnf-alpha), and 2 responded to all four cytokines (ew4 and ew5) (table 2). the overall responses to the cytokines used in our study reveal that the most efficient response was to tnfalpha. b possible reasons for the variation in sensitivity to il-1 beta, gm-csf, il-2 and tnf-alpha could be related to different batches of earthworms. our studies were carried out over a 7 month period and a b fig. 6 effects of tnf-alpha on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 2.5 ng/ml (black) or (b) 5 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. 131 used different batches of earthworms. seasonal variations may have also had an effect. in addition, the earthworm colony is an outbred population and genetic polymorphisms could contribute to cytokine sensitivities. it is also possible that the cytokine concentrations that were used in our studies were not in the appropriate range needed to stimulate immune responses in some of the earthworms. in addition, the fact that the cell populations were heterogeneous and included eleocytes, hyaline amebocytes and granular amebocytes, could explain variations due to different proportions of the three cellular subsets in each extruded sample. for example, a higher or lower number of eleocytes, known to contain riboflavin (plytycz et al., 2006) could influence the sensitivity of the amebocytes to the different cytokines used. methodologies are needed to improve subset purification, while preserving cell yield, to enable these types of studies to be conducted with hyaline amebocytes in isolation. perhaps the enhancement of phagocytosis observed in our experiments is due to the binding of human cytokines to primitive earthworm receptors. this may then stimulate the upregulation of prrs resulting in higher levels of bacteria being phagocytozed due to elevated levels of ligand receptors. kurt-jones et al. (2002) showed that gmcsf induced upregulated expression of tlr2 and cd14 on the surface of human neutrophils and also increased il-8 secretion and enhanced superoxide responses when gm-csf-primed neutrophils were stimulated with tlr2 ligands. further studies need to be conducted to determine whether a similar stimulatory pathway is induced by gm-csf in our model. selective pressures operating on the different evolutionary lines of descent include the pamps that make up the common pathogenic burden shared by both lineages. mandrioli et al. (2007), using a bioinformatic approach, suggested that invertebrate cytokine-like ancestral receptors may be promiscuous, and able to bind to different cytokines. their results favor the model which supports the conservation of receptor-coding, rather than ligand-coding genes between vertebrates and invertebrates, and that present-day receptors are more similar to their ancestor counterparts than the ligands for those receptors. gene duplications in vertebrates would permit receptor differentiation tailored for different ligands, with eventual specialization of a receptor towards a single ligand. the lack of gene duplication in invertebrates, however, would restrict receptor evolution, and favor the interaction of many ligands with a limited number of generalized receptors. this rationale would not only explain why molluscan immunocytes can bind to mammalian cytokines il-1alpha, il-1 beta, il-2, tnf-alpha and tnf-beta (reviewed in mandrioli et al., 2007), but would also explain the findings reported in this study for earthworm coelomocytes. it is interesting to note that although not yet reported in e. hortensis, the existence of a gene encoding a putative helical cytokine in drosophila melanogaster has been reported (malagoli et al., 2007) and shown to be upregulated following immune stimulation. these findings support the proposal that helical cytokines play a role in immune responses of invertebrates. perhaps similar genes will be identified in earthworms and other invertebrates. tnf-alpha, il-1 and tlr ligands have in common the ability to bind to cell surface receptors which share a conserved intracellular signaling motif. once engaged, the signal transduced culminates in the activation and nuclear translocation of nuclear factor-kappa b (nf-kb) and the subsequent upregulation of genes encoding tlrs, tnf-alpha, and il-1 (o’neill et al., 2000; muzio et al., 2000; haynes et al., 2001; hongxiu et al., 2006). in contrast to the nf-kb signaling pathway initiated by tnf-alpha and il-1 beta, gm-csf and il-2 stimulate the jak-stat pathway, which relies on janus kinases (jak) and signal transducers and activators of transcription (stat). interestingly, the jak-stat pathway components are molecularly and functionally conserved from the invertebrate drosophila to humans to a high degree (arbouzova et al., 2006). the signaling pathways operating in earthworms require elucidation to establish whether these conserved signal transduction mediators and adaptors are also involved in the signal transduction of innate immune responses in annelids. although not presented in this paper, we tested the effects of gm-csf in combination with il-1 beta on phagocytosis and preliminary evidence suggests a synergistic effect as seen by enhanced phagocytosis. these results need to include a larger number of respondents to verify synergistic effects of proinflammatory cytokines on phagocytosis. it would also be worthwhile to employ a wider variety of cytokines and different combinations. studies in our lab were also carried out to investigate whether il-1 beta, gm-csf, il-2 or tnf-alpha would have an effect on cell proliferation in vitro. the coelomocytes were incubated in vitro with cytokine for 48 h in order to provide sufficient time for any effects to be observed. cell proliferation was determined by measuring dna content using propidium iodide and flow cytometry. in contrast to the results obtained with phagocytosis, there was little to no effect on cell proliferation when coelomocytes were treated with the proinflammatory cytokines; only 4.1 % of cases exhibited increased proliferation in response to tnf-alpha, and no increased proliferation was observed in response to il-1 beta, il-2, or gm-csf (data not shown). therefore, our prelilminary results suggest that these proinflammatory cytokines do not enhance cell proliferation significantly in e. hortensis coelomocytes under the conditions used in this study. problems with fungal and bacterial contamination, despite the inclusion of a large number of antimicrobials in the culture medium, restricted our incubation period to a maximum of 48 h, however, longer incubation periods or increased temperature may be necessary to observe proliferative effects in vitro. enriching the different coelomocyte subpopulations may also lead to conditions warranting longer incubation periods needed to reveal proliferative responses to these cytokines. our lab also carried out preliminary experiments to study the in vitro effects of il-1 beta 132 and gm-csf on nk-like responses of small coelomocytes in e. hortensis using the nonradioactive flow cytometric procedure described by patel et al. (2007). interestingly the results obtained in these early experiments proved to be inhibitory, not stimulatory, to nk-activity with 3 out of 12 earthworms exhibiting statistically significant inhibition when pretreated with il-1beta, and 1 out of 12 exhibiting statistically significant inhibition when pretreated with gm-csf (data not shown). perhaps this difference can be attributed to the observation that nk-like activity in earthworms is mediated by sc, not lc which carry out phagocytosis (engelmann et al., 2004; salzet et al., 2006). perhaps these two different coelomocyte populations respond differently to proinflammatory cytokines through the use of distinct signal transduction pathways. it would be interesting to determine whether the signaling pathways associated with phagocytosis and proliferation in earthworms share signal transduction components which can be induced with similar cytokine or cytokine-like molecules. it would be worthwhile and of great importance to carry out a microarray analysis, or generate a cdna library to examine at the molecular level the changes occurring in earthworm coelomocytes in response to infection and exposure to human proinflammatory cytokines. for example, when highdensity oligonucleotide microarrays were generated in drosophila following microbial infection, 230 genes were induced and 170 were repressed, most of which had never before been associated with immune response, and many of the genes uncovered had unknown function (degregorio et al., 2001), providing new leads for innate immune activities in invertebrates. there is clearly a need to understand the signaling pathways operating in earthworms and microarrays would provide an invaluable tool to decipher the humoral, cellular and molecular interactions regulating innate immunity defenses in annelids. acknowledgements this work was supported by grants from the cabrini college faculty development grants committee (sf-e), and the pennsylvania academy of science (lg). references adamowicz a, wojtaszek j. morphology and phagocytotic activity of coelomocytes in dendrobaena veneta (lumbricidae). zoologica poloniae 46: 91-104, 2001. arbouzova ni, zeidler mp. jak/stat signaling in drosophila: insights into conserved regulatory and cellular functions. development 133: 26052616, 2006. axline sg, reaven ep. inhibition of phagocytosis and plasma membrane mobility of the cultivated macrophage by cytochalasin b. role of subplasmalemmal microfilaments. j. cell biol. 62: 647-659, 1974. bilej m, brys l, beschin a, lucas r, vercauteren e, hanušová r, et al. identification of a cytolytic protein in the coelomic fluid of eisenia foetida earthworms. immunol. lett. 45: 123-128, 1995. bilej m, rossmann p, šinkora m, hanušová r, beschin a, raes g, et al. cellular expression of the cytolytic factor in earthworms eisenia foetida. immunol. lett. 60: 23-29, 1998. beschin a, bilej m, brys l, torreele e, lucas r, magez s, et al. convergent evolution of cytokines. nature 400: 627-628, 1999. beschin a, bilej j, magez s, lucas r, debaetslier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on a similar lectin-like activity. prog. mol. subcell. biol. 34: 145-163, 2004. bruhn h, winkelmann j, andersen c, andrä j, leippe m. dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defence protein of the annelid eisenia fetida. dev. comp. immunol. 30: 597-606, 2006. carswell ea, old lj, kassel rl, green s, fiore n, williamson b. an endotoxin-induced serum factor that causes necrosis of tumors. proc. natl. acad. sci. usa 72: 3666-3670, 1975. cooper el. earthworm immunity. in: rinkevich b, műller weg (eds), invertebrate immunology, springer verlag, heidelberg, pp 10-45, 1996. cooper el, kauschke e, cossarizza a. digging for innate immunity since darwin and metchnikoff. bioessays 24: 319-333, 2002. cooper el, dvell k, engelmann p, nemeth p. still waiting for the toll? immunol. lett. 104: 18-28, 2006. cossarizza a, cooper el, suzuki mm, salvioli s, capri m, gri g, et al. earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. exp. cell res. 224: 174-182, 1996. cossarizza a, pinti m, troiano l, cooper el. flow cytometry as a tool for analyzing invertebrate cells. inv. surv. j. 2: 32-40, 2005. de gregorio e, spellman pt, rubin gm, lemaitre b. genome-wide analysis of the drosophila immune response by using oligonucleotide microarrays. proc. natl. acad. sci. usa 98: 12590-12595, 2001. engelmann p, pál j, berki t, cooper el, németh p. earthworm leukocytes react with different mammalian specific monoclonal antibodies. zoology 105: 257-265, 2002. engelmann p, molnár l, pálinkás l, cooper el, németh p. earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge. cell tissue res. 316: 391-401, 2004. engelmann p, palinkas l, cooper el, németh p. monoclonal antibodies identify four distinct annelid leukocyte markers. dev. comp. immunol. 29: 599-614, 2005. franceschi c, cossarizza a, monti d, ottaviani e. cytotoxicity and immunocyte markers in cells from the freshwater snail planorbarius corneus (l.) (gastropoda pulmonata): implications for the evolution of natural killer cells. eur. j. immunol. 21: 489-93, 1991. franchini a, ottaviani e. repair of molluscan tissue injury: role of pdgf and tgf-β. tissue cell 32: 312-321, 2000. franchini a, malagoli d, ottaviani e. cytokines and invertebrates: tgf-β and pdgf. curr. pharm. des. 12: 3025-3031, 2006. 133 haynes lm, moore dd, kurt-jones ea, finberg rw, anderson lj, tripp ra. involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. j. virol. 75: 1073010737, 2001. hongxiu l, masayuki k, maezenna b, yun y, xin l. ubiquitination of rip is required for tumor necrosis factor alpha-induced nf-kb activation. j. biol. chem. 281: 13636-13643, 2006. janssens s, beyaert r. role of toll-like receptors in pathogen recognition. clin. microbiol. rev. 16: 637-646, 2003. kalaç y, ayten k, gűlruh u, çotuk a. the role of opsonin in phagocytosis by coelomocytes of the earthworm dendrobaena veneta. j. cell mol. biol. 1: 7-14, 2002. kurt-jones ea, mandell l, whitney c, padgett a, gosselin k, newburger pe, et al. role of tolllike receptor 2 (tlr2) in neutrophil activation: gm-csf enhances tlr2 expression and tlr2-mediated interleukin 8 responses in neutrophils. blood 100: 1860-1868, 2002. lange s, nussler f, kauschke e, lutsch g, cooper el, herrmann a. interaction of earthworm hemolysin with lipid membranes requires sphingolipids. j. biol. chem. 272: 2088420892, 1997. lange s, kauschke e, mohrig w, cooper el. biochemical characteristics of eiseniapore, a pore-forming protein in the coelomic fluid of earthworms. eur. j. biochem. 262: 547-556, 1999. leulier f, lemaitre b. toll-like receptors taking an evolutionary approach. nat. rev. genet. 9: 165-178, 2008. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978. 2007. mandrioli m, malagoli d, ottaviani e. evolution game: which came first, the receptor or the ligand?. inv. surv. j. 4: 51-54, 2007. mosiman v, patterson b, canterero l, goolsby c. reducing cellular autofluorescence in flow cytometry: an in situ method. cytometry 30: 151-156, 1997. muzio m, bosisio d, polentarutti n, d’amico g, stoppacciaro a, mancinelli r, et al. differential expression and regulation of toll-like receptors (tlr) in human leukocytes: selective expression of tlr3 in dendritic cells. j. immunol. 164: 5998-6004, 2000. o’neill l. the toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defence. biochem. soc. trans. 28: 557-563, 2000. ottaviani e, caselgrandi e, franceschi c. cytokines and evolution: in vitro effects of il-1 alpha, il-1 beta, tnf-alpha and tnf-beta on an ancestral type of stress response. biochem. biophys. res. commun. 207: 288-292, 1995. ottaviani e, franchini a, malagoli d, genedani s. immunomodulation by recombinant human interleukin-8 and its signal transduction pathways in invertebrate hemocytes. cell mol. life sci. 57: 506-513, 2000. patel m, francis j, cooper el, fuller-espie sl. development of a flow cytometric, nonradioactive cytotoxicity assay in eisenia fetida: an in vitro system designed to analyze immunosuppression of natural killer-like coelomocytes in response to 7, 12 dimethylbenz[a]anthracene (dmba). eur. j. soil biol. 43 (suppl 1): s97-s103, 2007. plytycz b, homa j, koziol b, różanowska m, morgan aj. riboflavin content in autofluorescent earthworm coelomocytes is species-specific. folia histochem. cytobiol. 44: 275-280, 2006. prendergast r, lutty g, scott a. directed inflammation – the phylogeny of lymphokines. dev. comp. immunol. 7: 629-632, 1983. procházková p, šilerová m, felsberg j, josková r, beschin a, de baetselier p, et al. relationship between hemolytic molecules in eisenia fetida earthworms. dev. comp. immunol. 30: 381392, 2006. renzelli-cain r, kaloustian kv. evidence for the involvement of opioid peptides in phagocytosis, conformation, granulation and aggregation of immunocompetent lumbricus terrrestris amoebocytes. comp. biochem. physiol. 111: 205-211, 1995. roch p. invertebrate immune responses: cells and molecular products. in: cooper el (ed) advances in comparative & environmental physiology, spring-verlag, berlin, pp 116-145, 1996. salzet m, tasiemski a, cooper e. innate immunity in lophotrochozoans: the annelids. curr. pharm. des. 12: 3043-3050, 2006. 134 activation and regulation mechanisms of toll signaling cascades in insects isj 7: 181-191, 2010 issn 1824-307x review the molecular activation and regulation mechanisms of proteolytic toll signaling cascade in insect innate immunity kh ryu1, jw park1, k kurokawa1, m matsushita2, bl lee1 1the national research laboratory of defense proteins, college of pharmacy, pusan national university, busan, 609-735, korea 2department of applied biochemistry; tokai university, hiratsuka, kanagawa 259-1292, japan accepted september 2, 2010 abstract recently we biochemically determined the molecular recognition and regulatory mechanisms of how beetle’s larvae recognize gram-positive bacteria and fungi via toll signaling cascade. the biochemical analysis of newly identified molecules provides us how beetles recognize invading pathogenic microbes and how they defend their bodies using elegant innate immunity. here, we will focus on reviewing the biochemical analyses and biological functions of newly identified molecules involved in insect toll signaling cascade. key words: toll cascade; innate immunity; protease inhibitor; insects introduction innate immunity is an evolutionarily conserved first-line host defense that senses pathogenic microorganisms through “pattern-recognition” molecules that recognize the conserved molecular patterns on the surface of microbes (medzhitov and janeway, 1997). invertebrate’s innate immune response is a crucial host defense system to defend against microbial infection (hoffmann et al., 1999). the ability of a host to distinguish between self and non-self remains a central hallmark of innate immunity. the pathogenic microbes possess distinct pathogen-associated molecular patterns (pamps), such as peptidoglycans (pgs) of gram-positive bacteria and β-1,3-glucans of fungi. the recognition of pamps is achieved by a group of germ line-encoded receptors and soluble proteins (hoffmann et al., 1999). ___________________________________________________________________________ corresponding author: lee bok luel the national research laboratory of defense proteins, college of pharmacy, pusan national university, jangjeon dong, gumjeong gu, busan, 609-735, korea e-mail: brlee@pusan.ac.kr list of abbreviation: amp, antimicrobial peptides; sp, serine proteinase; pg, peptidoglycan; pgrp, peptidoglycan recognition protein; grp, β-1,3-glucan recognition protein; gnbp, gram-negative binding protein; msp, modular sp; modsp, modular sp; spe, spätzleprocessing enzyme; sae, spe activating enzyme; hp, hemolymph protease. in drosophila, the synthesis of antimicrobial peptides (amps) in response to microbial infections is under the control of the toll and immune deficiency (imd) signaling pathway (lemaitre and hoffmann, 2007). the toll signaling pathway responds mainly to the lysine (lys)-type pg of gram-positive bacteria and fungal β-1,3-glucan, whereas the imd pathway responds to mesodiaminopimelic acid (dap)-type pg of gramnegative bacteria and certain gram-positive bacilli. the biological significance of these two drosophila signaling pathways is demonstrated by the fact that mutations of the genes involved in these pathways dramatically decrease resistance to microbial infections, e.g., toll mutants are susceptible to fungal infections and relish, the nf-κb protein involved in imd pathway, mutants lose resistance to gram-negative bacterial infections ( lemaitre et al., 1996; hedengren et al., 1999). toll signaling cascade is amplified in hemolymph (insect blood) by a proteolytic serine protease (sp) cascade. the amplification of toll recognition signals results in an efficient host defense strategy in insects, which are devoid of an acquired immune system. drosophila genetic studies showed that lystype pg of gram-positive bacteria is recognized by two different protein complex consisted of the pg recognition protein-sa (pgrp-sa) and the gramnegative binding protein 1 (gnbp1) (michel et al., 2001; gobert et al., 2003; pili-floury et al., 2004). in contrast, fungal β-1.3-glucan is known to be recognized by gnbp3 (gottar et al., 2006). these lys-type pg and β-1.3-glucan recognition signals 181 that are mediated via pgrp-sa and gnbp3, respectively, are suggested to induce the activation of a sp proteolytic cascade, ultimately leading to the conversion of pro-spätzle into processed spätzle (lemaitre et al., 1996; weber et al., 2003; gay and gangloff, 2007). drosophila spätzle-processing enzyme (spe), a typical clip-domain-containing sp, has been identified as the terminal sp that induces the cleavage of pro-spätzle (jang et al., 2006; kambris et al., 2006). the processed spätzle functions as a native ligand for the toll receptor, and induces the expression of amp genes from the fat body (levashina et al., 1999; weber et al., 2003; gay and gangloff, 2007). therefore, drosophila toll signaling pathway is an elegant example showing of that pattern recognition proteins-mediated pathogen recognition signals are amplified by a proteolytic sp cascade. in addition, recent interesting study performed by shia et al. (2009) showed that knockdown of toll ligand spätzle in the drosophila hemocytes (insect blood cells) induced the inhibition of the expression of amp genes from fat body, suggesting that toll receptor-dependent amp responses in the fat body require spätzle secretion from hemocytes. also, this result indicates that the integration between humoral responses characterized by amp production by fat body and cellular response, such as phagocytosis, mediated by hemocytes depend on the cytokine-like spätzle production, a similar process with mammalian cytokine-mediated immune responses. the drosophila genetic studies are very powerful for characterizing and ordering the components in the drosophila toll signaling pathway (ligoxygakis et al., 2002a). however, there is a limit for this system in terms of determining the biochemical mechanisms involved in regulating this proteolytic cascade. drosophila has several alternative routes to the toll pathway, used in various developmental stages and infection protocols, and it seems difficult to determine the clear activation mechanism by the genetic approach only. for instance, drosophila persephone is another protease linked to the toll pathway and antifungal immunity, yet the biological functions of this molecule have been partially characterized by drosophila genetic studies. the proper identification of upstream or downstream factor(s) of drosophila persephone still awaits further investigations (levashina et al., 1999; gottar et al., 2006). to provide compelling biochemical data on how the lys-type pg recognition signal can be sequentially transferred to spätzle using purified pattern recognition proteins and sps, it is necessary to use a larger insect, which enables us to collect large amounts of hemolymph and to perform biochemical studies. recently, our group determined the activation and regulation mechanisms of tenebrio toll signaling cascade by approaching biochemical methods using a large beetle, tenebrio molitor (kim et al., 2008; jiang et al., 2009; roh et al., 2009). this large insect enabled us to collect large amounts of hemolymph for biochemical studies, allowing us to purify several different tenebrio sps. we demonstrated that the recognition of lys-type pg-mediated by the pgrp-sa/gnbp1 complex induces the processing of pro-spätzle to the cleaved spätzle through the sequential activation of three different tenebrio sps: modular serine protease (msp), spätzle-activating enzyme (sae) and spätzle-processing enzyme (spe) (kim et al., 2008). tenebrio spe has been shown as a terminal sp that cleaves pro-spätzle. additionally, we provided another biochemical evidences of the mechanism by which the gnbp3-mediated fungal β-1,3-glucan recognition signal is also converted pro-spätzle to spätzle after activation of the same three different sps that are involved in lys-type pg recognition signal (roh et al., 2009). therefore, it will be valuable to compare the biological diversity of toll signaling cascades in three different insects, fruit fly drosophila melanogaster, tobacco hornworm manduca sexta and mealworm t. molitor. this will shed further insight into biological diversity of the molecular mechanisms of pathogen recognition and amplification signals in insects. since excellent review papers focusing toll signaling and melanin synthesis cascades are available now (ferrandon et al., 2007; lemaitre and hoffmann, 2007; cerenius et al., 2008, 2010), we mainly focus on biochemical studies of toll signaling cascade using mealworm (t. molitor) and tobacco hornworm (m. sexta) larvae. pathogen recognition molecules of toll cascade pgrp family proteins are critical receptors in drosophila immune responses that are required for the recognition of pg and for subsequent activation of amp gene expression (lemaitre and hoffmann, 2007). pgrps were first characterized in the moths bombyx mori and trichoplusia ni (yoshida et al., 1996; kang et al., 1998) and proposed to be receptors that can trigger insect’s innate immune responses. pgrps share homology with nacetylmuramoyl-l-alanine amidases, which cleave pg at the lactylamide bond between the glycan backbone and the stem-peptides (kang et al., 1998). some non-catalytic pgrps, such as pgrp-lc, -le, -sa and -sd, lack a critical cysteine residue in the catalytic pocket and are not able to cleave pg (mellroth et al., 2003), but these pgrps can bind pgs and are necessary for the expression of amp genes, indicating that these pgrps directly recognize bacteria and activate innate immune responses. in contrast, catalytic pgrps, such as pgrp-sc1a, -lb and -sc2, include this cysteine residue in the active site and are potent enzymes that cleave pg. after digestion with pgrp-sc1b, staphylococcal pg exhibits less activation of the amp genes in a drosophila blood cell line, so it was hypothesized that catalytic pgrps may act as scavengers to limit an inflammatory response to free pg (mellroth et al., 2003). gnbp family, a 50-kda protein found in hemolymph of b. mori and named as a gramnegative binding protein (lee et al., 1996). however, the biochemical studies demonstrated that gnbp belong to the family of β-1,3-glucan recognition proteins (βgrp) that had first been purified on their ability to trigger the pro-phenoloxidase (melanin synthesizing enzyme in invertebrates) in response to fungal infections (yoshida et al., 1986). drosophila 182 genetic studies demonstrated that lys-type pg is recognized by a complex comprised of the pgrpsa and gnbp1 (gobert et al., 2003; pili-floury et al., 2004), while β-1,3-glucan from yeast is recognized by gnbp3 (gottar et al., 2006). both the pgrpsa/gnbp1 complex and gnbp3 are believed to mediate the activation of a sp cascade that ultimately leads to the cleavage of pro-spätzle into processed spätzle (lemaitre et al., 1996). native gnbp3 proteins were purified from three insects, silk worm (b. mori, ochiai and ashida, 2000), tobacco hormworm (m. sexta, ma and kanost, 2000) and mealworm (t. molitor, zhang et al., 2003). the purified proteins bound to 1,3-β-d-glucan but not to bacterial pg. subsequent molecular cdna clonings revealed that gnbp3 molecules contains a region with a sequence similar to bacterial glucanases. interestingly, two catalytically important residues in the glucanases had been replaced with nonhomologous amino acids in all three gnbp3, suggesting that gnbp3 molecules have evolved from an ancestral glucanase gene but retained only the ability to recognize β-1,3-glucan. native gnbp1 was only purified from mealworm and it was involved in pg-recognition reaction with pgrp-sa (kim et al., 2008). next, native pgrp-sa proteins were purified from three insects, silkworm, cabbage looper (t. ni) and mealworm (yoshida et al., 1996; kang et al., 1998; park et al., 2006). the purified pgrp-sa recognized bacterial pgs. in vitro reconstitution experiments demonstrated that tenebrio and bombyx pgrp-sas are recognition molecules of pg-dependent pro-phenoloxidase cascades (yoshida et al., 1996; park et al., 2006). until this time, pgrp proteins from tobacco hornworm were not purified yet. sp zymogens involved in the lys-type pgmediated toll signaling cascade drosophila genetic studies provided the evidence of that pgrp-sa and gnbp1 makes a complex upon recognition of lys-type pg and then recruits down-stream sps for the activation of toll signaling cascade (gobert et al., 2003). however, the identification of immediately downstream sps of the pgrp-sa/gnbp1 complex is not easily performed in drosophila system due to protein redundancy and a limitation of drosophila hemolymph collection. since tenebrio larvae also showed high antimicrobial activities to the challenge of gram-positive bacteria or fungi, it was supposed that tenebrio larvae may have all essential components necessary for the activation and regulation of tenebrio toll signaling cascade. therefore, we assumed that the immediate downstream sp of the pgrp-sa/gnbp1 complex can be purified using tenebrio hemolymph by biochemical approaches (park et al., 2007). also, we hypothesized that gnbp1 and an sp would be recruited to the lys-type pg/pgrp-sa complex when the soluble lys-type pg/tenebrio pgrp-sa complex was incubated with pgrp-sa-depleted tenebrio hemolymph. indeed, tenebrio pgrpsa/lys-type pg complex recruited a 50-kda protein and a 35-kda protein. the amino acid sequencing of the n-terminal residues identified the 50-kda protein as tenebrio gnbp1 and the 35-kda protein as tenebrio msp. interestingly, this msp protease does not contain a clip domain, which is commonly found in proteases upstream of the toll and pro-phenoloxidase cascades (piao et al., 2005). drosophila gnbp1 was known to physically interact with pgrp-sa and activates drosophila toll pathway. however, an interaction between gnbp1 and pgrp-sa has not been observed in vitro (gobert et al., 2003). our observation suggests that the binding of pgrp-sa to pg enhanced the interaction between pgrp-sa and gnbp1, and subsequently the active form of msp was recruited to the lys-type pg/pgrpsa/gnbp1 complex. subsequent cdna cloning demonstrated that t. molitor msp (tm-msp) contains four low-density lipoprotein receptor a repeat (ldl) domains, one complement control protein (ccp) domain and a chymotrypsin-like sp domain (kim et al., 2008). the substrate specificity pocket residues of the sp domain, ser-569 (c189; “c” for the chymotrypsinogen numbering), ser-596 (c216), and gly-610 (c226) indicate that tm-msp is a chymotrypsin-like sp. msp-like molecules was identified in other two insects, m. sexta hp (hemolymph protease)-14 (ms-hp-14) (ji et al., 2004) and the d. melanogaster msp protein (dmmsp, cg31217, buchon et al., 2009). the domain organizations of these three known msp orthologues are shown in fig. 1a and the biological functions of these three msp molecules are reported (ji et al., 2004; park et al., 2007; buchon et al., 2009). also, recently, bucheon et al reported that drosophila msp integrates signals originating from the circulating recognition molecules gnbp3 and pgrp-sa and connects them to the grass-spe-spätzle extracellular pathway upstream of the toll receptor (buchon et al., 2009). although we found that msp is recruited into the lys-type pg recognition complex, the lack of information regarding the identity of the immediate downstream factor(s) of msp limits our knowledge of the molecular details of pg recognition and the involvement of sps in the toll signaling pathway. therefore, the identification and characterization of the biological functions of sps involved in the tenebrio toll signaling pathway are essential for the elucidation of the molecular mechanism of innate host defense system. spe, a terminal sp that converts spätzle pro-protein into a processed form capable of binding the toll receptor, was identified in drosophila (jang et al., 2006). we purified the sps immediately downstream protease of tenebrio msp in order to obtain biochemical information regarding the lys-type pg recognition signaldependent activation of the toll cascade. two sps, tenebrio 41-kda and 44-kda protein, were purified to homogeneity by several column chromatographies and their cdnas were cloned (kim et al., 2008). in order to identify the spe molecule in tenebrio, we initially examined the active form of the 44-kda protease because the 44kda zymogen protein is similar to drosophila spe and easter. since the cdnas of tenebrio spätzle and the toll proteins have not been determined, we 183 fig. 1 the domain organizations of msp homologues (a) and tm-msp, tm-41 kda sp (tm-sae) and tm-44 kda sp (tm-spe) (b). a, comparison of domain organization between t. molitor msp (tm-msp), m. sexta hemolymph proteinase 14 (ms-hp14), and dm-msp homologue (cg31217). pink circles, rectangular symbols and boxes indicate the domains of ldla, ccp, and sp domains of msp homologues, respectively. b, green circles symbol indicates the clip domain. arrows represent the cleavage sites of sp zymogens during activation. the red and blue residues in the boxes indicate the specificity-related residue and catalytic triad ser residue, respectively. expressed the recombinant tribolium spätzle proprotein and the toll ectodomain in a baculovirus system. to address whether the purified active 44kda protease cleaves tribolium spätzle pro-protein in vitro, we incubated the pro-spätzle protein with trypsin, the active forms of tenebrio msp and the 41-kda and 44-kda proteases. only the active 44kda protease cleaved the spätzle pro-protein. under the same conditions, trypsin cleaved the prospätzle protein nonspecifically, and the active forms of tenebrio msp and the 41-kda protease did not cleave the pro-spätzle protein. after performing further biochemical experiments, we confirmed 44 kda protease as tenebrio spe. also, recombinant hp8 protease functioning as manduca spe was purified and its biological function was also determined (an et al., 2009). we next tried to identify the upstream activator of tenebrio spe. because tenebrio spe has a trypsin cleavage site, we hypothesized that the upstream sp is a trypsin-like sp. as mentioned above, the msp and the 41-kda zymogens were identified as chymotrypsin-like and trypsin-like sps, respectively, suggesting that the active form of the 41-kda protease may cleave the spe zymogen. to test this hypothesis, we incubated the active form of 41-kda protein with the purified spe zymogen. as expected, the spe zymogen was hydrolyzed into a 35-kda sp domain and a 15-kda clip domain. the 35-kda band was identified as the sp domain of spe. therefore, we designated the 41-kda protease as tenebrio spe-activating enzyme (sae). 184 because the sequence of the cleavage site in the sae zymogen is leu-124-ile-125, the upstream sp of sae is probably similar to chymotrypsin. this result suggests that the sae zymogen is cleaved by the msp. therefore, active tm-msp was incubated with the recombinant sae zymogen. the sae zymogen was hydrolyzed into a 35-kda sp domain and an 11-kda clip domain. the n-terminal amino acid sequence of the 35-kda band was ile-val-glygly-thr-asn. this sequence is identical to the amino acid sequence of the sae zymogen from ile-125 to asn-130, demonstrating that the msp induced a limited proteolytic cleavage between the clip domain and the catalytic sp domain of the sae zymogen. thus, the sae protease is an immediate downstream target of msp. the domain organization and the cleavage sites by upstream sps were summarized in figure 1b. recently, sae orthologue from manduca system is identified and its biochemical properties are demonstrated (an et al., 2009). drosophila sps required for the activation of toll signaling cascade were screened using rnai technology by kambris et al. (2006). they suggested that two d. melanogasrter catalytic sps (dm-grass and dmspirit) and two noncatalytic sp homologs (sphs), such as dm-spheroide and dm-sphinx1/2, have been involved in the pg-dependent drosophila toll pathway. by homology research with known sps, it turned out that dm-grass and dm-spirit are clip domain-containing trypsin-like sps. dm-spheroide and dm-sphinx1/2 each have a non-catalytic sp domain but no clip domains at the n-terminus. they also have gly and ile residues, respectively, instead of a ser residue in the catalytic site of sps. the reason why we do not identify similar sps and sphs in our studies is unclear, and further studies are necessary to answer this question. however, one plausible explanation for this can be that tenebrio sphs may exist with serine protease inhibitors (serpins) in the hemolymph and are not directly involved in the toll pathway activation. by performing rnai experiments against drosophila sps or sphs, there is a possibility that serpins can be released to the hemolymph by lack of sph and that catalytic sps, such as dm-grass and dm-spirit might be trapped by the released serpins leading to inhibition of the toll pathway activation. lys-type pg recognition signal is amplified via a three step proteolytic cascade finally, to confirm whether the spätzle proprotein is cleaved, lys-type-pg, pgrp-sa, gnbp1, zymogens of msp, sae, spe and pro-spätzle were incubated together in the presence of ca2+, and the cleavage of pro-spätzle was detected by western blot analysis. as predicted, a 14-kda band corresponding to cleaved spätzle was observed (kim et al., 2008). however, if any one of the components was omitted from the incubation mixture, no cleavage of the pro-spätzle occurred (kim et al., 2008). in conclusion, these experiments demonstrated that the pgrp-sa/gnbp1-mediated lys-type pg recognition signal is transferred by three different sps; the initiating enzyme is the 82kda chymotrypsin-like msp, and the other two enzymes are the 41-kda sae and 44-kda spe clip domain-containing trypsin-like sps. sp zymogens involved in the β-1,3-glucanmediated toll signaling cascade to identify the immediate downstream molecule(s) that is recruited by the β-1,3glucan/gnbp3 complex, insoluble β-1,3-glucan was incubated with the native tenebrio gnbp3. when gnbp3/insoluble β-1,3-glucan complex was incubated with the tenebrio hemolymph fraction, a 35-kda band was specifically enriched in the gnbp3/insoluble β-1,3-glucan complex. the nterminal amino acid sequence of the 35-kda protein perfectly matched that of the catalytic sp domain of activated tenebrio msp, suggesting that activated msp was recruited to the β-1,3-glucan/gnbp3 complex and that msp is an apical sp that functions as the immediate downstream molecule of the β1,3-glucan/gnbp3 complex (roh et al., 2009) as like the apical downstream sp of pgrp-sa/gnbp1 in response to lys-type pg (kim et al., 2008). because tenebrio pro-msp is activated in the presence of either the β-1,3-glucan/gnbp3 complex or the lys-type pgn/pgrp-sa/gnbp1 complex, we assumed that the downstream sps of the β-1,3glucan/gnbp3 complex would be identical to tenebrio sae and spe that are activated in response to lys-type pg. β-1,3-glucan recognition signal is also amplified via three-step proteolytic cascade we performed in vitro reconstitution experiments by using five purified proteins: gnbp3, pro-msp, pro-sae, pro-spe, and prospätzle (roh et al., 2009). western blot analysis revealed that processed spätzle was generated upon incubation of the five proteins, β-1,3-glucan, and ca2+ (roh et al., 2009). depletion of any of the components resulted in the loss of cleavage of the pro-spätzle. under the same conditions, the processing of prospätzle to the cleaved spätzle was also observed with pgrp-sa, gnbp1, msp, sae, spe, and spätzle in the presence of lys-type pg and ca2+ (roh et al., 2009). these results clearly demonstrate that gnbp3, in the presence of β-1,3glucan, induces the activation of a three-step proteolytic cascade involving msp, sae, and spe sequentially. this activation leads to the processing of pro-spätzle into the mature form that functions as a ligand for the toll receptor. in addition, these data show that the β-1,3glucan and lys-type pg recognition signals are sharing a common three step proteolytic cascade to transduce their recognition signals to the toll receptor (fig. 2). the effector molecules after toll signaling cascade activation although we have provided biochemical evidence elucidating the mechanism by which the lys-type pg and β-1,3-glucan recognition signals are transferred to the toll receptor, we have not demonstrated whether this sp cascade is present in vivo. we hypothesized that if this cascade is present 185 fig. 2 model summarizing the molecular events in the regulation of the tenebrio toll signaling cascade. the molecular activation mechanism of the tenebrio toll cascade leading to production of amps. processed spätzle induces the production of endogenous spn40 and spn55 as a negative feedback regulator of the toll cascade. in vivo, the same amp(s) will be produced in the insect hemolymph when the pathway molecules are injected into the tenebrio larvae since tenebrio toll signaling pathway is using a common proteolytic cascade. to address this hypothesis, we injected β1,3-glucan, lys-type pg, activated sae and processed spätzle into the tenebrio larvae. the hemolymphs collected after injection of above four molecules had high antimicrobial activities against s. aureus, e. coli and s. cerevisiae. we purified two amps, tenecin 1 and tenecin 2, by column chromatography from these four hemolymphs. tenecin 1 had a bactericidal activity against grampositive bacteria and was previously identified by our group (moon et al., 1994). the amino acid sequence of tenecin 1 and its disulfide bond arrangement are similar to the defensin protein from drosophila (bulet et al., 1999). tenecin 2 is highly homologous (65 % identity) to coleoptericin (bulet et al., 1991), which was purified from the coleopteran insect, zophobas atratus. tenecin 2 showed bactericidal activity against e. coli. taken together, tenecin 1 and tenecin 2 are induced by treatment with β-1,3-glucan, lys-type pg, sae and spätzle suggesting that β-1,3-glucan and lys-type pg activate toll receptors by the same three-step proteolytic cascade, which results in the production of tenecin 1 and 2 (fig. 2). 186 fig. 3 a comparison with drosophila, manduca and tenebrio extracellular proteolytic toll signaling pathways involving sp cascades. arrows indicate the activation of downstream components or steps in cascade. dashed arrows indicate steps that have not been experimentally verified or in which components of the pathway have not yet been determined. pg, peptidoglycan; pgrp, peptidoglycan recognition protein; grp, β-1,3-glucan recognition protein; gnbp, gram-negative binding protein; propo, prophenoloxidase; po, phenoloxidase; msp, modular sp; modsp, modular sp; spe, spätzle-processing enzyme; sae, spe activating enzyme; psh, persephone; sph, sp homologue; hp, hemolymph protease; pap, pro-po activating protease. diversity of toll proteolytic signaling cascades in drosophila, manduca and tenebrio system the outline of activation mechanism of toll signaling cascades in three different insects are summarized in figure 3. intensive biochemical studies performed in t. molitor and m. sexta, together with genetic analysis in d. melanogaster, reveal striking similarities in the mechanisms underlying sp activation by pattern recognition proteins. all involve the sequential activation of typical sps, such as modular sp and clip-domain containing sps. interestingly, tenebrio and drosophila used msp as an apical sp in the toll cascade, but, manduca hp14 corresponding to msp did not activate hp6 zymogen corresponding to tenebrio sae, suggesting that another direct downstream sp of pgrp-sa/gnbp1 complex might be existing in hemolymph in manduca system. in drosophila, epistasis analysis has demonstrated that an drosophila msp named modular sp (modsp), acts down stream of pgrp-sa and upstream of the sp grass (buchon et al., 2009). moreover, drosophila modsp does not participate in the persephone-dependent branch of the toll pathway, but is instead part of a linear pathway of sps connecting microbe recognition by pattern recognition proteins to the activation of spätzle by spe. tenebrio msp, manduca hp14 and drosophila modsp proteases all share a common structure comprising four or five ldl domains followed by a complement control domain and a c-terminal sp domain. the upstream pathogen recognition features of insect’s toll cascade are also reminiscent of the complement activation by the lectin pathway in mammals in which the recognition of carbohydrate by the mannose binding lectin (mbl) leads to the autoactivation of mbl-associated sps (masps, matsushita and fujita, 1992). masps also showed similar domain organization with those of insect msps. manduca toll cascade summarized that prohp6 becomes activated in response to microbial infection and participates in two immune pathways (fig. 3); activation of pap1, which leads to prophenoloxidase activation and melanin synthesis, and activation of hp8, which stimulates a toll-like pathway (an et al., 2009). hp8 activates spätzle to induce amp synthesis via toll receptor. in melanin synthesis cascade, an initiation proteinase precursor, prohp14, is autoactivated in response to grampositive bacterial or fungal infection. hp14 activates prohp21; hp21 activates propap2 or propap3; pap2 or pap3 then cleaves pro-phenoloxidase to form active phenoloxidase in the presence of sph1 and sph2. activation of pro-phenoloxidase can also be catalyzed by pap1 when the high mr sph complex is present simultaneously. pap1 also activates prosph2 directly and can indirectly lead to prohp6 activation (wang and jiang, 2007, 2008). tenebrio toll cascade support a model in which lys-type pg and β-1,3-glucan activate a common set of three sp zymogens sequentially (fig. 3). this three-step proteolytic cascade-dependent 187 processing of the extracellular pro-spätzle produces active spätzle, which then binds to the toll receptor, resulting in the induction of amp expression in the tenebrio larval hemocytes. each of the three sps has unique biochemical properties to regulate activation of the tenebrio toll pathway. the initial enzyme pro-msp is an 82-kda sp zymogen with an n-terminal chymotrypsin-like cleavage site and a cterminal chymotrypsin-like catalytic sp domain. prosae is a 41-kda sp with an n-terminal chymotrypsin cleavage site and a c-terminal trypsinlike catalytic sp domain, and pro-spe is a 44-kda protein with an n-terminal trypsin-like cleavage site and a c-terminal trypsin-like catalytic sp domain. these three different sp combinations enhance the specificity of the proteolytic sp cascade and prevent nonspecific cleavage of these sp zymogens. three serpins functioning as regulatory molecules of toll signaling cascade serpins act as suicide substrates by binding covalently to their target proteases and belong to a superfamily of sp inhibitors (gettins, 2002). serpins regulate various physiological processes and molecular defense systems, such as blood coagulation, fibrinolysis, inflammation and complement activation in mammals (gooptu and lomas, 2009). to date, four drosophila serpins are known to be involved in innate immunity (spn43ac, spn27a, spn77ba and spn28d) and have been extensively studied using a genetic approach. spn43ac mutant flies accumulate cleaved spätzle, resulting in the constitutive activation of the toll signaling pathway and expression of amps (levashina et al., 1999). spn27a and spn28d regulate the toll pathway during early development (hashimoto et al., 2003; ligoxygakis et al., 2003; scherfer et al., 2008) and are involved in the melanin biosynthesis.(de gregorio et al., 2002; ligoxygakis et al., 2002b) another serpin, spn77ba, was identified as a negative regulator of melanization in the drosophila respiratory system (the trachea) (tang et al., 2008). in manduca system, kanost’s group reported elegant results regarding serpin splicing: 12 different copies of manduca serpin 1 undergo mutually exclusive alternative splicing to produce 12 putative protein isoforms, which differ in their carboxyl-terminal 3946 residues including the p1 residue. these serpins inhibited manduca sps with different specificities (jiang and kanost, 1997; ragan et al., 2010). these serpins were characterized and suggested as negative regulators of the pro-phenoloxidase and toll signaling cascades (kanost et al., 2004). however, molecular regulatory mechanisms of how serpins regulate invertebrate’s innate immune responses are not well understood due to the uncertainty of the identity of the target sps by the serpins. we purified three novel serpins (spn40, spn55 and spn48) from the hemolymph of t. molitor (jiang et al., 2009) (fig. 2). these tenebrio serpins made specific serpin-sp complexes with three toll signaling cascade-activating sps, such as msp, sae, and spe and cooperatively blocked tenebrio toll signaling cascade and β-1,3-glucanmediated melanin biosynthesis. also, the protein expression levels of spn40 and spn55 were dramatically increased in vivo by the injection of a toll ligand, processed spätzle, into tenebrio larvae. this increase in spn40 and spn55 protein levels indicates that these two serpins function as inducible negative feedback inhibitors. surprisingly, tenebrio spn55 and spn48 were cleaved after tyr and glu residues of reactive center loops, respectively, despite being targeted by trypsin-like sae and spe proteases. these unexpected cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, indicating that they may contribute to highly specific and timely inactivation of detrimental sps during innate immune responses. taken together, our results showed the specific regulatory mechanisms of innate immune responses by three tenebrio novel serpins. a balance between activation and inhibition of the sp-mediated innate immune response must be maintained to avoid damage to the host (ferrandon et al., 2007). as described above, tenebrio three serpins targeting three toll cascade-activating sps act as negative regulators of toll signaling, indicating that each sp in a cascade may be regulated by a specific serpin. this is first biochemical evidences that sp-serpin pairs directly regulate the pattern recognition protein-dependent toll signaling cascade. to date, several cascade reactions including drosophila toll signaling cascade have been hypothesized to be regulated by a single “bottleneck” protease. however, our data show that the control of tenebrio toll proteolytic signaling cascades is more precisely regulated. furthermore, we have demonstrated that spn40 and spn55 function as inducible negative feedback regulators in vivo. these results, in combination with our other reports (kan et al., 2008; kim et al., 2008; roh et al., 2009) support a model in which the lys-type pg and β-1,3-glucan-dependent toll signaling and prophenoloxidase cascades are negatively regulated by three endogenous serpins (fig. 2). finally, our studies highlight the elaborate regulatory mechanism of invertebrate innate immune defense systems. conclusion the developments of genetics and molecular biology enable us to screen the drosophila mutants that had deficient immune responses against bacterial and fungal infection, subsequently to discover two conserved innate immune signaling cascades-toll and imd-both of which lead to the activation of nuclear factor kb (nf-kb) transcription factors (lemaitre and hoffmann, 2007). in this review, the activation and regulation mechanisms of toll cascade were discussed. however, molecular mechanism by microbe-derived protease-mediated toll cascade (so called danger signaling pathway) is not determined yet. the exact molecular activation and regulation mechanism of microbial proteasemediated toll pathway should be determined, such as how pro-persephone is cleaved by microbial proteinase and identity of tenebrio or manduca counter part protease of drosophila persephone proteinase, and whether really active form of 188 persephone specifically cleave pro-spe for the activation of spätzle in tenebrio or manduca system. also, dap-type pg-mediated imd signal pathway is unclear. even though the identities of pattern recognition receptors of drosophila imd pathway and recognition mechanism between tracheal cytotoxin (tct) and pgrp-le or pgrp-lc were determined (chang et al., 2006; lim et al., 2006), tct will not be generated naturally by all gramnegative bacteria and bacilli species. therefore, we should find a natural ligand of imd pathway and also should elucidate the dap-type pg recognition mechanism and extracellular signaling pathway of imd pathway in future. another unexploited area is how mycoplasma bacteria, which are deficient of pg in cell membrane, are recognized in insects. understanding the signaling pathway triggered by mycoplasma and how their recognition activates invertebrate innate immune responses will be big homework for invertebrate immunologist and biochemists. also, further challenging theme in insect immunity is the determination of recognition and activation mechanisms against intracellular pathogenic microbes, such as listeria monocytogenes. recently, kurata’s group nicely discussed the relationship between autophagy and insect innate immunity (yano et al., 2008; kurata, 2010). drosophila pgrp-lc and -le are known to sense the dap-type pg of extracellular and intracellular-infective bacteria and then to induce several innate immune responses, such as amp production, via activation of imd pathway. yano et al observed pgrp-le-dependent autophagy when drosophila adults were infected with l. monocytogenes, which is independent of the imd pathway (yano et al., 2008). it will be worthwhile to investigate the identification of agonist molecule that induces pgrp-le-mediated autophagy and to determine the molecular mechanism of dap-type pg-dependent autophagy. acknowledgements this work was supported for two years by pusan national university research grant. references an c, ishibashi j, ragan ej, jiang h, kanost mr. functions of manduca sexta hemolymph proteinases hp6 and hp8 in two innate immune pathways. j. biol. chem. 284: 1971619726, 2009. buchon n, poidevin m, kwon hm, guillou a, sottas v, lee bl, et al. a single modular serine protease integrates signals from patternrecognition receptors upstream of the drosophila toll pathway. proc. natl. acad. sci. usa 106: 12442-12447, 2009. bulet p, cociancich s, dimarcq jl, lambert j, reichhart jm., hoffmann d, et al, insect immunity. isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family. j. biol. chem. 266: 24520-24525, 1991. bulet p, hetru c, dimarcq jl, hoffmann d. antimicrobial peptides in insects; structure and function. dev. comp. immunol. 23: 329-344, 1999. cerenius l, kawabata si, lee bl, nonaka m, söderhäll k. proteolytic cascades and their involvement in invertebrate immunity. trends. biochem. sci., 2010 [in press]. cerenius l, lee bl, söderhäll k. the proposystem: pros and cons for its role in invertebrate immunity. trends immunol. 29: 263-271, 2008. chang ci, chelliah y, borek d, mengin-lecreulx d, deisenhofer j. structure of tracheal cytotoxin in complex with a heterodimeric patternrecognition receptor. science 311: 1761-1764, 2006. de gregorio e, han sj, lee wj, baek mj, osaki t, kawabata s, et al. an immune-responsive serpin regulates the melanization cascade in drosophila. dev. cell 3: 581-592, 2002. ferrandon d, imler jl, hetru c, hoffmann ja. the drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. nat. rev. immunol. 7: 862874, 2007. gay nj, gangloff m. structure and function of toll receptors and their ligands. annu. rev. biochem. 76: 141-165, 2007. gettins pg. serpin structure, mechanism, and function. chem. rev. 102: 4751-4804, 2002. gobert v, gottar m, matskevich aa, rutschmann s, royet j, belvin m, et al. dual activation of the drosophila toll pathway by two pattern recognition receptors. science 302: 2126-2130, 2003. gooptu b, lomas da. conformational pathology of the serpins: themes, variations, and therapeutic strategies. annu. rev. biochem. 78: 147-176, 2009. gottar m, gobert v, matskevich aa, reichhart jm, wang c, butt tm, et al. dual detection of fungal infections in drosophila via recognition of glucans and sensing of virulence factors. cell 127: 1425-1437, 2006. hashimoto c, kim dr, weiss la, miller jw, morisato d. spatial regulation of developmental signaling by a serpin. dev. cell 5: 945-950, 2003. hedengren m, asling b, dushay ms, ando i, ekengren s, wihlborg m, et al. relish, a central factor in the control of humoral but not cellular immunity in drosophila. mol. cell 4: 827-837, 1999. hoffmann ja, kafatos fc, janeway ca, ezekowitz ra. phylogenetic perspectives in innate immunity. science 284: 1313-1318, 1999. jang ih, chosa n, kim sh, nam hj, lemaitre b, ochiai m, et al. a spatzle-processing enzyme required for toll signaling activation in drosophila innate immunity. dev. cell 10: 45-55, 2006. ji c, wang y, guo x, hartson s, jiang h. a pattern recognition serine proteinase triggers the prophenoloxidase activation cascade in the tobacco hornworm, manduca sexta. j. biol. chem. 279: 34101-34106, 2004. jiang h, kanost mr. characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from manduca 189 sexta. j. biol. chem. 272: 1082-1087, 1997. jiang r, kim eh, gong jh, kwon hm, kim ch, ryu kh, et al. three pairs of protease-serpin complexes cooperatively regulate the insect innate immune responses. j. biol. chem. 284: 35652-35658, 2009. kambris z, brun s, jang ih, nam hj, romeo y, takahashi k, et al. drosophila immunity: a large-scale in vivo rnai screen identifies five serine proteases required for toll activation. curr. biol. 16: 808-813, 2006. kan h, kim ch, kwon hm, park jw, roh kb, lee h, et al. molecular control of phenoloxidaseinduced melanin synthesis in an insect. j. biol. chem. 283: 25316-25323, 2008. kang d, liu g, lundstrom a, gelius e, steiner h. a peptidoglycan recognition protein in innate immunity conserved from insects to humans. proc. natl. acad. sci. usa 95:10078-10082, 1998. kanost mr, jiang h, yu xq. innate immune responses of a lepidopteran insect, manduca sexta. immunol. rev. 198: 97-105, 2004. kim ch, kim sj, kan h, kwon hm, roh kb, jiang r, et al. a three-step proteolytic cascade mediates the activation of the peptidoglycaninduced toll pathway in an insect. j. biol. chem. 283: 7599-7607, 2008. kurata s. extracellular and intracellular pathogen recognition by drosophila pgrp-le and pgrp-lc. int. immunol. 22: 143-148, 2010. lee wj, lee jd, kravchenko vv, ulevitch rj, brey pt. purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, bombyx mori. proc. natl. acad. sci. usa 93: 7888-7893, 1996. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. lemaitre b, nicolas e, michaut l, reichhart, jm, hoffmann ja. the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults. cell 86: 973-983, 1996. levashina ea, langley e, green c, gubb d, ashburner m, hoffmann ja, et al. constitutive activation of toll-mediated antifungal defense in serpin-deficient drosophila. science 285: 19171919, 1999. ligoxygakis p, pelte n, hoffmann ja, reichhart jm. activation of drosophila toll during fungal infection by a blood serine protease. science 297: 114-116, 2002a. ligoxygakis p, pelte n, ji c, leclerc v, duvic b, belvin m, et al. a serpin mutant links toll activation to melanization in the host defence of drosophila. embo j. 21: 6330-6337, 2002b. ligoxygakis p, roth s, reichhart jm. a serpin regulates dorsal-ventral axis formation in the drosophila embryo. curr. biol. 13: 2097-2102, 2003. lim jh, kim ms, kim he, yano t, oshima y, aggarwal k, et al. structural basis for preferential recognition of diaminopimelic acidtype peptidoglycan by a subset of peptidoglycan recognition proteins. j. biol. chem. 281: 8286-8295, 2006. ma c, kanost mr. a beta1,3-glucan recognition protein from an insect, manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade. j. biol. chem. 275: 7505-7514, 2000. matsushita m, fujita t. activation of the classical complement pathway by mannose-binding protein in association with a novel c1s-like serine protease. j. exp. med. 176: 1497-1502, 1992. medzhitov r, janeway ca. innate immunity: the virtues of a nonclonal system of recognition. cell 91: 295-298, 1997. mellroth p, karlsson j, steiner h. a scavenger function for a drosophila peptidoglycan recognition protein. j. biol. chem. 278: 70597064, 2003. michel t, reichhart jm, hoffmann ja, royet j. drosophila toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. nature 414: 756-759, 2001. moon hj, lee sy, kurata s, natori s, lee bl. purification and molecular cloning of cdna for an inducible antibacterial protein from larvae of the coleopteran, tenebrio molitor. j. biochem. 116: 53-58, 1994. ochiai m, ashida m. a pattern-recognition protein for beta-1,3-glucan. the binding domain and the cdna cloning of beta-1,3-glucan recognition protein from the silkworm, bombyx mori. j. biol. chem. 275: 4995-5002, 2000. park jw, je br, piao s, inamura s, fujimoto y, fukase k, et al. a synthetic peptidoglycan fragment as a competitive inhibitor of the melanization cascade. j. biol. chem. 281: 7747-7755, 2006. park jw, kim ch, kim jh, je br, roh kb, kim sj, et al. clustering of peptidoglycan recognition protein-sa is required for sensing lysine-type peptidoglycan in insects. proc. natl. acad. sci. usa 104: 6602-6607, 2007. piao s, song yl, kim jh, park sy, park jw, lee bl, et al. crystal structure of a clip-domain serine protease and functional roles of the clip domains. embo j. 24: 4404-4414, 2005. pili-floury s, leulier f, takahashi k, saigo k, samain e, ueda r, et al. in vivo rna interference analysis reveals an unexpected role for gnbp1 in the defense against grampositive bacterial infection in drosophila adults. j. biol. chem. 279: 12848-12853, 2004. ragan ej, an c, yang ct, kanost mr. analysis of mutually-exclusive alternatively spliced serpin-1 isoforms and identification of serpin-1 proteinase complexes in manduca sexta hemolymph. j. biol. chem. 2010 [in press]. roh kb, kim ch, lee h, kwon hm, park jw, ryu jh, et al. proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component. j. biol. chem. 284: 1947419481, 2009. scherfer c, tang h, kambris z, lhocine n, hashimoto c, lemaitre b. drosophila serpin-28d regulates hemolymph phenoloxidase activity and adult pigmentation. dev. biol. 323; 189-196, 2008. 190 shia ak, glittenberg m, thompson g, weber an, reichhart jm, ligoxygakis p. toll-dependent antimicrobial responses in drosophila larval fat body require spatzle secreted by haemocytes. j. cell. sci. 122: 4505-4515, 2009. and establishes signaling. nat. immunol. 4: 794-800, 2003. yano t, mita s, ohmori h, oshima y, fujimoto y, ueda r, et al. autophagic control of listeria through intracellular innate immune recognition in drosophila. nat. immunol. 9: 908-916, 2008. tang h, kambris z, lemaitre b, hashimoto c. a serpin that regulates immune melanization in the respiratory system of drosophila. dev. cell 15: 617-626, 2008. wang y, jiang h. reconstitution of a branch of the manduca sexta prophenoloxidase activation cascade in vitro: snake-like hemolymph proteinase 21 (hp21) cleaved by hp14 activates prophenoloxidase-activating proteinase-2 precursor. insect biochem. mol. biol. 37: 1015-1025, 2007. yoshida h, kinoshita k, ashida m. purification of a peptidoglycan recognition protein from hemolymph of the silkworm, bombyx mori. j. biol. chem. 271: 13854-13860, 1996. yoshida h, ochiai m, ashida m. beta-1,3-glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system. biochem. biophys. res. commun. 141: 1177-1184, 1996. wang y, jiang h. a positive feedback mechanism in the manduca sexta prophenoloxidase activation system. insect biochem. mol. biol. 38: 763-769, 2008. zhang r, cho hy, kim hs, ma yg, osaki t, kawabata s, et al. characterization and properties of a 1,3-beta-d-glucan pattern recognition protein of tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation. j. biol. chem. 278: 42072-42079, 2003. weber an, tauszig-delamasure s, hoffmann ja, lelievre e, gascan h, ray kp, et al. binding of the drosophila cytokine spatzle to toll is direct 191 apoptosis in molluscan immune defense isj 6: 49-58, 2009 issn 1824-307x review apoptosis in molluscan immune defense im sokolova department of biology, university of north carolina at charlotte, charlotte nc, usa accepted april 20, 2009 abstract apoptosis, or type i programmed cell death, is a fundamental biological process involved in cellular homeostasis in metazoans. apoptosis plays a key role in immune system homeostasis and function, defense against parasite and pathogens and self/non-self recognition. in this review, i present our current knowledge of the mechanisms and signaling pathways underlying apoptosis in mollusks and its roles in host-pathogen interactions and immune defense. both signaling and execution pathways of apoptosis appear to be highly conserved in mollusks, although there is evidence that some apoptotic mechanisms (e.g., caspase-independent cell death) may differ from model invertebrates such as caenorhabditis and drosophila and more closely resemble those seen in vertebrates. apoptosis is important for the functioning of the molluscan immune system as indicated by the high baseline apoptosis rates observed in circulating and resident hemocytes. apoptosis also plays a role in host protection against parasites by limiting the spread of the pathogen while preventing inflammatory damage of surrounding tissues. in molluscs, interaction between immune cells and parasites or pathogens usually triggers apoptosis; however, some pathogens (especially obligatory intracellular parasites that depend on the host cell for their survival and proliferation) can inhibit this response and prevent host cell death. currently, the mechanisms underlying pathogen-induced modulation of apoptosis in molluscs are not well understood. summarizing our current knowledge of the immune functions of apoptosis in molluscs, and comparing them with the more widely studied vertebrate systems, this review will delineate the gaps in our understanding of this critical cellular process in molluscs and assist in the ongoing search for evolutionary novel mechanisms of apoptosis in immune defense and self/non-self-recognition. key words: immune response; apoptosis; parasites; pathogens; mollusca introduction molluscs, the second most diverse group of animals (next only to arthropods) with about 93,000 extant species, are abundant in most marine, brackish, freshwater and terrestrial habitats. molluscs play vital roles in these ecosystems and are often used as bioindicators of the ecosystem health. in some ecosystems (most notably in marine and estuarine habitats), molluscs can function as ecosystem engineers creating and modifying habitats for other species and affecting the habitat quality by eliminating suspended particles or participating in the top-down control of algal blooms. molluscs have also been used by humans since the dawn of the human history as food resources or to ___________________________________________________________________________ corresponding author: inna m sokolova department of biology university of north carolina at charlotte 9201 university city blvd., charlotte nc, 28223 usa e-mail: isokolov@uncc.edu provide other valuable commodities such as pearls and mother-of-pearl, tyrian purple from muricid gastropods used to dye royal robes, non-opioid pain-killers derived from cone snails’ toxins and anticancer drugs from some gastropod peptides (mcfadden et al., 2003; faircloth and cuevas, 2006; han et al., 2008). moreover, molluscs serve as important vectors in the transmission of parasites and pathogens to humans and/or domestic animals. given the important ecological, economical and medicinal roles of molluscs, understanding the immune defenses of these animals is critical for improving disease resistance and increasing the survival of ecologically and economically important molluscs, and for improving their aquaculture production and/or sanitation to reduce parasite/pathogen transmission. studies of the molluscan immunity are also important from the viewpoint of evolutionary and comparative immunology. the study of the immune defense of molluscs may reveal immunobiological novelties 49 mailto:isokolov@uncc.edu thereby providing insights into the evolution of immunity, and may offer new solutions for the treatment of infectious and parasitic diseases or immunological disorders in humans and domestic animals. although molluscan immunity has been actively studied in the past few decades, our knowledge about the cellular and molecular mechanisms of molluscan immune defenses and their interactions with pathogens and parasites is still far from complete. significant progress has been made in our understanding of the phagocytic and bactericidal responses of molluscan immune cells and of the humoral factors involved in non-self recognition and pathogen killing; due to space limitations, i will only briefly mention these immune mechanisms in this review (see “overview of the molluscan immunity” below) and further refer the reader to a number of excellent reviews focusing on these topics (see anderson, 1996; kennedy et al., 1996; glinski and jarosz, 1997; canesi et al., 2002; paul, 2003; tiscar and mosca, 2004; vasta and ahmed, 2008 and references therein). the main goal of the present review is to draw attention of invertebrate immunologists to an important but currently understudied aspect of molluscan immunity – namely, the role of apoptosis, or type i programmed cell death (pcd i), in molluscan immune defense and host-pathogen interactions. given that the study of molluscan apoptosis is still in its infancy, i will draw on relevant examples from vertebrate literature to illustrate molecular mechanisms and possible immunological roles of apoptosis in protection against parasites, pathogens, and tumors in mollusks. it is my hope that by providing a comprehensive overview of our current knowledge of molluscan apoptosis, this review will provide a roadmap for future studies to understand this critical cellular process in mollusks and to potentially reveal novel mechanisms and roles of apoptosis in the immune defense and self/non-self-recognition in animals. overview of molluscan immunity the immune system of molluscs, as all other invertebrates, consists only of innate immunity and is lacking the adaptive immunity components. while the innate immune system is often regarded as an evolutionary more ancient and hence more primitive form of immunity than the adaptive responses seen in vertebrates, it is actually a surprisingly complex and efficient form of protection against many parasites and pathogens encountered by molluscs. indeed, successful adaptive radiation of molluscs and their ability to colonize a broad range of aquatic and terrestrial habitats clearly speaks to their ability to efficiently combat infections and parasitic invasions. external barriers (such as shells, mucus and epithelia) of mollusks constitute the first line of defense against pathogens and parasites; when these barriers are breached, the second, internal line of defense involving cellular and soluble (humoral) hemolymph components come into play. humoral components of the molluscan immune defense include lysosomal enzymes (such as βglucuronidase, acid and alkaline phosphatase, lipase, aminopeptidase and lysozyme), lectins including agglutinins, fibrinogen-related proteins (freps), and c-type lectins, and antimicrobial peptides that aid in recognition of pathogens and parasites by marking them for destruction via opsonizing or directly killing (review in anderson, 1996; kennedy et al., 1996; canesi et al., 2002; paul, 2003; tiscar and mosca, 2004; vasta and ahmed, 2008). agglutinins, freps and other lectins along with pattern recognition receptors (prrs, which in mollusks include toll-like receptors, imd pathway receptors and intracellular nucleotideoligomerization domain (nod) proteins) are responsible for the surprising degree of specificity shown by the innate immune system for pathogens and parasites (paul, 2003; yeretssian et al., 2008). fig. 1 transmission electron micrograph (tem) of resident hemocytes in gills (a) and hepatopancreas (b) of oysters c. virginica. hemocytes, h. 50 while humoral immunity is very important for host defense, it is indisputable that the cellular components of the hemolymph, hemocytes, play a central role in the innate immune responses of mollusks (fig. 1). hemocytes are the main effector components of the molluscan immune system and are responsible for phagocytosis of parasites, pathogens, and foreign particles (which represents a complex process including recognition, adhesion, ingestion, destruction or encapsulation and final elimination of foreign cells or materials). hemocytes are also directly engaged in pathogen and parasite killing via the oxidative burst reaction a rapid generation of toxic reactive oxygen species (ros) by a membrane bound enzyme nadh-oxidase. ros (especially hydrogen peroxide) can be further converted by hemocyte myeloperoxidase to hypochloric acid (hocl) that has strong bactericidal and viricidal properties (tiscar and mosca, 2004). molluscan hemocytes are comprised of two main types: hyalinocytes small cells with cytoplasm containing few or no granules, and granulocytes large phagocytic cells containing abundant numbers of cytoplasmic granules (kennedy et al., 1996; tiscar and mosca, 2004). these two major cell types are further classified into subtypes based on their size, morphology and (to the degree that it is known) function (e.g., acidophilic, basophilic and neutrophilic granulocytes, natural killer-like cells, etc.) (kennedy et al., 1996; takahashi and mori, 2000). overall, molluscan granulocytes strongly resemble vertebrate monocytes and macrophages both in structure and function and share with the latter such key properties as avid phagocytosis, pathogen-induced oxidative burst, production and release of nitric oxide and lysosomal enzymes (canesi et al., 2002). recent studies have revealed subpopulations of molluscan hemocytes that possess lymphocyte-like morphology and physiological properties similar to mammalian natural killer (nk) cells (monti et al., 1992). molecular mechanisms of apoptosis in molluscs the term “apoptosis” was coined by kerr, wyllie and curie in their seminal 1972 paper to describe a distinctive form of programmed cell death (pcd) defined by characteristic morphological features such as chromatin condensation, membrane blebbing and cell shrinkage (kerr et al., 1972 cited after feig and peter, 2007). since then, the study of apoptosis using both vertebrate and invertebrate model systems has led to the recognition that this is a multifunctional process, highly conserved evolutionarily, and plays a critical role in cellular and tissue homeostasis, embryonic development, and immune defense in multicellular organisms. unlike necrosis that may be considered as “accidental” cell death in response to major mechanical or chemical injuries, apoptosis is a highly orchestrated cellular process in which intracellular components are sequestered and disposed of in an orderly manner without the induction of inflammation. mechanisms of apoptosis have been based predominantly on the studies of vertebrates and invertebrate models such as caenorhabditis and drosophila. while these findings have been extensively reviewed previously (see reviews in hengartner, 1996, 2000; vermeulen et al., 2005; circu and aw, 2008; ow et al., 2008; suen et al., 2008 and references therein) and their detailed analysis is beyond the scope of the present review, a brief account of the major apoptotic pathways is in order to set the stage for a discussion of the mechanisms underlying molluscan apoptosis. briefly, the apoptotic cell death can be triggered through two major pathways – intrinsic (or mitochondrial), in response to internal cellular damage and extrinsic, in response to death clues received from the environment (fig. 2). although both pathways can function independently, there is substantial cross-talk that allows for amplification of the death signal (feig and peter, 2007). a key characteristic of the majority of apoptotic pathways is the involvement of a family of proteases called caspases that cleave target proteins at specific sites typically containing aspartic acid residues followed by a caspase-specific three amino acid sequence (creagh et al., 2003). caspases involved in apoptosis include initiator caspases (e.g., caspases 2, 8, 9 and 10) that cleave and activate the effector caspases (e.g., caspase 3, 6 and 7), which in turn act upon intracellular targets ranging from cytoskeleton proteins to specific cell structures such as mitochondria, nuclear lamina or chromatin (hengartner 2000; creagh et al., 2003). both extrinsic and intrinsic apoptotic pathways converge on the activation of caspases although it should be noted that caspase-independent apoptosis can also occur. in vertebrates, a hallmark of the intrinsic apoptotic pathway is a release of cytochrome c from mitochondria that is usually accompanied by the formation of a large-molecular size pore in the mitochondrial membrane (the mitochondrial permeability transition (mpt) pore) and collapse of the mitochondrial membrane potential (mignotte and vayssiere, 1998). factors that bring about activation of apoptosis via the mitochondrial pathway include dna damage, oxidative damage of mitochondrial or cytosolic proteins and lipids, viral stimulation and/or removal of pro-mitogenic signals (growth factor withdrawal) (circu and aw, 2008). once in the cytoplasm, cytochrome c interacts with apoptotic peptidase activating factor 1 (apaf-1), procaspase-9 and datp to form a complex called the apoptosome that activates the initiator caspase 9, which in turn activates the effector molecule, caspase 3. caspase 3 activation causes degradation of proteins, dna and other cellular components culminating in cell death (zimmermann et al., 2001). mitochondrial permeability transition and cytochrome c release are tightly regulated by the bcl-2 family of proteins that includes both proor anti-apoptotic members (circu and aw, 2008). the extrinsic pathway of apoptosis is initiated via binding of specific protein ligands to so called death receptors on the cell surface. in vertebrates, six major types of death receptors have been described including fas, trail (tnf-related apoptosis inducing ligand) receptor -1 and -2, tumor necrosis factor (tnf) receptor-1, tnf receptorrelated apoptosis-mediating protein (tramp), and death receptor-6 (dr6). activation of death receptors 51 fig. 2 schematic representation of major apoptotic signaling and execution pathways. extrinsic (death receptoractivated) pathways are shown on the right, and intrinsic (mitochondrial) including caspase-dependent and caspase-independent pathways are shown on the left of the diagram. note considerable cross-talk between the pathways. arrows indicate activation, and lines with black dots inhibition. red circles with the letter “m” indicate parts of the pathways that have been demonstrated in molluscs. abbreviations: permeability transition pore, ptp; cytochrome c, cyt c; procaspase, pro-casp; fas-associated death domain, fadd; apoptotic peptidase activating factor 1, apaf-1; apoptosis inducing factor, aif; inhibitor of apoptosis family of proteins, iap. causes rapid formation of a death-inducing signaling complex (disc) that initiates a cascade of caspase activation (notably initiated by the activation of caspase 8) leading to induction of apoptosis (zimmermann et al., 2001; schultz and harringto, 2003). homologues of death receptors and fasl have been also found in the genome of an ascidian ciona intestinalis (dehal et al., 2002; terajima et al., 2003). although previous studies have isolated several proteins containing death domains from other invertebrates, there are no unequivocal death receptor orthologs reported in drosophila, c. elegans or porifera genomes despite concerted efforts to find them (muzio, 1998; bridgham et al., 2003). this suggests that death receptor-mediated apoptosis may have evolved within the chordate lineage and may not be functional in non-chordate invertebrates. despite the central role of caspases in the intrinsic and extrinsic apoptotic pathways, caspase activation is not an absolute requirement for the induction of programmed cell death. the most prominent caspase-independent death effector molecules are apoptosis-inducing factor (aif), a phylogenetically conserved mitochondrial flavoprotein, and endonuclease g, a mitochondrionspecific nuclease (van loo et al., 2001; li et al., 2001; cande et al., 2002; tait and green, 2008). endonuclease g and aif are released from mitochondria upon a death signal and translocate directly into the nucleus where they bind to dna and induce caspase-independent chromatin condensation and dna fragmentation leading to cell death (van loo et al., 2001; li et al., 2001; cande et al., 2002). some non-caspase proteases such as cathepsins, calpains and serine proteases like granzyme a/b and omi/htr a can also trigger caspase-independent apoptosis (vermeulen et al., 2005). overall, the considerable redundancy (i.e. potential triggering of the multiple apoptotic pathways by a single stimulus), cross-talk, and mutual amplification between caspase-dependent 52 and -independent pathways of apoptosis stresses the critical importance of this cellular process in metazoans; it appears that once the death signal has been received, apoptosis proceeds at all costs to eliminate the damaged, malignant, infected or otherwise dangerous cells. programmed cell death with all the characteristic hallmarks of apoptosis including cell shrinkage and blebbing, chromatin condensation, dna fragmentation and translocation of a phospholipid phosphatydilserine into the outer leaflet of the cell membrane, have been described in a variety of mollusks (sunila and labanca, 2003; sokolova et al., 2004; buckland-nicks and tompkins, 2005). moreover, many of the molecular components of apoptotic cellular machinery have also been found in mollusks and appear to be highly structurally and functionally conserved. molluscan cells (including hemocytes) possess caspase 3-like activity (pirger et al., 2008), and similar to vertebrates, molluscan caspase 3 can be activated by cytochrome c and datp (sokolova et al., 2004). molluscan caspase-3 activity can also be specifically stimulated by staurosporin although, unlike in mammals, the mechanism of this activation in mollusks does not involve the activating cleavage of pro-caspase 3 (bravarenko et al., 2006). recent studies have demonstrated that the signaling pathways involved in the regulation of molluscan apoptosis share significant molecular and functional similarity with those seen in vertebrates. two central players in cell death and survival, tnfα and nf-κb transcription factors, appear to play a key role in cell signaling in mollusks. molluscan tnf-α shares considerable molecular and functional similarity with vertebrate homologs (terahara and takahashi, 2008). for example, its expression increases in response to bacterial infections and results in the induction of apoptosis in molluscan hemocytes (terahara and takahashi, 2008). in contrast to tnf-α, nf-κb transcription factor typically exhibits pro-survival, anti-apoptotic effects in vertebrates (aggarwal, 2004; dey et al., 2008). in mollusks, all the key components of nf-κb signaling cascade have been described and bear considerable similarity to the mammalian nf-κb signaling pathway (ouwe-missi-oukem-boyer et al., 1994; montagnani et al., 2004; zhu and wu, 2008). however, the exact role of nf-κb in molluscan apoptosis has not been studied and requires further investigation. many downstream elements of apoptotic signaling cascades show significant similarity between molluscan and vertebrate cell death. thus, similar to mammals, mitogen-activated protein kinases and rho, a member of the ras gtpase family, are involved in regulation of molluscan apoptosis and exhibit anti-apoptotic effects in molluscan hemocytes (lacoste et al., 2002). protein kinase a (but not protein kinase c) also appears to be involved in molluscan hemocyte apoptosis; in the case of noradrenaline-induced apoptosis, inhibition of pka but not pkc activity attenuated apoptosis levels (lacoste et al., 2002). another potential key effector of molluscan apoptosis is cyclic amp (camp); however, its role may be proor antiapoptotic, depending on the circumstances. for example, in oyster (crassostrea gigas) hemocytes, camp appears to promote apoptosis since the adenylate cyclase inhibitor 2’, 5’-dideoxyadenosine (dda) reduces levels of noradrenaline-induced pcd i (lacoste et al., 2002). in contrast, elevated levels of camp induced by pituitary adenylate cyclase activating polypeptide (pacap) have antiapoptotic effects in terrestrial snails (helix pomatia), significantly attenuating dopamineand colchicineinduced apoptosis in the salivary gland (pirger et al., 2008). further research is needed to determine whether these disparate effects of camp are speciesor cell type-specific, or whether they are differentially expressed depending on the physiological condition of an organism and/or the nature of the stimulus. a tumor suppressor p53 homolog also plays an important role in apoptosis signaling in mollusks. in normal mammalian cells, p53 suppresses the formation of tumors by arresting the cell cycle or by apoptosis in response to genotoxic stress-induced dna damage (böttger et al., 2008). apoptosis can be induced by p53 either via translocation into the nucleus where it upregulates transcription of proapoptotic genes or by translocation to the mitochondria where it binds to and inactivates bcl-2 and other antiapoptotic proteins (böttger et al., 2008). in clams (mya arenaria), p53 protein shares significant sequence and structural similarity to human p53 and localizes to the nucleus in response to apoptotic signals (holbrook et al., 2009). overexpression of mortalins (hsp70 family proteins) that bind p53 and prevent its translocation to the nucleus has been shown to inhibit the expression of pro-apoptotic p53-dependent genes and decrease levels of apoptosis in molluscan cells in a similar manner to that seen in vertebrate species (böttger et al., 2008). there is also evidence that the mitochondrial pathway of p53dependent apoptosis activation is functional in mollusks (böttger et al., 2008) but further studies are needed to confirm this suggestion. nitric oxide (no) is another signaling molecule that is involved in the regulation of apoptosis. the effects of no on apoptosis vary greatly depending upon the dose of no used, the cell type, and the physiological status of the cell, and can be either proor anti-apoptotic (brune et al., 1999). molluscan cells including hemocytes contain nitric oxide synthase and produce no (terahara and takahashi, 2008). however, the role of no in apoptotic regulation has not been extensively studied in mollusks. the only documented study on the effects of no on molluscan apoptosis shows that inhibition of nitric oxide synthase (nos) activity during ilyanassa obsoleta larvae metamorphosis induces apical ganglion cell apoptosis (gifondorwa and leise, 2006). in molluscan hemocytes, no production is elevated following exposure to parasites or pathogens, or following exposure to the cytokine, il-2 (barsia and ramos-martinez, 2008; terahara and takahashi, 2008), but it is not known whether this induction has an effect on the level of apoptosis in these cells or if yes, whether it involves a down-stream caspase activation. overall, the exact mechanisms and roles of no in mollusc cell apoptosis remain to be fully elucidated. 53 in contrast to some of the highly conserved elements of the caspase-dependent apoptotic machinery, little is known about the caspaseindependent apoptotic pathway in mollusks although its existence has been proposed. this is based on the observation that cadmium (cd)-induced apoptosis in molluscan hemocytes occurs in the absence of mitochondrial permeability transition or caspase 3 activation (sokolova et al., 2004). a recent study from our laboratory has also shown that a pancaspase inhibitor, z-val-ala-asp-fluoromethylketone (z-vad-fmk), fails to prevent apoptosis of crassostrea virginica hemocytes following infection with the intracellular parasite perkinsus marinus, suggesting the involvement of caspase-independent pathways (grewal and sokolova, unpublished data). in mammalian cells, caspase-independent apoptosis induced by trace metals (mn2+ and cd2+) or a toxic plant metabolite (allicin) is associated with protein kinase a-dependent increases in the expression and nuclear translocation of aif in the absence of caspase 3 activation or poly(adp-ribose) polymerase (parp) cleavage (ouabrahim et al., 2001; shih et al., 2003; park et al., 2005). the molecular mechanisms underlying caspaseindependent apoptosis in molluscs are not known and their determination would be an exciting avenue for future investigations. notably, a partial gene sequence of an aif homolog isolated from oysters c. virginica has been recently published in marine genomics database (www.marinegenomics.org, accession # mgid89694) that has significant similarity at the protein level to vertebrate aifs (55 % amino acid identity, e=10-74-10-72 by blastx, altschul et al., 1997). studies of caspaseindependent pcd i in molluscs hold an especially high promise of novelty because current information from model invertebrates (drosophila and caenorhabditis) suggest that caspase-independent apoptosis does not occur in these organisms (tait and green, 2008) raising a possibility that this pathway was either lost to ecdysozoa, or evolved independently in lophotrochozoa (including molluscs) and vertebrates ancestors. exposure to environmental stressors, such as high temperatures, changes in salinity, and the presence of pollutants has been shown to induce apoptosis in molluscan cells, presumably via the intrinsic pathway. for example, high salinities result in elevated levels of oyster (c. virginica) hemocyte apoptosis (goedken et al., 2005). similarly, exposure to elevated temperatures (28 ºc) stimulate apoptosis in oyster hemocytes whereas moderate temperature changes in the near-optimum range (between 10 and 25 ºc) has no effect (goedken et al., 2005; cherkasov et al., 2007). pollutants such as heavy metals, tributyltin (tbt), and pah, can also induce apoptosis in molluscan immune and non-immune cells, with the degree of induction depending on the dose and time of exposure to the pollutants, and on the exposure mode (in vitro or in vivo) (barsiene et al., 2008). given that most of the apoptosis-inducing environmental stressors described above are also known to induce elevated ros production and oxidative stress in mollusks (pruski and dixon, 2002; abele et al., 2002; lannig et al., 2006), it is reasonable to suggest that this stress-induced apoptotic cell death could be triggered by oxidative damage. however, the precise molecular mechanisms responsible for stress-induced apoptosis in mollusks are presently not known and require further study. apoptosis in molluscan cells can also be triggered by activation of surface receptors including hormone receptors and integrins. in oysters (c. gigas), activation of integrins (either by specific ligands or by integrin-binding rgd (arg-gly-asp)containing peptides) promotes apoptosis in a similar manner to that seen in mammalian neutrophils (terahara and takahashi, 2008). notably, unlike mammalian cells where rgd-induced apoptosis is due to anoikis (i.e. prevention of attachment of anchorage-dependent cells to extracellular matrix), the ability of rgd-containing peptide to induce apoptosis in non-adherent, adherent, and spreading molluscan hemocytes alike, indicates that the signaling pathways engaged by integrin activation are independent of hemocyte attachment (terahara and takahashi, 2008). finally, noradrenaline, a catecholamine produced by the neuroendocrine system and by the immune cells of mollusks, also can induce apoptosis of molluscan hemocytes via βadrenergic signaling pathways (lacoste et al., 2002). overall, current data suggests that the mechanisms underlying apoptosis in mollusks (to the degree that they are known) closely resemble those seen in vertebrates. this includes similarity of the major biochemical and molecular steps of apoptotic pathways, as well as redundancy and flexibility of the apoptotic program that can switch between caspase-dependent and -independent pathways in response to specific environmental or developmental stimuli. moreover, there is also evidence that some of the apoptotic pathways in mollusks may be sufficiently divergent from other model invertebrates (such as drosophila and caenorhabditis) so as to warrant further study in the hopes that the determination of the specific regulatory and execution pathways of molluscan apoptosis will discover novel mechanisms of pcd and shed light on the evolution of this crucial cellular process in metazoans. apoptosis as an immunomodulatory mechanism apoptosis has increasingly become a focus of study for biomedical immunologists with the discovery of the immunomodulatory and defense roles of apoptotic cell death and the recognition that high levels of apoptosis are essential for the normal functioning of immune system (hildeman et al., 2007; feig anf peter, 2007; birge and ucker, 2008). in molluscs and other non-model invertebrates, studies of the roles of apoptosis in immunity have been hampered by the scarcity of genetic information and available molecular tools. however, the high degree of evolutionary conservation seen in the apoptotic pathways between vertebrates and invertebrates provide both research tools and motivation for the students of molluscan immunity to venture into this exciting new field and to study the role of apoptosis in immune defense and hostpathogen relationships in mollusks. 54 http://www.marinegenomics.org/ the importance of cell death in the immune defenses of molluscs has long been proposed, even before the discovery of programmed cell death. thus, in an early paper by michelson (1963), cell death (which at that time was considered to be exclusively due to necrosis) is cited as an important immune defense mechanism of gastropods against a variety of pathological agents including trematodes, protozoa, and bacteria (cited by glinski and jarosz, 1997). the importance of apoptosis in the functioning of the molluscan immune system is reflected by the detection of high baseline apoptosis rates that range from 5 to 25 % in circulating hemocytes and can reach to up to 50 % in infiltrating tissue hemocytes (sunila and labanca, 2003; sokolova et al., 2004; goedken et al., 2005; cherkasov et al., 2007). typically, granulocytes show higher levels of apoptosis than hyalinocytes possibly due to the higher phagocytic and oxidative respiratory burst activity in granulocytes (sunila and labanca, 2003; goedken et al., 2005). apoptosis of immune cells can play an important role in protection against parasites and pathogens by the innate immune system. apoptosis is immunologically silent and does not induce inflammation (birge and ucker, 2008; yeretssian et al., 2008). thus, apoptosis of the infected cells is thought to dampen pathogen spread yet protect the integrity of surrounding tissues by limiting potentially damaging inflammation. recent studies in vertebrate models has shown that some pathogens, especially those obligate intracellular parasites that depend on survival of the host cells for their persistence, have evolved strategies to inhibit apoptotic cell death (review in böttger et al., 2008). inhibition of apoptosis in mammalian cells has been observed following infection with intracellular pathogens including leishmania donovani, trypanosoma spp. and theileria spp. (luder et al., 2001; bruchhaus et al., 2007). this parasite-induced inhibition of apoptosis is associated with decreases in caspase-3 activity, inhibition of pro-apoptotic protein kinase c–mediated c-fos gene expression, tnf-α induction and/or changes in nf-κb expression in host cells (nash et al., 1998; heussler et al., 2001; goebel et al., 2001; aga et al., 2002). bacteria, such as chlamidia, pneumococci, or rikketsia, and viruses can also prevent apoptosis of the infected host cells via interference with nf-κb signaling, caspase activation, or balance of proand anti-apoptotic members of bcl-2 family of proteins (böttger et al., 2008). furthermore, pharmaceutical or genetic inhibition of apoptosis renders hosts more susceptible to intracellular pathogens indicating that apoptosis triggered by these pathogens is protective for the host and could play a beneficial role in eliminating the infection (böttger et al., 2008). in molluscs, induction of apoptosis upon contact with pathogens or parasites, and parasite-induced inhibition of apoptosis have both been described. for example, hemocytes of the pacific oyster (crassostrea gigas) show an induction of apoptosis during phagocytosis of live or heat-killed marine bacteria planococcus citraeus (terahara and takahashi, 2008). elevated levels of apoptosis have been associated with the oxidative burst of hemocytes and are abolished by treatment with antioxidants suggesting that apoptosis may be induced by oxidative damage to hemocytes during bacterial killing (terahara and takahashi, 2008). symbiotic bacteria of some cephalopods (vibrio spp.) can also induce apoptosis in host cells (mcfall-ngai, 1999). importantly, our earlier studies have also shown an early increase in apoptosis of oyster hemocytes upon infection by the intracellular protozoan parasite, perkinsus marinus, which then returns back basal levels, perhaps due to parasiteinduced inhibition of the apoptotic response (fig. 3; sokolova, grewal and hughes, unpublished data). apoptosis levels in resident tissue hemocytes of oysters has also been shown to be dramatically reduced from approximately 50 % to 10-11 % in oysters naturally infected by p. marinus or another obligate intracellular protozoan parasite, haplosporidium nelsoni (sunila and labanca, 2003). notably, infection with p. marinus results in a significantly faster induction of apoptosis in hemocytes derived from p. marinus-resistant pacific oysters (c. gigas) than those from p. marinussusceptible eastern oysters (c. virginica) suggesting that faster induction of apoptosis may be an effective defense mechanisms against this intracellular parasite (goedken at al., 2005). overall, it is apparent that while apoptosis induction and the possible subversion of this response by successful parasites and pathogens may play a critical role in both disease resistance and parasite/pathogen development and transfer, our knowledge about the occurrence of, and mechanisms responsible for, apoptosis in infected mollusks is currently limited. clearly, further studies are urgently needed to determine how specific and wide-spread the apoptotic response to infections is fig. 3 parasite-induced apoptosis in molluscan hemocytes. hemocytes of oysters crassostrea virginica were infected with an intracellular pathogen perkinsus marinus, and tem obtained 30 min postinfection. (hemocyte, h; p. marinus cells, pm). arrows show apoptotic bodies of dying hemocytes. 55 in molluscs, and whether it differs in response to bacterial and viral pathogens, and to uniand multicellular eukaryotic parasites (especially schistosoma and other trematodes). summary and future directions recent advances in the fields of molluscan immunology and fundamental cell biology have brought about significant breakthroughs in our understanding of the mechanisms underlying apoptotic cell death in molluscan cells and, in particular, their immune cells. these studies reveal a high degree of evolutionary conservation of key signaling and execution pathways of apoptosis and indicate that programmed cell death likely plays a key role in homeostasis and functioning of the molluscan immune system. however, our knowledge about the molecular mechanisms of molluscan apoptosis and its immunomodulatory and immune defense roles is currently far from complete. further studies are urgently needed to delineate the mechanisms underlying apoptosis in molluscs. despite the high degree of evolutionary conservatism (attesting to the key role of this process in this organisms’ survival), some molluscan apoptotic pathways and induction mechanisms appear to be sufficiently different from those defined in invertebrate models such as drosophila and caenorhabditis, or in vertebrates, to expect that studies of molluscan apoptosis will yield important discoveries of the novel and evolutionarily distinct ways in which “molluscs do it”. our current studies of the abilities of parasites and pathogens to dysregulate molluscan apoptosis have also barely scratched the surface, and many exciting discoveries await researchers in the field. molluscan genome sequencing projects (such as those proposed for biomphalaria glabrata and c. gigas) and molluscan est libraries may provide essential new molecular tools for probing these mechanisms and their role(s) in the molluscan immune system. more studies are needed to determine the molecular mechanisms underlying pathogen-induced modulation of apoptosis in molluscan cells and these will provide significant insights into immune avoidance and the evolution of the host-parasite arms race that is apparent between molluscs and their parasites/pathogens. such studies will also help to identify the crucial physiological and biochemical pathways that may prove therapeutic targets to prevent proliferation and spread of the mollusc diseases. another interesting immunological aspect of apoptosis that has not been addressed in molluscs (or any other invertebrate model) is the suppression of the immune responses by apoptotic cells, and the role of parasite-induced apoptosis dysregulation in intra-host competition between parasites. in mammals, interaction of macrophages with apoptotic cells inhibits inflammatory cytokine and chemokine secretion thereby limiting immune responsiveness (birge and ucker, 2008). this immunosuppressive effect of apoptotic cell bodies appears to be conserved across metazoan evolution (birge and ucker, 2008) and so it would be interesting to determine whether parasite-induced suppression of apoptosis in molluscan hemocytes (such as seen in perkinsus spp. or haplosporidium spp. infections) not only assists in proliferation and spread of the parasites responsible for this inhibition, but may also prevent secondary infections by subsequent invaders thus reducing intraand interspecific competition between parasites within a host individual. unlike homeostatic mechanisms in mammals that closely regulate the organism’s body temperature, osmolarity, and ph, these parameters are allowed much wider variations in molluscs following changes in their environment. the ramifications of extrinsic and intrinsic changes in apoptosis on the immune system of mollusks are poorly understood and require further investigation. while excessive apoptosis due to an environmental stress may suppress the immune system, moderate stimulation may modulate host-parasite relationships and alter disease outcome. in either case, understanding these interactions will be crucial to predict the proliferation and dissemination parasites and pathogens in natural populations of molluscs and potentially, their role as vectors in the diseases of domestic animals and humans. knowledge of the molluscan apoptotic mechanisms could also provide critical information for the development of immortalized mollusk cell lines. currently, no such lines are available for marine mollusks, and the existing cell lines of biomphalaria are notorious for their difficulty to maintain in culture. development of new molluscan cell lines by suppressing apoptotic cell death in culture would provide critical new tools for advancement of experimental cell biology and physiology of mollusks. important as it is, apoptosis is not the only type of the programmed cell death in multicellular organisms. other highly conserved forms of pcd such as autophagy and pyroptosis (a proinflammatory, caspase-1-dependent pcd) have been described and may play a role in immune defense. currently, the role of the alternative forms of pcd in the immune defense has been underexplored, and their mechanisms, or even occurrence, in mollusks awaits further investigation. acknowledgements this work was supported by funds provided the national science foundation career award (ibn0347238), north carolina sea grant (award # 20062175-01), faculty research grant from unc charlotte and a unc charlotte advance program bonnie cone fellowship (through an nsf advance institutional transformation program grant, nsf-0548401). the author is grateful to drs marriott, gorbushin and sukhotin for their helpful comments on an earlier draft of this manuscript, and to mr cherkasov for his invaluable assistance with tem microscopy. references abele d, heise k, portner ho, puntarulo s. temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam mya arenaria. j. exp. biol. 205: 1831-1841, 2002. 56 aga e, katschinski dm, van zandbergen g, laufs h, hansen b, muller k, et al., inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite leishmania major. j. immunol.169: 898-905, 2002. aggarwal bb. nuclear factor-κb:the enemy within. cancer cell 6: 203-208, 2004. altschul sf, madden tl, schaffer aa, zhang j, zhang z, miller, w, et al. gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids res. 25: 3389-3402, 1997. anderson rs. production of reactive oxygen intermediates by invertebrate hemocytes: immunological significance. in: k söderhäll, g vasta, s iwanaga (eds), new directions in invertebrate immunology, sos publications, fair haven, nj, pp 109-129, 1996. barcia r, ramos-martinez ji. effects of interleukin-2 on nitric oxide production in molluscan innate immunity. inv. surv. j. 5: 43-49, 2008. barsiene j, andreikenaite l, garnaga g, rybakovas, a. genotoxic and cytotoxic effects in the bivalve mollusks macoma balthica and mytilus edulis from the baltic sea. ekologija 54: 44-50, 2008. birge rb, ucker ds. innate apoptotic immunity: the calming touch of death. cell death diff. 15: 1096-1102, 2008. böttger s, jerszyk e, low b, walker c. genotoxic stress-induced expression of p53 and apoptosis in leukemic clam hemocytes with cytoplasmically sequestered p53. cancer res. 68: 777-782, 2008. bravarenko ni, onufriev mv, stepanichez my, lerusalimsky vn, blaban pm, gulyaeva nv. caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail helix. eur. j. neurosci. 23: 129-140, 2006. bridgham jt, wilder ja, hollocher h, johnson al. all in the family: evolutionary and functional relationships among death receptors. cell death diff. 10: 19-25, 2003. bruchhaus i, roeder t, rennenberg a, heussler v. protozoan parasites: programmed cell death as a mechanism of parasitism. trends parasitol. 23: 376-383, 2007. brune b, knethen av, sandau kb. nitric oxide (no): an effector of apoptosis. cell death diff. 6: 969-975, 1999. buckland-nicks j, tompkins g. paraspermatogenesis in ceratostoma foliatum (neogastropoda): confirmation of programmed nuclear death. j. exp. zoolog. a comp. exp. biol. 303: 723-741, 2005. cande c, cecconi f, dessen p, kroemer g. apoptosis-inducing factor (aif): key to the conserved caspase-independent pathways of cell death. j. cell sci. 115: 4727-4734, 2002. canesi l, gallo g, gavioli m, pruzzo c. bacteriahemocyte interactions and phagocytosis in marine bivalves. microsc. res. tech. 57: 469476, 2002. cherkasov aa, overton rajr, sokolov ep, sokolova im. temperaturedependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation. j. exp. biol. 210: 46-55, 2007. circu ml, aw ty. glutathione and apoptosis. free radic. res. 42: 689-706, 2008. creagh em, conroy h, martin sj. caspaseactivation pathways in apoptosis and immunity. immunol. rev. 193: 10-21, 2003. dehal p, satou y, campbell rk, chapman j, degnan b, de tomaso a, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157-2167, 2002. dey a, tergaonkar v, lane dp. double-edged swords as cancer therapeutics: simultaneously targeting p53 and nf-κb pathways. nature 7: 1031-1040, 2008. faircloth g, cuevas c. kahalalide f and es285: potent anticancer agents from marine molluscs. prog. mol. subcell. biol. 43: 363-379, 2006. feig c, peter me. how apoptosis got the immune system in shape. eur. j. immunol. 37: 61-70, 2007. gifondorwa dj, leise em. programmed cell death in the apical ganglion during larval metamorphosis of the marine mollusc ilyanassa obsoleta. biol. bull. 210: 109-120, 2006. glinski z, jarosz j. molluscan immune defense. arch. immunol. ther. exp. (warsz) 45: 149155, 1997. goebel s, gross u, luder cgk. inhibition of host cell apoptosis by toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(adpribose) polymerase expression. j. cell sci. 114, 3495-3505, 2001. goedken m, morsey b, sunila i, dungan c, de guise s. the effects of temperature and salinity on apoptosis of crassostrea virginica hemocytes and perkinsus marinus. j. shellfish res. 24: 177-183, 2005. han ts, teichert rw, olivera bm, bulaj g. conus venoms a rich source of peptide-based therapeutics. curr. pharm. des. 14: 2462-2479, 2008. hengartner mo. programmed cell death in invertebrates. curr. opin. genet. develop. 6: 34-38, 1996. hengartner mo. the biochemistry of apoptosis. nature, 407: 770-776, 2000. heussler vt, kuenzi p, rottenberg s. inhibition of apoptosis by intracellular protozoan parasites. inter. j. parasitol. 31: 1166-1176, 2001. hildeman d, jorgensen t, kappler j, marrack p. apoptosis and the homeostatic control of immune responses. curr. opin. immunol. 19: 516-521, 2007. holbrook lac, butler ra, cashon re, van beneden rj. soft-shell clam (mya arenaria) p53: a structural and functional comparison to human p53. gene 433: 81-87, 2009. kennedy, vs, newell, rie, eble, af. the eastern oyster crassostrea virginica. college park,maryland: a maryland sea grant book, 1996. 57 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22faircloth%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22cuevas%20c%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'prog%20mol%20subcell%20biol.'); http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22han%20ts%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22teichert%20rw%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22olivera%20bm%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22bulaj%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'curr%20pharm%20des.'); lacoste a, cueff a, poulet sa. p35-sensitive caspases, map kinases and rho modulate {beta}-adrenergic induction of apoptosis in mollusc immune cells. j. cell sci. 115: 761-768, 2002. lannig g, flores jf, sokolova im. temperaturedependent stress response in oysters, crassostrea virginica: pollution reduces temperature tolerance in oysters. aquat. toxicol. 79: 278-287, 2006. li ly, luo x, wang x. endonuclease g is an apoptotic dnase when released from mitochondria. nature 412: 27-29, 2001. luder cgk, gross ug, lopes mf. intracellular protozoan parasites and apoptosis: diverse strategies to modulate parasite-host interactions. trends parasitol. 17: 480-486, 2001. mcfadden dw, riggs dr, jackson bj, vona-davis l. keyhole limpet hemocyanin, a novel immune stimulant with promising anticancer activity in barrett's esophageal adenocarcinoma. am. j. surg. 186: 552-555, 2003. mcfall-ngai mj. consequences of evolving with bacterial symbionts: insights from the squidvibrio associations. annu. rev. ecol. syst. 30: 235-256, 1999. mignotte b, vayssiere j-l. mitochondria and apoptosis. eur. j. biochem. 252: 1-15, 1998. montagnani c, kappler c, reichhart jm, escoubas jm. cg-rel, the first rel/nf-κb homolog characterized in a mollusk, the pacific oyster crassostrea gigas. febs lett. 561: 75-82, 2004. monti d, salvioli s, cossarizza a, franceschi c, ottaviani e. cytotoxicity and cell death: studies on molluscan cells and evolutionary considerations. acta biol. hung. 43: 287-291, 1992. muzio m. signalling by proteolysis: death receptors induce apoptosis. int. j. clin. lab. res. 28: 141-147, 1998. nash pb, purner mb, leon rp, clarke p, duke rc, curiel tj. toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis. j. immunol. 160: 1824-1830, 1998. oubrahim h, stadtman er, chock pb. mitochondria play no roles in mn(ii)-induced apoptosis in hela cells. proc. natl. acad. sci. usa 98: 9505-9510, 2001. ouwe-missi-oukem-boyer o, porchet e, capron a, dissous c. characterization of immunoreactive tnf alpha molecules in the gastropod biomphalaria glabrata. dev. comp. immunol. 1994 18: 211-218, 1994. ow yp, green dr, hao z, mak tw.cytochrome c: functions beyond respiration. nat. rev. mol. cell biol. 9: 532-542, 2008. park s-y, cho s-j, kwon h-c, lee k-r, rhee d-k, pyo s. caspase-independent cell death by allicin in human epithelial carcinoma cells: involvement of pka. cancer lett. 224: 123-132, 2005. paul, we. fundamental immunology. lippincott, williams and wilkins, 5th edition, philadelphia, 2003. pirger z, nemeth j, hiripi l, toth g, kiss p, lubics a, et al. pacap has anti-apoptotic effect in the salivary gland of an invertebrate species, helix pomatia. j. mol. neurosci. 36: 105-114, 2008. pruski am, dixon dr. effects of cadmium on nuclear integrity and dna repair efficiency in the gill cells of mytilus edulis l. aquat. toxicol. 57: 127-137, 2002. schultz dr, harringto wjjr. apoptosis: programmed cell death at a molecular level. semin. arthritis rheum. 32: 345-369, 2003. shih cm, wu js, ko wc, wang lf, wei yh, liang hf, et al. mitochondria-mediated caspaseindependent apoptosis induced by cadmium in normal human lung cells. j. cell. biochem. 89: 335-347, 2003. sokolova im, evans s, hughes fm. cadmiuminduced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition. j. exp. biol. 207: 3369-3380, 2004. suen df, norris kl, youle rj. mitochondrial dynamics and apoptosis. genes dev. 22: 15771590, 2008. sunila i, labanca j. apoptosis in the pathogenesis of infectious diseases of the eastern oyster crassostrea virginica. dis. aquat. organ. 56: 163-170, 2003. tait swg, green dr. caspase-independent cell death: leaving the set without the final cut. oncogene 27: 6452-6461, 2008. takahashi kg, mori k. functional profiles of hemocytes in the bio-defense process of the pacific oyster, crassostrea gigas. tohoku j. agricul. res. 51: 15-27, 2000. terahara k, takahashi kg. mechanisms and immunological roles of apoptosis in molluscs. curr. pharm. des. 14: 131-137, 2008. terajima d, yamada s, uchino r, ikawa s, ikeda m, shida k, et al. identification and sequence of seventy-nine new transcripts expressed in hemocytes of ciona intestinalis, three of which may be involved in characteristic cell-cell communication. dna res.10: 203-212, 2003. tiscar pg, mosca f. defense mechanisms in farmed marine molluscs. vet. res. comm. 28 (suppl. 1): 57-62, 2004. van loo g, schotte p, van gurp m, demol h, hoorelbeke b, gevaert k, et al. endonuclease g: a mitochondrial protein released in apoptosis and involved in caspase-independent dna degradation. cell death diff. 8: 1136-1142, 2001. vasta gr, ahmed h. animal lectins: a functional view. crc press, boca raton fl, 2008. vermeulen k, van bockstaele d, berneman z. apoptosis: mechanisms and relevance in cancer. ann. hematol. 84: 627-639, 2005. yeretssian g, labbe k, saleh m. molecular regulation of inflammation and cell death. cytokine 43: 380-390, 2008. zimmermann kc, bonzon c, green dr. the machinery of programmed cell death. pharmacol. ther. 92: 57-70, 2001. zhu b, wu x. identification of outer membrane protein ompr from rickettsia-like organism and induction of immune response in crassostrea ariakensis. mol. immunol. 45: 3198-3204, 2008. 58 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22li%20ly%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22luo%20x%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22wang%20x%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22mcfadden%20dw%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus 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http://www.ncbi.nlm.nih.gov/pubmed/18568041?ordinalpos=101&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/18568041?ordinalpos=101&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/18559474?ordinalpos=102&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/18559474?ordinalpos=102&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22zhu%20b%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22wu%20x%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'mol%20immunol.'); minireview isj 6: s35-s45, 2009 issn 1824-307x review genetic perspectives on the ascidian central nervous system a locascio*, f ristoratore*, a spagnuolo, l zanetti, m branno cellular and developmental biology laboratory, stazione zoologica “anton dohrn”, villa comunale, napoli, italy *equal contribution accepted march 13, 2009 abstract in 2002, date of publication of the ciona intestinalis genome, ascidians entered the post-genomic era. this tool had a fundamental role and has become the starting point for a series of new functional and genomic studies. recently, great efforts have been done to characterize the genetic cascades of genes having a key role in early embryonic development and to draw the regulatory networks in which they are involved. in this review, we focused our attention on the last advances obtained in the attempt to clarify the complex molecular events governing ascidian central nervous system development with a special interest for anterior neural and sensory structures. we discussed the more recent theories on its early induction and late regionalization. in particular, we used some conserved genes fully or partially characterized as examples to compare ascidian and vertebrate central nervous system (cns). by integrating the various results obtained with microarray, morpholino loss of function and promoter analyses, we showed that many progresses have been done to unravel the gene networks controlling early cns induction and formation. unfortunately, fewer advances have been done in the identification of the regulatory cascades controlling late cns regionalization and sensory organs differentiation. some results are discussed to point out the importance of fully characterizing also these specific regulatory cascades. key words: ascidian; nervous system; genetic pathway; genome; regulatory network   introduction situated at the base of the chordate lineage, ascidians possess many features shared with vertebrates; the larval form, in particular, has been recognized as evolutionarily significant because it reveals features of the early evolution of the vertebrate body plan. due to their relative simplicity and their crucial phylogenetic position, ascidians have the unique potential to illuminate molecular mechanisms underlying the ancestral body plan from which modern chordates diversified. in particular, the larval nervous system of ciona intestinalis, consisting of a brain vesicle, a visceral ganglion and a tail nerve chord, is simple but well differentiated. the ciona central nervous system (cns) can thus be viewed as a miniature prototype of the chordate brain and represents an exciting experimental model to understand ancestral features of the chordate nervous system, its development and physiology. ___________________________________________________________________________ corresponding author: annamaria locascio cellular and developmental biology laboratory stazione zoologica “anton dohrn” villa comunale, 80121, napoli, italy e-mail: anny@szn.it thanks to their transparent embryos, ascidians have been a model system for embryological studies for over a century. a number of new methodologies, developed in recent years and ranging from molecular, genomic, and physiological approaches, are greatly contributing to the knowledge of the complex regulatory networks underlying ascidian cns development. the sequencing of c. intestinalis (dehal et al., 2002) and ciona savignyi (http://www.broad.mit.edu/annotation/ciona/) genomes and the accumulation of molecular resources, that rival those available for fruit flies and mice, introduced ascidians into the post-genomic era. ciona embryos are readily amenable to experimentation in the laboratory and in vitro fertilization can produce thousands of synchronously dividing embryos that develop rapidly. ascidians are hermaphrodites, and the ability of ciona to self-fertilize provides a rapid means for identifying recessive zygotic mutations (moody et al., 1999; sordino et al., 2001). a screening for mutations naturally occurring in a large percentage of wild ciona populations (sordino et al., 2008), identified more than 40 % of the   s35 mailto:anny@szn.it mutations lead to morphological alterations at level of brain and sensory vesicle formation. this natural genetic polymorphism constitutes a valuable source of phenotypes for studying embryonic development. developmental mutants have also been obtained in both ciona species through chemical mutagenesis (moody et al., 1999; sordino et al., 2000; sordino et al., 2001) and, more recently, through insertional mutagenesis using the minos transposable element (sasakura et al., 2003). additionally, recently generated stable transgenics expressing gfp represent useful tools for investigations on mechanisms of tissue formation during embryogenesis and morphogenesis. both mutagenesis and stable transgenesis in ciona require the rearing of animals in the laboratory for several generations. this requirement has been assisted by the development of new culture techniques and maintenance protocols (cirino et al., 2002; hendrickson et al., 2004), and recently improved by introducing the use of closed, recalculating sea water systems to permit ciona culture in inland laboratories (joly et al., 2007). using electroporation, transient transgenesis is successfully performed in ascidians allowing characterization of cis-regulatory dnas (alfano et al., 2007; corbo et al., 1997; erives et al., 1998; ristoratore et al., 1999). the wide number of tissuespecific enhancers currently available allows both the production of mutant phenotypes, via ectopic expression of regulatory genes (spagnuolo and di lauro, 2002; takahashi et al., 1999), and disruption of gene activity through the overexpression of dominant negative proteins (corbo et al., 1998). injection of morpholino antisense oligonucleotides (satou et al., 2001) provides an alternative method for the analysis of loss of gene function. the use of comprehensive microarrays has revealed extensive temporal patterns of gene activity (azumi et al., 2007a) and assisted in the determination of spatial patterns of gene expression within individual blastomeres in sequentially staged embryos (yamada et al., 2005). recently, a simple method allowing the maintenance of dissociated cells from ciona in primary cultures has been developed (zanetti et al., 2007). cells that conserve their functionality have been successfully cultured, opening the possibility that this method could be used in a wide range of experiments in this animal model. in particular, this method allowed the identification of two types of neurons resembling motorneurons and large eminens cells (imai and meinertzhagen 2007a, b). taking into account that the ascidian nervous system is largely inaccessible to neurophysiological studies because of the tough outer tunic of the larvae, primary cell culture represents a useful system to overcome these limitations. collectively, all these technical advancements in ciona provide powerful tools for the study of the gene networks controlling chordate development. here we summarise the more recent advances on the genetic mechanisms underlying the development of nervous system structures, highlighting the similarities as well as the differences observed across chordates. we will focus primarily on c. intestinalis and, where appropriate, we discuss also findings from other ascidian species. ascidian cns in chordate evolution six years have passed from the publication in science of the c. intestinalis genome sequence (dehal et al., 2002) and since then much progress has been made in the knowledge of both genomic and developmental processes. the genomes of another ciona species, c. savignyi (http://www.broad.mit.edu/annotation/ciona/) and of the appendicularian oikopleura dioica (http://www.genoscope.cns.fr/spip/oikopleuradioica-whole-genome.html) have been sequenced and assembled. advances in genome annotation (ensembl), fish analysis (shoguchi et al., 2008) and identification of aflp markers (kano et al., 2006) greatly contributed to the construction of ciona chromosomal maps. shoguchi et al. (2008) mapped about 82 % of the genome sequence information on all arms of the ciona chromosomes. what emerged from these studies is that regulatory genes and their targets, belonging to the same developmental gene networks, are distributed over all fourteen chromosomes. thus, genes whose genomic regulation and functions are interconnected do not need physical clustering to be coordinately activated. all together, this new piece of information makes ascidians a very powerful model system for functional genomic studies. among the main novelties there is the new ascidian phylogenetic position as sister group to the vertebrates, a position long thought to be held by the cephalochordates. taking advantage of the genome sequence of the tunicate oikopleura dioica, a set of 146 genes from 14 deuterostome species have been aligned and phylogenetically analyzed. the results suggest that tunicates do not represent the earliest chordate lineage, as previously inferred, but in fact they are the closest living relatives of vertebrates (delsuc et al., 2006). recently the ciona genome release has been complemented by large scale cdna and est projects accompanied by expression analyses (http://ghost.zool.kyoto-u.ac.jp; imai et al., 2004; satou and satoh, 2005). cdna and oligo-dna microarrays allowed the construction of new urochordate expression map databases (azumi et al., 2007b; yamada et al., 2005) and detailed expression profiles are now available for most of the genes encoding cell signaling and regulatory proteins. a three dimensional computational software organizes and integrates gene expression data with the complex single cell developmental programs (http://crfb.univ-mrs.fr/aniseed/). a three dimensional ascidian body atlas database shows interactive developmental tables of 26 newly defined embryonic stages, accompanied by cell lineage descriptions and time laps photos (http://chordate.bpni.bio.keio.ac.jp/faba/1.2/top.html). all these data, together with the key position of ascidians at the base of vertebrate origin, give this system the power to clarify the complex gene networks governing chordate evolution and   s36 http://www.genoscope.cns.fr/spip/oikopleura-dioica-whole-genome.html http://www.genoscope.cns.fr/spip/oikopleura-dioica-whole-genome.html vertebrate development. the regionalized expression patterns in ascidians of specific markers of vertebrate forebrain, midbrain, mid-hindbrain boundary (mhb) and hindbrain, such as otx, pax2/5/8, en, and the hox genes, suggest a tripartite organization of the ascidian cns. in particular, the sensory vesicle, marked by the otx gene, is considered homologous to the vertebrate fore-midbrain; the neck region marked by pax2/5/8 and en is considered to be homologous to the vertebrate mhb; finally the visceral ganglion marked by hox genes corresponds to the vertebrate hindbrain (fig. 1) (imai et al., 2002; wada et al., 1998). the evolution of the cns and in particular of its anterior structures is a crucial point in the chordate lineage. vertebrates have a complex, segmented and highly organized cns that evolved from the same chordate ancestor of ascidians. many complex and still poorly defined regulatory networks governing nervous system development and differentiation can be clarified by taking advantage of recent progress in functional genomic studies and the compact and non redundant genome of ascidians. the ascidian larva is composed of only ~2600 cells of which about 330 cells constitute the cns. following the basic chordate body plan, neurulation gives rise to a sensory vesicle of ~215 cells, including two pigmented sensory organs and, in sequence along the antero-posterior axis, to the neck, the visceral ganglion and dorsal hollow nerve cord (nicol and meinertzhagen, 1991). more recently, the presence in ciona of a midbrain homologous region has been reconsidered based on a comparative analysis of the expression pattern of dmbx, a marker of the midbrain in vertebrates (takahashi and holland, 2004). takahashi and holland (2004) demonstrated that the ascidian dmbx gene is expressed only in the visceral ganglion, in a region that is posterior to the pax2/5/8 expression territory (mhb homologue) and coincident with rostral limit of hox gene territories (hindbrain homologue). the presence of the ciona dmbx gene only in the visceral ganglion could correspond to the expression of vertebrate dmbx in the hindbrain (fig.1), thus reinforcing visceral ganglion homology with the vertebrate hindbrain and suggests that a midbrain homologue is missing in ciona. data from amphioxus, where no dmbx expression is observed in the neural tube (takahashi and holland, 2004), further support the notion that the midbrain is a novelty that evolved specifically in the vertebrate lineage. despite the evident morphological differences between ascidian and vertebrate cns structures, there are clear homologies in nervous system patterning as deduced from the expression patterns of many developmental genes. the otx gene defines the forebrain of vertebrates, and marks also the ascidian larva sensory vesicle (fig. 1). this region has been compared to the vertebrate rostral cns and is considered analogous to the forebrain (wada et al., 1998). a series of vertebrate genes, foxh1, nkx2.1 and otp, are markers of the developing hypothalamus and specifically are involved in cell fate restriction, hypothalamus primordium delineation and neuroendocrine hypothalamic nuclei differentiation. the ascidian homologues (ci-foxha, ci-nkx2.1 and ci-otp) were found to be expressed in regionalized patterns within the ciona ventro-lateral sensory vesicle. these data suggest a possible correspondence between these vertebrate and ascidians structures (moret et al., 2005). supporting this suggestion of homology, the coronet cells on the left side of the sensory vesicle in ascidians have been structurally compared to the cells of saccus vasculosus in the vertebrate hypothalamus (katz, 1983). similarities can also be observed at the level of cns patterning, exemplified by the regionalized expression domains of hox genes. nine hox genes have been identified in the ciona genome (dehal et al., 2002; spagnuolo et al., 2003), which have undergone a significant reorganization, including rearrangement of the gene positions, dispersion, and breakage on separate chromosomes. despite the extensive shuffling of ciona hox gene cluster, some of the gene members maintain a coordinated expression in the larval cns. ci-hox1, 3, 5 and 10, indeed, show a restricted and colinear expression profile along the antero-posterior axis at the level of the visceral ganglion and nerve cord, which are considered homologous to vertebrate hindbrain and spinal cord respectively (gionti et al., 1998; ikuta et al., 2004; locascio et al., 1999). recently, a detailed analysis in ciona at various embryonic stages of the dynamic expression profiles of ciotx, pax2/5/8, cifgf8/17/18, en and hox genes in part supports the tripartite model but in part indicates that also a dipartite model, lacking the mhb region, may be consistent (ikuta and saiga, 2007). the expression profiles of pax2/5/8 and en in the neck region and the presence of an intervening gap between otx and hox gene expression territories in larval stages support the tripartite organization (fig.1). expression of fgf8/17/18 seems to be in contrast with this model. in vertebrates, fgf 8 is expressed in the mhb and play a pivotal role in the organizer activity (the capacity to change the fate of surrounding tissues when transplanted in other cns territory) of this region (liu and joyner, 2001). in ciona, the absence of fgf8/17/18 expression in the neck region and its presence only more posteriorly in the visceral ganglion (fig.1) could indicate that ascidians possess an ancestral mhb lacking organizer activity. on the other hand, the lack of a gap between otx and hox domains at earlier stages of development supports a dipartite organization. dufour (2006) proposed a third model based on ciphox2 expression at larva and adult stages. in vertebrates phox2 genes (phox2a and phox2b) mark the hindbrain and define cranial motorneurons of the branchiovisceral class. in ascidians, ciphox2 is restricted to the neck region at larva stage and will   s37 fig. 1 comparison of gene expression domains in the central nervous system of ascidians and vertebrates. homologous genes are indicated by the same colours. the most anterior regions are marked by otx (blue) genes and correspond to the sensory vesicle in ascidians and the forebrain and midbrain in vertebrates. dmbx gene (dark blue) is expressed in the midbrain only in vertebrates, while it is present in the hindbrain (visceral ganglion) in both chordate organisms. expression of pax2/5/8 (light blue) and en (green) in the intervening region between otx and hox genes (hox1, red; hox3, orange; hox 5, yellow; hox10, dark green) is common to both vertebrates and ascidians. fgf8/17/18 (lilac) is a marker of the mhb region, at the junction between midbrain and hindbrain, in vertebrates while it is expressed more posterior in the visceral ganglion in ascidians. d, dorsal; v, ventral. give rise to the adult motorneurons located in the cerebral ganglion. these results, together with hox genes expression profiles, suggested a possible homology of the posterior half of the neck with vertebrate hindbrain and of the visceral ganglion with spinal cord (dufour et al., 2006). unfortunately these controversial data, together with the absence in ascidian genome of another marker of vertebrate mhb, the gbx gene (castro et al., 2006), make it difficult to clearly establish which model most appropriately reflects the ascidian condition and thus, the appearance during chordate evolution of the mhb organizer. gene expression analyses in additional deuterostome organisms (eg. hemichordate) could help to elucidate the ancestral situation of the basic patterning of cns and the subsequent evolutionary steps that led to the differentiation of the highly complex vertebrate nervous system. cns regulatory networks ascidian neural specification is organized via conserved chordate gene networks. these networks have been expanded during vertebrate evolution to create more complex neural structures. in ascidian embryos, blastomere divisions are initially synchronous, and become partially asynchronous later in development. invariant cleavages and cell lineages give rise in ciona to about 330 neural cells, of which about 2/3 form the sensory vesicle, 47 the visceral ganglion and 95 the caudal nerve cord. at the 110 cell stage, most of the blastomere fates have been already determined; descendents of the   s38 a-line form the anterior neural structures, while the neural tube derives from the b8.19 and a7 cell lines (cole and meinertzhagen, 2004). it was already known that fgf9/16/20 has a fundamental role in inducing anterior neural differentiation in the a-line, while nodal and erk signaling are involved in caudal neural tube formation (imai et al., 2006; lemaire et al., 2002). imai et al. (2006) contributed much to our knowledge of gene networks involved in ciona neurogenesis. large scale morpholino assays and subsequent characterization by rt-qpcr of altered gene expression in the mutant phenotypes permitted the establishment of a series of regulatory relationships at the level of a single cell. in particular, they focused their attention on regulatory genes specifically localized in the blastomeres determined to acquire a particular cell fate. the analysis and comparison of significant changes in the expression profiles of genes permitted the description of the early networks that lead to the induction of different tissues and to the establishment of restricted cell fates (imai et al., 2006). unfortunately, morpholino studies do not permit the observer to unequivocally distinguish between direct and indirect target genes. the combination of morpholino “loss of function studies” of genes of interest together with promoter analyses of their candidate downstream genes has a great potential for the elucidation of regulatory networks and reconstruction of the precise genetic cascades leading to a specific tissue differentiation. ascidians represent indeed a very suitable model system to perform regulatory element assays given their compact genomes and the frequent location of minimal promoters approximately <1 kb upstream of the transcription start sites of the genes of interest. moreover, the opportunity to compare specific genomic fragments between closely related species represents a further advantage that these organisms offer. additionally, electroporation is a relatively simple and efficient method that gives hundreds of transgenic embryos (corbo et al., 1997). an interesting example of the results that can be achieved combining loss of function and gene promoter studies is offered by the reconstruction of the neural induction and otx genetic cascade during early embryonic development. ascidian ectodermal induction starts at the 8cell stage when ci-gataa expression is repressed in vegetal territories by β-catenin accumulation, and thus restricted to the animal hemisphere. at 816 cell stage, ci-gataa activates pan-animal pole genes, such as ci-fog, while fgf9/16/20 is active as a neural inducer in the a-line. at the 32 cell stage brain specification begins with the activation of the otx gene through fgf9/16/20 signaling. studies of the ci-otx promoter revealed the presence of seven ci-gataa sites for animal activation and two ets binding sites for its restriction to neural cells induced by fgf (bertrand et al., 2003; rothbacher et al., 2007). in particular, fgf9/16/20 activates otx and dmrt1 and both of them, on their own, activates transcription factor genes such as six1/2 and six3/6 (imai et al., 2006). nodal is a signaling molecule of the tgfβ factor superfamily, and acts as an organizing signal in the development of the nerve cord. at the 32 cell stage, fgf9/16/20 activates also nodal whose expression is restricted to the b6.5 cells through the activity of both soxc and foxa. nodal activates msxb, pax3/7, snail, delta-like and chordin in the b6.5 cell line and induces roof nerve cord differentiation. in the a7.8 lineage, nodal induces snail, delta-like and neurogenin to form the lateral ependymal cells of the nerve cord (imai et al., 2006). these studies have extended our understanding of the first phases of neural induction and formation; on the other hand, our knowledge on the gene networks acting later in development, during nervous system regionalization and differentiation, is still very poor. in most cases the roles of genes temporally expressed at various stages of development have been studied in detail only at early embryonic stages by morpholino loss of function experiments; their roles at later stages are still largely unknown. only few genes specifically expressed later in development, and potentially involved in neural structure differentiation, have been studied in detail. in most cases their function, and the genetic cascades in which they are involved, are only partially defined and remain unconnected to other known regulatory networks. to analyze the function of genes involved in late events of cns differentiation, a morpholino approach may give results difficult to interpret; studies of their transcriptional regulation could be more informative on the regulatory networks in which these genes are involved. here we offer three examples of genes expressed in the cns at late stages of ciona development; dissection of their promoter regions has already given some clues to the elements controlling their expression. further studies are needed to identify their upstream regulators in order to define some steps of the genetic circuits at the bases of cns regionalization. hox genes represent a good example of conserved regulatory genes involved in late cns regionalization. several ascidian hox genes show conserved expression patterns, indicating a role in patterning the antero-posterior axis, however most studies treat their expression profiles, genomic, and chromosomal organization adding little functional data. the study of cihox3 promoter, the only one deeply analysed, led to the characterization of an 80-bp element found to be sufficient to recapitulate the endogenous expression pattern of cihox3 (locascio et al., 1999). unfortunately this sequence did not show any likely recognition sequence for known transcription factors, this requires a deeper bioinformatics analysis to identify putative binding sites. the 80-bp cihox3 promoter fragment however served to address whether hox3 regulatory elements have been conserved during chordate evolution. the 80-bp cihox3 promoter fragment, when tested in mouse embryos, was unable to reproduce any neural-specific expression in transgenic mice, thus suggesting that the elements regulating hox3 expression are not conserved   s39 between ciona and mouse. nevertheless, it is interesting to note that, when a slightly larger enhancer fragment was tested, a reproducible segmental pattern of expression in the mouse hindbrain was obtained, suggesting that some elements acting in ciona are recognised by mouse transcriptional machinery (locascio et al., 1999). these early interesting results give rise to the possibility of identifying hox specific enhancer elements responsible for antero-posterior regionalization that have been conserved during chordate evolution. another example of an important regulatory gene, whose function has been remarkably well conserved during evolution, is the pax6 gene. pax 6 encodes a transcription factor that has been implicated in the development of eyes and portions of anterior nervous systems, throughout the animal kingdom. recently the regulatory region of the c. intestinalis pax6 has been analyzed and compared with that of mouse and drosophila pax6 (irvine et al., 2008). the results showed a similar level of complexity in cis-regulatory elements on a gross scale. the three species have regulatory elements located in a large intron near the 5' end of the gene, able to drive transcription in photoreceptors, brain and nerve cord. both drosophila and ciona have major enhancers for brain and nerve cord upstream of the transcription start site. despite these similarities in the genomic organization, no sequence similarities have been found using blast alignment, suggesting that after divergence of a common ancestry sequence similarities have been obscured by binding site turnover and rearrangements (irvine et al., 2008). a final example derives from studies of the msx family of transcription factors. members of the msx family are among the regulatory genes expressed from early to late embryonic stages and showing several different functions during development. msx genes show highly conserved functions during evolution in neural patterning and dorso-ventral subdivision of the embryonic neuroectoderm (d'alessio and frasch, 1996; bendall and abateshen, 2000). only one msx gene is present in ciona genome and it shows an interesting expression pattern in the mesenchyme and nervous system precursors starting from the early gastrula stage (aniello et al., 1999). in larval stages, the expression is maintained in the sensory vesicle and in the visceral ganglion. loss of function experiments, via morpholino oligonucleotide microinjection, gave some information on the role played by msx in muscle differentiation, but they failed to give crucial information on msx function in nervous system development, probably because this later function was masked by early expression (imai et al., 2006). in this case, the characterization of the regulatory region could give important insights on the regulation of this gene, in different tissues and at different developmental stages, and could help to place msx gene in the network leading to anterior cns regionalization. it has been demonstrated that the specific expression of msx in the neural precursors is regulated by a 30-bp region that is able to recapitulate the endogenous expression in the nervous system both at neurula and tailbud stages. comparison of the ci-msx promoter with that of murine msx-1 did not show any sequence homology, suggesting that these genes are regulated by different networks. unfortunately, although some putative binding sites have been identified in the 30-bp region, the factors responsible for msx activation have not yet been identified (russo et al., 2004). further studies are necessary to reconstruct its specific role in the nervous system regionalization and the genetic cascade in which it is involved. it is possible to argue from the fore mentioned examples that we have only begun to exploit the enormous potential of regulatory region analyses in ciona. recently, a database has been created containing information on regulation of tunicate genes collected from literature. it includes information regarding the minimal promoter length, the transcription factors involved and their binding sites, as well as the localization of the gene expression (sierro et al., 2006). further improvements, such as the inclusion of information on the regulation of halocynthia roretzi, c. savignyi and o. dioica genes, and continuous updating will greatly contribute to reconstruct genetic networks. cns sensory organ differentiation the brain vesicle of ascidian larvae contains two distinct pigmented sensory organs clearly visible through its transparent body. the more anterior pigmented sensory organ, the otolith, is involved in the perception of gravity, whereas the more posterior one, the ocellus, is involved in the perception of light stimuli. the two sensory organs are responsible for the swimming behaviour of the larva (tsuda et al., 2003). the otolith is composed of a large spherical cell attached to the ventral wall of the sensory vesicle by a narrow stalk. it contains a single pigment granule that occupies most of the cell body. the ocellus is composed of a cup-shaped pigmented cell, a number of photoreceptors and three lens cells (dilly, 1969; nicol and meinertzhagen, 1991). the ascidian ocellus is considered a simple eye. the presence of photoreceptors, of 3 lens cells and of a pigmented cell in the ocellus, suggests some similarity with vertebrate eye. the ascidian “simple eye”, with its close association of the pigment cell and photoreceptors, may thus offer a “unique possibility” to study the developmental programs bringing to both melanization and photoreceptors differentiation. the “unique possibility” is related not only to the simplicity of the system but also to the very well characterized lineages of photoreceptor and pigment cells. the molecular mechanisms regulating ascidian pigment cell development are not yet clear. the two pigment cells arise from the paired a8.25 blastomeres that are positioned bilaterally in the gastrula ascidian embryo and will give rise to the a9.49 pair at neural plate stage (fig. 2a, b). it is known that determination of a8.25 cells as pigment cell precursors requires direct inductive influence from the nerve cord precursor cells at the gastrula stage. at this stage the a8.25 blastomeres constitute   s40 fig. 2 cell lineage of pigment cell precursors (light blue) in ascidian embryos at 110 cell, neural plate and tailbud stages (a-c). expression pattern of chordin gene in halocynthia roretzi (d) and ciona intestinalis (h) embryos at tailbud stage. white arrow in d indicates chordin expressing cell in the sensory vesicle. image d is from darras et al., 2001. an equivalence group in the sense that both have the potential to form either an ocellus or otolith (darras and nishida, 2001). the choice to adopt either otolith or ocellus cell fate is made after neural tube closure at early tailbud stage and requires cellcell interactions. as the neural tube closes, the four cells derived from the division of the two a8.25 cells, the a10.97 and a10.98 pairs, converge and intercalate along the anterior-posterior axis in order to align along the midline (fig. 2c). the a10.98 pair gives rise to part of the brain vesicle, the anterior a10.97 forms the otolith while the posterior one differentiates into the ocellus pigment cell. in the ascidian h. roretzi, the choice to become ocellus or otolith seems to be influenced by the antagonistic effect of bmp, expressed at tailbud stage in the four tyrosinase positive cells (a10.98 and a10.97 pairs), and chordin, secreted from the adjacent posterior cell (fig. 2d) (darras and nishida, 2001). it seems that high doses of bmp inhibit pigmentation (as in the a10.98 pair), intermediate doses allow the development of an otolith (in the anterior a10.97) and low levels lead to ocellus development (in posterior a10.97) (darras and nishida, 2001). nevertheless this mechanism seems not to be universal, as suggested by the wide expression pattern of chordin in the anterior cns of c. intestinalis tailbud embryos (fig. 2e; personal unpublished data). this evident difference render it unlikely that in ciona bmp/chordin could be the sole responsible for the induction of the posterior a10.97 blastomere to form the ocellus. further studies are necessary to elucidate the molecular mechanisms underling early and late regulation of pigment cell formation. these studies will take advantage of the analysis of molecular markers specific for the pigment cell precursors at different stages of development. some genes involved in the process of melanization have been identified. tyrosinase expression, as well as its enzyme activity, has been detected in the two types of pigment cells in several ascidian species, suggesting that this enzyme is conserved as key enzyme for melanin synthesis and that melanin has similar chemical characteristics to the melanin of vertebrates. moreover, the expression pattern of a trp (tyrosinase related protein) has been analysed as well as a mitf gene (microphthalmia transcription factor); both show an expression pattern correlated, in some developmental stages, with restriction of pigment cell fate indicating their potential involvement in this process as in higher chordates. in the ascidian h. roretzi, as in higher chordates (camacho-hubner and beermann, 2000; camacho-hubner et al., 2002; camp et al., 2003; goding, 2000), tyrosinase gene family (tygf) has been used as a model to approach studies on “pigmentation programs” (kusakabe et al., 2001). firstly it has been identified a hrtyr promoter fragment able to direct lineage-specific and developmentally correct expression of hrtyr during embryogenesis. a search for conserved recognition sequences in this promoter has revealed the presence of several putative pax3-binding consensus sites; the data that over-expression of hrpax-3/7 is able to induce ectopic expression of hrtyr (halocynthia roretzi tyrosinase) have reinforced the hypothesis of a direct relation between hrpax-3/7 and hrtyr (toyoda et al., 2000). a parallel approach on the hrtrp (halocinthia roretzi thyrosinase related protein) promoter has permitted the identification of two otx binding consensus sites involved in hrtrp expression; here too, hroth overexpression can transactivate this promoter in an otx site-dependent manner,   s41 confirming a direct function exerted by hroth on hrtrp expression (dehal et al., 2002). the mechanisms of tyrosinase/trp activation seem therefore to be conserved on most aspects during chordate evolution (for a review see murisier and beermann, 2006). in higher chordates, members of the microphthalmia (mitf) transcription factor family play a central role in specification of pigment cell lineage (aksan and goding, 1998; hotta et al., 2000). the absence of mitf binding sites in hrtyr and hrtrp minimal promoter elements, compared with higher chordates, is a little bit surprising, given that molecular data from h. roretzi strongly supports the antiquity of the association of the mitf family members with pigment cells (yajima et al., 2003). one possibility is that mitf, at least in h. roretzi, is not involved in the regulation of hrtyr and hrtrp expression; or that hrmitf boxes, located in the far upstream region, are not necessary but can contribute to a full efficient expression of tyrosinase(s) during halocynthia embryogenesis; or that hrmitf/hrtyr-hrtrp interaction is not direct but mediated by other factors. the scenario emerging from the data collected so far in ascidians point to an evolutionary conservation of most factors involved in pigment cells differentiation. what about photoreceptors? the question is very intriguing given that photoreceptor and pigment cell precursors, in the ascidian c. intestinalis, share at the late gastrula stage common factors, as mitf and bmp5/7, that slightly later, at the neurula stage, become localized specifically in pigment cell precursors (data not shown). studies on photoreceptor differentiation in ascidians have produced very few data so far. opsin (ci-opsin1) (kusakabe et al., 2001) and arrestin (ci-arr) (nakagawa et al., 2002) genes have been isolated and characterized as photoreceptorspecific markers from the ascidian c. intestinalis; a 3kb ci-arr promoter region has been demonstrated to recapitulate the expression of the endogenous gene (ikuta et al., 2004). these genes are directly involved in the visual cycle; studies on their transcriptional regulation could therefore help to clarify the mechanisms involved in terminal differentiation of photoreceptors. only recently a key factor required for the differentiation of vertebrate eye, the rx gene, has been cloned from ciona (ci-rx). “loss of function” experiments have indicated that it is required for ocellus development. in particular, larvae lacking rx function do not develop ocellus pigment cell, lack photoreceptors and are unable to response to light stimuli (d'aniello et al., 2006). furthermore, a ci-rx regulatory region, that recapitulate the expression of the endogenous gene, has been identified (d'aniello et al., 2006). this ci-rx “eye enhancer” could provide interesting keys to unravel the genetic circuits controlling a step just prior to the terminal differentiation of photoreceptors. studies on the transcriptional regulation of genes as bmp and mitf in ascidians could finally be instrumental to shed light on the mechanisms/factors involved in the initial choice pigment cell-photoreceptor. recent work has suggested that pigmented cells of the ocellus and otolith are necessary for sensing light and gravity, respectively (sakurai et al., 2004; tsuda et al., 2003). the behaviour of ascidian larva has long been studied illustrating that ascidian larvae present phototactic and geotactic responses. the larvae become sensitive to light about 4 hours after hatching, responding to decreased light intensity by swimming more actively. a direct proof for the role of pigmentation in ascidian larval physiology have been obtained only recently, taking advantage of two mutant lines of c. savignyi that are unable to make melanin and thus lack pigment in the larval sensory structures. behavioural studies on non pigmented offspring demonstrated that unpigmented larvae are unable to detect source of light and consequently are unable to seek out the shaded location preferred by their wild type siblings (jiang et al., 2005). moreover, the c. savignyi mutant larvae lacking pigmentation do not behave properly in response to gravity. other information could come from the physiological studies of other naturally occurring mutants of c. intestinalis isolated in the gulf of naples, presenting several defects in pigmentation ranging from total absence of pigmented cells to presence of only one pigmented cell (otolith or ocellus) (sordino et al., 2008). conclusions the new molecular tools and the great progresses in deciphering ascidian genome and chromosomal mapping, allowed moving to a further and more advanced step in the comprehension of the complex chordate body organization. from the study of single genes or single genetic cascades, it is now possible to integrate and interconnect the regulatory networks that underlie the organization, function and development of the ascidian nervous system. these progresses refer overall to early steps of neural induction but represent the milestone for future reconstruction of late cns regionalization and differentiation. despite the obvious morphological differences and the divergence of regulatory sequences that, in some cases, have been observed, it seems that most of the main cns developmental programs are conserved between ascidians and vertebrates. this result strongly spurs to use ascidians, with their compact genome and relatively simple cns, as model system to characterize some of the conserved and fundamental steps of chordate neurogenesis. to this aim, it will be very important in the next years to integrate the functional studies (eg. morpholino loss of function) of genes involved in a specific patterning with the characterization of the corresponding regulatory elements. acknowledgements we would like to thank ag cole for careful reading of the manuscript. references aksan i, goding cr. targeting the microphthalmia basic helix-loop-helix-leucine zipper transcription factor to a subset of e-box elements in vitro and in vivo. mol. cell biol. 18: 6930-6938, 1998.   s42 alfano c, russo mt, spagnuolo a. developmental expression and transcriptional regulation of cipans, a novel neural marker gene of the ascidian, ciona intestinalis. gene 406: 36-41, 2007. aniello f, locascio a, villani mg, di gregorio a, fucci l, branno m. identification and developmental expression of ci-msxb: a novel homologue of drosophila msh gene in ciona intestinalis. mech. dev. 88: 123-126, 1999. azumi k, sabau sv, fujie m, usami t, koyanagi r, kawashima t, et al. gene expression profile during the life cycle of the urochordate ciona intestinalis. dev. biol. 308: 572-582, 2007a. azumi k, usami t, kamimura a, sabau sv, miki y, fujie m, et al. cdna microarray analyses reveal candidate marker genes for the detection of ascidian disease in korea. zool. sci. 24: 12311240, 2007b. bendall aj, abate-shen c. roles for msx and dlx homeoproteins in vertebrate development. gene 247: 17-31, 2000. bertrand v, hudson c, caillol d, popovici c, lemaire p. neural tissue in ascidian embryos is induced by fgf9/16/20, acting via a combination of maternal gata and ets transcription factors. cell 115: 615-627, 2003. camacho-hubner a, beermann f. cellular and molecular features of mammalian pigmentationtyrosinase and trp. pathol. biol. 48: 577-583, 2000. camacho-hubner a, richard c, beermann f. genomic structure and evolutionary conservation of the tyrosinase gene family from fugu. gene 285: 59-68, 2002. camp e, badhwar p, mann gj, lardelli m. expression analysis of a tyrosinase promoter sequence in zebrafish. pigment cell res. 16: 117-126, 2003. castro lf, rasmussen sl, holland pw, holland nd, holland lz. a gbx homeobox gene in amphioxus: insights into ancestry of the antp class and evolution of the midbrain/hindbrain boundary. dev. biol. 295: 40-51, 2006. cirino p, toscano a, caramiello d, macina a, miraglia v, monte a. laboratory culture of the ascidian ciona intestinalis: a model sistem for molecular developmental biology research. mar. mod. elec. rec. at: http://www.mbl.edu/biologicalbulletin/mmer/ciri no/cirtit.html, 2002. cole ag, meinertzhagen ia. the central nervous system of the ascidian larva: mitotic history of cells forming the neural tube in late embryonic ciona intestinalis. dev. biol. 271: 239-262, 2004. corbo jc, fujiwara s, levine m, di gregorio a. suppressor of hairless activates brachyury expression in the ciona embryo. dev. biol. 203: 358-368, 1998. corbo jc, levine m, zeller rw. characterization of a notochord-specific enhancer from the brachyury promoter region of the ascidian, ciona intestinalis. development 124: 589-602, 1997. d'alessio m, frasch m. msh may play a conserved role in dorsoventral patterning of the neuroectoderm and mesoderm. mech. dev. 58: 217-231, 1996. d'aniello s, d'aniello e, locascio a, memoli a, corrado m, russo mt, et al. the ascidian homolog of the vertebrate homeobox gene rx is essential for ocellus development and function. differentiation 74: 222-234, 2006. darras s, nishida h. the bmp/chordin antagonism controls sensory pigment cell specification and differentiation in the ascidian embryo. dev. biol. 236: 271-288, 2001. dehal p, satou y, campbell rk, chapman j, degnan b, de tomaso a, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157-2167, 2002. delsuc f, brinkmann h, chourrout d, philippe h. tunicates and not cephalochordates are the closest living relatives of vertebrates. nature 439: 965-968, 2006. dilly pn. studies on the receptors in ciona intestinalis. 3. a second type of photoreceptor in the tadpole larva of ciona intestinalis. z. zellforsch mikrosk. anat. 96: 63-65, 1969. dufour hd, chettouh z, deyts c, de rosa r, goridis c, joly js, et al. precraniate origin of cranial motoneurons. proc. natl. acad. sci. usa 103: 8727-8732, 2006. erives a, corbo jc, levine m. lineage-specific regulation of the ciona snail gene in the embryonic mesoderm and neuroectoderm. dev. biol. 194: 213-225, 1998. fanelli a, lania g, spagnuolo a, di lauro r. interplay of negative and positive signals controls endoderm-specific expression of the ascidian cititf1 gene promoter. dev. biol. 263: 12-23, 2003. gionti m, ristoratore f, di gregorio a, aniello f, branno m, di lauro r. cihox5, a new ciona intestinalis hox-related gene, is involved in regionalization of the spinal cord. dev. genes evol. 207: 515-523, 1998. goding cr. mitf from neural crest to melanoma: signal transduction and transcription in the melanocyte lineage. genes dev. 14: 17121728, 2000. hendrickson c, christiaen l, deschet k, jiang d, joly js, legendre l, et al. culture of adult ascidians and ascidian genetics. methods cell biol. 74: 143-170, 2004. hotta k, takahashi h, asakura t, saitoh b, takatori n, satou y, et al. characterization of brachyury-downstream notochord genes in the ciona intestinalis embryo. dev. biol. 224: 6980, 2000. ikuta t, saiga h. dynamic change in the expression of developmental genes in the ascidian central nervous system: revisit to the tripartite model and the origin of the midbrain-hindbrain boundary region. dev. biol. 312: 631-643, 2007. ikuta t, yoshida n, satoh n, saiga h. ciona intestinalis hox gene cluster: its dispersed structure and residual colinear expression in development. proc. natl. acad. sci. usa 101: 15118-15123, 2004.   s43 http://www.mbl.edu/biologicalbulletin/mmer/cirino/cirtit.html http://www.mbl.edu/biologicalbulletin/mmer/cirino/cirtit.html imai jh, meinertzhagen ia. neurons of the ascidian larval nervous system in ciona intestinalis: i. central nervous system. j. comp. neurol. 501: 316-334, 2007a. imai jh, meinertzhagen ia. neurons of the ascidian larval nervous system in ciona intestinalis: ii. peripheral nervous system. j. comp. neurol. 501: 335-352, 2007b. imai ks, hino k, yagi k, satoh n, satou y. gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. development 131: 4047-4058, 2004. imai ks, levine m, satoh n, satou y. regulatory blueprint for a chordate embryo. science 312: 1183-1187, 2006. imai ks, satoh n, satou y. region specific gene expressions in the central nervous system of the ascidian embryo. gene expr. patterns 2: 319-321, 2002. irvine sq, fonseca vc, zompa ma, antony r. cisregulatory organization of the pax6 gene in the ascidian ciona intestinalis. dev. biol. 317: 649659, 2008. jiang d, tresser jw, horie t, tsuda m, smith wc. pigmentation in the sensory organs of the ascidian larva is essential for normal behavior. j. exp. biol. 208: 433-438, 2005. joly js, kano s, matsuoka t, auger h, hirayama k, satoh n, et al. culture of ciona intestinalis in closed systems. dev. dyn. 236: 1832-1840, 2007. kano s, satoh n, sordino p. primary genetic linkage maps of the ascidian, ciona intestinalis. zool. sci. 23: 31-39, 2006. katz m. comparative anatomy of the tunicate ciona intestinalis. biol. bull. 164: 1-27, 1983. kusakabe t, kusakabe r, kawakami i, satou y, satoh n, tsuda m. ci-opsin1, a vertebrate-type opsin gene, expressed in the larval ocellus of the ascidian ciona intestinalis. febs lett. 506: 69-72, 2001. lemaire p, bertrand v, hudson c. early steps in the formation of neural tissue in ascidian embryos. dev. biol. 252: 151-169, 2002. liu a, joyner al. early anterior/posterior patterning of the midbrain and cerebellum. annu. rev. neurosci. 24: 869-896, 2001. locascio a, aniello f, amoroso a, manzanares m, krumlauf r, branno m. patterning the ascidian nervous system: structure, expression and transgenic analysis of the cihox3 gene. development 126: 4737-4748, 1999. moody r, davis sw, cubas f, smith wc. isolation of developmental mutants of the ascidian ciona savignyi. mol. gen. genet. 262: 199-206, 1999. moret f, christiaen l, deyts c, blin m, vernier p, joly js. regulatory gene expressions in the ascidian ventral sensory vesicle: evolutionary relationships with the vertebrate hypothalamus. dev. biol. 277: 567-579, 2005. murisier f, beermann f. genetics of pigment cells: lessons from the tyrosinase gene family. histol. histopathol. 21: 567-578, 2006. nakagawa m, orii h, yoshida n, jojima e, horie t, yoshida r, et al. ascidian arrestin (ci-arr), the origin of the visual and nonvisual arrestins of vertebrate. eur. j. biochem. 269: 5112-5118, 2002. nicol d, meinertzhagen ia. cell counts and maps in the larval central nervous system of the ascidian ciona intestinalis. j. comp. neurol. 309: 415-429, 1991. ristoratore f, spagnuolo a, aniello f, branno m, fabbrini f, di lauro r. expression and functional analysis of cititf1, an ascidian nk-2 class gene, suggest its role in endoderm development. development 126: 5149-5159, 1999. rothbacher u, bertrand v, lamy c, lemaire p. a combinatorial code of maternal gata, ets and beta-catenin-tcf transcription factors specifies and patterns the early ascidian ectoderm. development 134: 4023-4032, 2007. russo mt, donizetti a, locascio a, d'aniello s, amoroso a, aniello f, et al. regulatory elements controlling ci-msxb tissue-specific expression during ciona intestinalis embryonic development. dev. biol. 267: 517-528, 2004. sakurai d, goda m, kohmura y, horie t, iwamoto h, ohtsuki h, et al. the role of pigment cells in the brain of ascidian larva. j. comp. neurol. 475: 70-82, 2004. sasakura y, awazu s, chiba s, satoh n. germ-line transgenesis of the tc1/mariner superfamily transposon minos in ciona intestinalis. proc. natl. acad. sci. usa 100: 7726-7730, 2003. satou y, imai ks, satoh n. action of morpholinos in ciona embryos. genesis 30: 103-106, 2001. satou y, satoh n. cataloging transcription factor and major signaling molecule genes for functional genomic studies in ciona intestinalis. dev. genes evol. 215: 580-596, 2005. shoguchi e, hamaguchi m, satoh n. genome-wide network of regulatory genes for construction of a chordate embryo. dev. biol. 316: 498-509, 2008. sierro n, kusakabe t, park kj, yamashita r, kinoshita k, nakai k. dbtgr: a database of tunicate promoters and their regulatory elements. nucleic acids res. 34: 552-555, 2006. sordino p, andreakis n, brown er, leccia ni, squarzoni p, tarallo r, et al. natural variation of model mutant phenotypes in ciona intestinalis. plos one 3: e2344, 2008. sordino p, belluzzi l, de santis r, smith wc. developmental genetics in primitive chordates. philos. trans. r. soc. lond. b biol. sci. 356: 1573-1582, 2001. sordino p, heisenberg c, cirino p, toscano a, giuliano p, marino r, de santis r. a mutational approach to studiyng development of the protochordate ciona intestinalis (tunicata; chordata). sarsia 85: 173-176, 2000. spagnuolo a, di lauro r. cititf1 and endoderm differentiation in ciona intestinalis. gene 287: 115-119, 2002. spagnuolo a, ristoratore f, di gregorio a, aniello f, branno m, di lauro r. unusual number and genomic organization of hox genes in the tunicate ciona intestinalis. gene 309: 71-79, 2003.   s44 takahashi h, hotta k, erives a, di gregorio a, zeller rw, levine m, et al. brachyury downstream notochord differentiation in the ascidian embryo. genes dev. 13: 1519-1523, 1999. organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian pax-2/5/8, hox and otx genes. development 125: 1113-1122, 1998. yajima i, endo k, sato s, toyoda r, wada h, shibahara s, et al. cloning and functional analysis of ascidian mitf in vivo: insights into the origin of vertebrate pigment cells. mech. dev. 120: 1489-1504, 2003. takahashi t, holland pw. amphioxus and ascidian dmbx homeobox genes give clues to the vertebrate origins of midbrain development. development 131: 3285-3294, 2004. toyoda r, sato s, ikeo k, gojobori t, numakunai t, goding cr, et al. pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates. gene 259: 159-170, 2000. yamada l, kobayashi k, satou y, satoh n. microarray analysis of localization of maternal transcripts in eggs and early embryos of the ascidian, ciona intestinalis. dev. biol. 284: 536550, 2005. tsuda m, sakurai d, goda m. direct evidence for the role of pigment cells in the brain of ascidian larvae by laser ablation. j. exp. biol. 206: 14091417, 2003. zanetti l, ristoratore f, francone m, piscopo s, brown er. primary cultures of nervous system cells from the larva of the ascidian ciona intestinalis. j. neurosci. methods 165: 191-197, 2007. wada h, saiga h, satoh n, holland pw. tripartite   s45 cns sensory organ differentiation review isj 6: 163-174, 2009 issn 1824-307x review specificity and signaling in the drosophila immune response n silverman, n paquette, k aggarwal university of massachusetts medical school, worcester, usa accepted december 07, 2009 abstract the drosophila immune response is characterized by the rapid and robust production of a battery of antimicrobial peptides immediately following infection. the genes encoding these antimicrobial peptides are controlled by two nf-κb signaling pathways that respond to microbial infection. the imd pathway is triggered by dap-type peptidoglycan, from the cell wall of most gram-negative and certain gram-positive bacteria, and activates the nf-κb precursor protein relish. the toll pathway, on the other hand, is stimulated by lysine-type peptidoglycan from many gram-positive bacteria, β 1,3 glucans from many fungi, as well as by microbial proteases. toll signaling leads to the activation and nuclear translocation of dif or dorsal, two other nf-κb homologs. this review presents our current understanding of the molecular mechanisms involved in microbial recognition and signal transduction in these two innate immune pathways. key words: toll; imd; pgrp; peptidoglycan; antimicrobial peptides overview of drosophila immunity insects, such as drosophila, thrive in microberich environments. not surprisingly, they have evolved complex mechanisms to combat microbial infection. these defenses include structural barriers to infection, such as the cuticle and peritrophic membrane. insects also rely on inducible responses such as phagocytosis, the production of antimicrobial compounds, and homeostatic mechanisms that help repair the damage caused by infection (lemaitre and hoffmann, 2007). together, these defense mechanisms allow insects to be broadly resistant to a large range of pathogens without an acquired immune response. the inducible humoral insect immune response has been most widely studied in the favorite model system drosophila melanogaster, where microbial challenge leads to the rapid and robust production of a battery of antimicrobial peptides (amps). several families of amps have been described in drosophila, with antifungal and anti-bacterial (both anti-gram-negative or anti-gram-positive) activities. some of these amps appear to be unique to insects, e.g., diptericin, while others have homologs in mammals, e.g., defensins, cecropins and drosomycin (lee et al., 1989; simon et al., 2008). ___________________________________________________________________________ corresponding author: neal silverman divison of infectious diseases, department of medicine university of massachusetts medical school 364 plantation st, worcester, ma 01605, ma, usa e-mail: neal.silverman@umassmed.edu as best we know, production of amps is regulated at the transcriptional level. to date, nearly all amp genes have been found to be controlled by nf-κb family transcription factors. drosophila have two distinct pathways which activate nf-κb factors and drive transcription of amp genes following infection. the toll pathway responds to several different types of microbes, including fungi and many gram-positive bacteria, and leads to the activation of the nf-κb family members dif and dorsal. on the other hand, the imd pathway is activated by gram-negative and certain types of gram-positive bacteria and leads to the activation of the nf-κb precursor relish. the details of the how different microbes are detected and discriminated by these two pathways is the main focus of this review. first, the basic outline of these two signaling pathways will be summarized. toll and imd signaling as mentioned above, the toll pathway is able to activate two nf-κb homologs, dif and dorsal. both of these proteins are similar to mammalian p65, and are held in the cytoplasm of unstimulated cells by the drosophila iκb homolog cactus. like mammalian iκbs, cactus is phosphorylated and degraded upon stimulation (fernandez et al., 2001). one outstanding question within this pathway is the identity of the cactus kinase. to date, only the kinase pelle, homologous to the mammalian irak family of kinases, has been shown to function in the 163 mailto:neal.silverman@umassmed.edu figure 1 toll signaling pathway. the toll signaling pathway and its multiple modes of activation in the drosophila immune response. three distinct mechanisms for microbial recognition, leading to the cleavage of spätzle and activation of toll, are illustrated. the mechanisms include peptidoglycan detection, by pgrp-sa, pgrp-sd and gnbp1, β-glucan detection through gnbp3, and protease activity sensing via the serine protease persephone. all these detection modalities lead to the activation of the spätzle processing enzyme (spe) which converts this cytokine into its active form, for binding and activating toll. the intracellular signal transduction downstream of toll is very similar to the myd88-dependent pathway, which functions downstream of most mammalian tlrs. the key features of this pathway include a trimeric receptor associated complex, containing myd88, tube and pelle, which ultimately lead to the phosphorylation and degradation of the iκb homology cactus and the nuclear translocation of nf-κb homologs dif and dorsal. 164 toll pathway. in mammals, irak kinases are indirectly involved in the phosphorylation of iκbs, being required for the initiation of a kinase cascade that culminates in the activation of the iκb kinase (ikk) complex, which directly phosphorylates iκbα (skaug et al., 2009). in flies, a similar kinase cascade may be involved, although the components are not yet identified. alternatively, it remains possible that pelle directly phosphorylates cactus on the residues necessary for its degradation. this issue remains unresolved. it is clear that pelle is a component of a trimeric complex that associates with the active form of the transmembrane receptor toll. like all the mammalian toll-like receptors and the mammalian il-1 receptor, the cytoplasmic domain of drosophila toll contains a tir domain. this tir domain interacts with the drosophila myd88 homolog via a homotypic tir:tir interaction (tauszig-delamasure et al., 2002; charatsi et al., 2003; kambris et al., 2003). myd88 also contains a death domain (dd), which interacts with the protein tube. through its other face, the tube dd also interacts with pelle (towb et al., 1998; sun et al., 2002; sun et al., 2004). this trimeric myd88/tube/pelle complex is thought to interact transiently with the cytosolic tir domain of toll. this association likely leads to the auto-phosphorylation and activation of pelle, which in turn leads, directly or indirectly, to the phosphorylation of cactus. once cactus is degraded, the nf-κb factors dif and dorsal translocate to the nucleus where they control the transcription of target genes. in the adult fly, dif is critical for the activation of amp gene transcription (meng et al., 1999; rutschmann et al., 2000a; de gregorio et al., 2001, 2002; irving et al., 2001). in larvae, redundancy is observed between dif and dorsal, and only a double mutant fails to induce amp genes. in addition to controlling amp gene expression, dif and dorsal together also seem to regulate the survival of hemocytes in larvae (qiu et al., 1998; matova and anderson, 2006). dorsal (and much of the rest of the toll pathway) also plays a critical role in early embryonic development, in patterning the dorso-ventral axis. during development, phosphorylaztion of dorsal is linked to enhanced nuclear localization and transcriptional activation (drier et al., 1999, 2000). dif is not required in development but can partially substitute for dorsal in this process when expressed in the embryo (stein et al., 1998). the role of phosphorylation of dif, in the context of the immune response, has not been examined (for an overview of the toll signaling pathway, see fig. 1). the imd pathway culminates in the activation of a third nf-κb homolog, known as relish. like the mammalian nf-κb precursor proteins p100 or p105, relish contains an n-terminal rel homology domain and c-terminal iκb-like ankyrin repeats. in unstimulated cells, the c-terminal iκb-like domain is believed to hold full length relish in the cytoplasm (stöven et al., 2000). after immune stimulation relish is endoproteolytically cleaved, and the nterminal transcription factor module translocates into the nucleus while the c-terminal iκb-like domain remains in the cytoplasm. in addition to this proteolytic cleavage, full activation of relish also requires phosphorylation on two residues, serines 528 and 529, (erturk-hasdemir et al., 2009). this phosphorylation is not required for relish cleavage, but instead seems to be crucial for the transcriptional activation of some relish target genes. another protein, known as akirin, also functions in the nucleus for the induction of amp gene expression, but how it interacts with relish, if at all, is unclear (goto et al., 2008). relish cleavage and phosphorylation are controlled by two interconnected branches of the imd signaling pathway. serines 528 and 529 are directly phosphorylated by the drosophila ikk complex. this kinase complex includes drosophila ikkβ and ikkγ homologs, also known as ird5 and kenny, respectively (rutschmann et al., 2000b; silverman et al., 2000; lu et al., 2001). ikk activation, in turn, requires the map3k tak1 and its binding partner tab2. interestingly, both tab2 and ikkγ include conserved k63-polyubiquitin binding domains. these non-degratory ubiquitin chains are thought to function in the imd pathway, however the exact molecular mechanisms involved are not yet clear (zhou et al., 2005). the putative e3 ubiquitin ligase diap2 is also required in the imd pathway and may promote k63-chain formation (kleino et al., 2005; leulier et al., 2006; huh et al., 2007). further upstream, kinase activation also requires the imd protein, the drosophila fadd homolog, and the caspase-8 like dredd (leulier et al., 2000; georgel et al., 2001; leulier et al., 2002; naitza et al., 2002). these three proteins may form a trimeric complex as fadd can interact with both imd and dredd. how this complex signals the activation of tak1 is still under investigation. in addition to a poorly defined role in the activation of tak1, dredd is also required for the cleavage of relish. interestingly, the ikk complex is also required for relish cleavage, but its kinase activity is not involved in this function (stöven et al., 2003; erturkhasdemir et al., 2009). instead, the ikk complex may function as a scaffold facilitating the cleavage of relish by dredd (for an overview of the imd signaling pathway, see fig. 2). in addition to playing a key role in activation of the ikk complex and relish, tak1 also activates drosophila jnk signaling through hemipterous (mkk7) and basket (jnk) (sluss et al., 1996; holland et al., 1997; chen et al., 2002). thus, tak1 plays a crucial role at the nexus of jnk and nf-κb signaling in this innate immune signaling pathway. nf-κb (relish) plays a critical role in the induction of amp genes; without relish, no amp gene expression is detected. however, the role of the jnk pathway in amp regulation remains controversial. several reports have argued that jnk signaling actually down-modulates amp gene expression (kim et al., 2005, 2007), while another report has argued that jnk signaling is required for amp induction (delaney et al., 2006). more studies are required to resolve these conflicting conclusions. in addition to the possible inhibitory activity of jnk on nf-κb-responsive amp expression, the relish branch of the pathway also seems to generate an inhibitor of jnk signaling (park et al., 2004). the mechanism by which relish-induced gene products interfere with jnk 165 figure 2 imd signaling pathway. this pathway is preferentially triggered by dap-type peptidoglycan, common to gram-negative bacteria and certain gram-positives, especially bacillus spp. dap-type peptidoglycan can be recognized by different receptors, depending on its location and size. large insoluble peptidoglycan is recognized on the cell surface by pgrp-lcx, while smaller peptidoglycan fragments, like tct, can be recognized at the cell surface by a ligand-induced dimer of pgrp-lcx and pgrp-lca. in addition, dap-type peptidoglycan that reaches the cytosol can trigger another receptor, pgrp-le. both pgrp-lc and pgrp-le trigger a similar intracellular signal transduction pathway, as outlined here, that culminates in the activation of the nf-κb precursor relish. in addition, recognition of intracellular dap-type peptidoglycan by pgrp-le also triggers an autophagic response, which is important in the protection against intracellular bacteria. signaling has been suggested to involve the ubiquitin e3 ligase posh. posh is thought to target tak1 for degradation (tsuda et al., 2005), however it is not clear how this would preferentially inhibit jnk but not relish dependent responses. in addition to activating jnk and nf-κb/relish signaling, the imd pathway also induces the activation of the drosophila p38 pathway, which requires imd protein but not tak1 or any of the downstream components (zhuang et al., 2006). little is known about p38 signaling in the drosophila immune response, however it does appear to play a critical role in regulating the ros generating enzyme doux in the gut, thereby controlling the intestinal microflora (ha et al., 2009). microbial recognition both the toll and imd pathways are stimulated by bacterial peptidoglycans (leulier et al., 2003; kaneko et al., 2004). in addition, the toll pathway can be triggered by β-glucans, from fungal cell wall, or by proteases directly released from pathogens (gottar et al., 2006; el chamy et al., 2008). peptidoglycan (pgn) is the major structural component of the bacterial cell wall. pgn structures display a great deal of diversity, varying widely among different classes of bacteria. however, all pgns include a carbohydrate backbone, usually consisting of alternating nacetyl-glucosamine and n-acetyl-muramic acid residues and short stem-peptides containing both l and d amino acids. these stem-peptides are often cross-linked to each other to stiffen the cell wall; the precise structure of these cross-linking structures is highly variable. the stem-peptides also display a great deal of variation in their amino acid constituents. the carbohydrate backbone is more constant but also can be modified by various chemical substitutions, such as acetylation (schleifer and kandler, 1972; mengin-lecreulx and lemaitre, 2005) (see fig. 3 for a diagram of pgn structures). both the toll and imd pathways rely on peptidoglycan recognition proteins (pgrps) for sensing pgns (table 1). this family of proteins is structurally similar to type 2 amidases (nacetylmuramyl-l-alanine amidases), a class of enzymes that hydrolyze the bond between the lactyl group in acetylmuramic acid and l-alanine in the stem-peptide of pgn. in fact, some pgrps are type 2 amidases, while others lack the catalytic cysteine 166 figure 3 peptidoglycan structure. (a) as shown, peptidoglycan has a common core structure with a great deal of inherent variation. most notably, the constituent of the third position of the stem-peptide can vary, and are most commonly l-lysine or meso-dap. in addition, the amount and exact chemical nature of the crosslinking bridges can vary, with some examples noted in the box below. tct is a monomeric unit of the dap-type peptidoglycan chain, and is indicated in the dashed box. (b) structures of lysine and dap. and instead function by binding pgn (mellroth et al., 2003). drosophila encode for 13 pgrp genes, making about 17 distinct proteins through alternative splicing (werner et al., 2000). six of the drosophila pgrps (pgrp-sb1, -sb2, sc1a/b, sc2, -lb) are known or predicted type 2 amidases, that are involved in degrading pgn and dampening immune activation (mellroth et al., 2003; bischoff et al., 2006; zaidman-remy et al., 2006). the other seven lack type 2 amidase activity but function through binding pgn. in particular, 4 pgrps (pgrp-sa, -sd, -lc, and le) function as receptors in the imd or toll pathways, as detailed below. pgrp-lf seems to function as a decoy receptor, binding pgn but not activating immune signaling (persson et al., 2007; maillet et al., 2008), while the functions of pgrp-la and -ld remain elusive (royet and dziarski, 2007). initial studies suggested that the imd pathway was activated preferentially by gram-negative bacteria, which lead many to assume that lps, the most potent activator of the mammalian innate immune response, would be the main agonist of this pathway (samakovlis et al., 1990; werner et al., 2003). however, a careful analysis of published results suggested otherwise. in addition to gramnegatives, certain gram-positive bacteria, e.g., bacillus spp, were also imd pathway activators (lemaitre et al., 1997). subsequently, lemaitre’s group showed that diaminopimelic acid (dap)-type pgn, from escherichia coli or bacillus thurengensis, activated the imd pathway, while the silverman group demonstrated that purified lps samples were unable to trigger the imd pathway, and imd agonistic activity could be traced to dap-type pgn (leulier et al., 2003; kaneko et al., 2004). lemaitre’s group also showed that the toll pathway was activated by pgn, but in this case lysine-type pgn from gram-positives like m. luteus and e. fecalis was more potent. following these discoveries, a great deal has been learned about the molecular mechanisms involved in detecting various types of pgn. pgrpsa and pgrp-sd are required for the recognition of pgn and the activation of toll signaling (see below for more detail on the toll pathway) (michel et al., 2001; bischoff et al., 2004), while in the imd pathway either of two receptors, pgrp-lc or pgrp-le, are capable of recognizing dap-type pgn (gottar et al., 2002; ramet et al., 2002; kaneko et al., 2004, 2006; takehana et al., 2004; choe et al., 2005). pgn binding to either pgrp-lc or -le triggers the imd signaling pathway, described 167 table 1 functions and specificity of the pgrps retrieved in d. melanogaster * predicted specificity, based on the presence of arginine residue in key position for dap recognition. above, via a short conserved domain found in the nterminus of both receptors (kaneko et al., 2006). this conserved signaling domain has some similarity to the rhim domain found in the mammalian proteins rip1, rip3 and trif (meylan et al., 2004). however the molecular mechanisms involved in signaling by the pgrp-lc/le rhim-like domain still remain to be determined. regardless of the mechanisms involved, activation of these receptors leads to activation of both relish cleavage and relish phosphorylation. one key discovery, which enabled detailed molecular and biophysical analyses of pgrp-lc and pgrp-le, was that a monomeric fragment of pgn from gram-negative bacteria, known as trachael cytotoxin (tct), potently activates the imd pathway (kaneko et al., 2004; stenbak et al., 2004). tct is a disaccharide-tetrapeptide, featuring dap at the third position of its stem-peptide, isolated from culture supernatants of b. pertussis (fig. 3a). synthetic lactyl-tetrapeptides (substructures of tct) are able to serve as weak agonists of the imd pathway only if they contain dap at this third position, providing further demonstration that the dap residue is key to triggering the imd pathway (kaneko et al., 2004). pgrp-lc and pgrp-le preferentially bind dap containing pgn (takehana et al., 2002; swaminathan et al., 2006). the crystal structures of both pgrp-lc and pgrp-le bound to tct have been solved and show that a key arginine, arg254 in pgrp-le, provides the critical dap-specific interaction (chang et al., 2006; lim et al., 2006). compared to lysine, dap contains an additional carboxylate group (figure 3b), and the guanidinium group of arg254 forms a bidentate salt bridge with this moiety . in fact, this key arginine residue is common to all known dap-type recognizing pgrps and is found in the base of a deep cleft in which the pgn stem-peptide binds. in addition, dap binding pgrps also contain nearby glycine and tryptophan residues that are involved in other dap-specific interactions (swaminathan et al., 2006). pgrp-lc and pgrp-le are found in different subcellular compartments and detect microbes in these distinct environments. pgrp-lc is a single pass, type 2 transmembrane receptor that is found primarily on the cell surface (kaneko et al., 2004; mellroth et al., 2005). through alternative splicing, pgrp-lc encodes for 3 different receptors (pgrplca, -lcx, and -lcy) each with an identical cytosolic domain but distinct extracellular ligand binding pgrp domains. on the other hand, pgrple encodes for only one protein isoform, which lacks a transmembrane domain and functions as a cytosolic receptor (werner et al., 2000). while pgrp-lc is critical for recognizing extracellular bacteria (and extracellular pgn), pgrp-le surveils the cytosol for intracellular bacteria and/or small fragments of pgn that enter cells (kaneko et al., 2006; yano et al., 2008). rnai-based studies showed that the different splice isoforms of pgrplc are involved in recognizing different types of pgn. in particular, the recognition of polymeric name function pgn specificity pgrp-sb1 amidase daptype pgn pgrp-sb2 amidase daptype pgn* pgrp-sc1a/b amidase lys/daptype pgn pgrp-sc2 amidase daptype pgn* pgrp-lb amidase daptype pgn pgrpsa seml receptor for toll signaling lystype pgn pgrp-sd receptor for toll signaling daptype pgn pgrp-lc ird7 receptor for imd signaling daptype pgn pgrp-le receptor for imd signaling daptype pgn pgrp-lf decoy receptor daptype pgn pgrp-la unknown daptype pgn* pgrp-ld unknown daptype pgn* 168 pgn, as isolated from e. coli, requires only pgrplcx. moreover, pgrp-lcx mutants do not induce amp genes in response to e. coli infection. however, the response to extracellular monomeric pgn (tct) requires both pgrp-lcx and pgr-lca, while intracellular tct triggers pgrp-le. the finding that polymeric and monomeric dap-type pgns trigger distinct pgrp-lc receptors was explained, in part, by the crystal structure of pgrp-lca/x bound to tct. as mentioned above, most pgrps contain a deep cleft in which peptidoglycan fragments (sometimes referred to as muropeptides) bind. pgrp-lca is the exception; it contains two unique dipeptide sequences that disrupt the pgn binding cleft and occlude pgn binding. conversely, pgrp-lcx has a typical daptype pgn binding cleft and can avidly bind tct or polymeric pgn. upon binding tct, pgrp-lcx and pgrp-lca heterodimerize (chang et al., 2005; mellroth et al., 2005). the crystal structure of this ligand bound dimeric complex shows that the carbohydrate portion of tct makes key contributions to the dimerization interface, providing a clear explanation for the tct-induced dimerization (chang et al., 2006). it is reasonable to hypothesize that the ligand-induced heterodimerization of pgrplca and pgrp-lcx is critical for activation of downstream signaling, although this has not yet been demonstrated. this model of dimerizationinduced signaling does not explain how pgrp-lcx alone is sufficient for imd signaling triggered by polymeric pgn. the crystal structures suggest that pgrp-lcx is unlikely to form homo-multimers upon binding polymeric pgn, because of a steric clash at the putative dimerization interface. therefore, a distinct model must be proposed for signaling by polymeric pgn and pgrp-lcx. in this case, the ligand is polyvalent and likely binds to multiple individual pgrp-lcx receptors, perhaps creating a ‘cluster’ of receptors, thereby generating a density of pgrp-lcx cytosolic domains. this clustering, per se, may be sufficient to activate signal transduction, or perhaps the intracellular domains actually form higher order protein-protein interactions while the extracellular domains remain clustered on one large fragment of pgn, but not in direct contact with each other. future studies are required to examine these possibilities. like the pgrp-lca/x heterodimers, pgrp-le also multimerizes upon binding tct. however, the pgrp-le-tct complex forms very high order multimers in a ‘head to tail’ fashion. like the pgrplc structures, the pgrp-le structure was solved with the isolated pgrp domain, and we cannot be certain what quaternary structure the holo-receptor forms upon tct binding. however, these biophysical studies clearly demonstrate that tct causes pgrp-le to multimerize into large complexes (lim et al., 2006). as mentioned above, pgrp-le detects dap-type pgn that enters the cytosol. pgn may enter the cytosol from infection with intracellular bacteria, like listeria monocytogenes, or small pgn fragments, such as tct, appear to directly enter into cells. upon binding these pgns, pgrp-le likely forms higher order multimers and triggers imd signaling. in addition, pgrp-le activation can also induce an autophagic response that helps protect against intracellular microbes (yano et al., 2008). the role of ligand-induced receptor multimerization in the activation of imd signaling pathway, via pgrp-lcs and pgrp-le, requires further study. as mentioned above, these receptors signal through a rhim-like domain in their n-terminal domains. the mechanism by which the rhim-like domain functions in the context of dimerized, multimerized or clustered pgrp receptors is unknown. toll activation by pgn and beyond unlike the imd pathway, which is activated in a fairly specific manner by dap-type pgn, toll activation occurs indirectly by a wider array of immune stimuli. toll functions more like a cytokine receptor, binding a processed form of the cytokine spätzle, a member of the cysteine knot family of growth factors and cytokines (weber et al., 2003; hu et al., 2004; hoffmann et al., 2008). spätzle is made as a pro-protein that is found circulating in the hemolymph. upon immune activation (or developmental cues), serine protease cascades are triggered that culminate in the cleavage of spätzle. once processed, mature spätzle binds to and dimerizes the transmembrane receptor toll, initiating the intracellular signaling pathway described above. four different serine protease cascades appear to converge on the cleavage of spätzle. in early development, the protease easter is responsible for cleaving spätzle. during the immune response, bacterial pgn, fungal β-glucans, and microbial proteases are sensed by three distinct mechanisms, but converge upon activation of one serine protease, known as the spätzle processing enzyme (spe), which in turn cleaves and activates spätzle (fig. 1). the serine protease persephone appears to function as a sensor for proteases secreted by both fungal and bacterial pathogens (gottar et al., 2006; el chamy et al., 2008). persephone is likely activated by cleavage after a histidine residue, unlike the other proteases involved in the toll signaling pathways, and maybe a good target for subtilisin-like proteases produced by microbial pathogens (el chamy et al., 2008). the activation of the persephone-toll pathway by microbial proteases occurs independently of recognition of microbial cell wall material, which can also stimulate the toll pathway through more classical receptormediated recognition. for example, β-glucans from the cell wall of yeast are recognized by gram-negative binding protein 3 (gnbp3) (gottar et al., 2006), while two secreted pgrp receptors and gnbp1 are involved together in pgn recognition (gobert et al., 2003; bischoff et al., 2004; wang et al., 2006, 2008). [despite their name, none of the gnbps have been linked to the response to gram-negative bacteria, but gnbp1 and gnpb3 are involved in the recognition of fungal or gram-positive bacterial cell walls]. the n-terminus of gnbp3 binds to long β1,3 glucans, common to the cell walls of many types of fungi, especially yeast (mishima et al., 2009). gnbp1, on the other hand, is involved in pgn recognition, although its role is controversial. 169 lysine-type pgns, common to many grampositive bacteria, are potent agonist of the toll pathway. as in the imd pathway, pgrp receptors are critical for the recognition of lysine-type pgn. in particular, two secreted pgrps, pgrp-sa and -sd, are involved in the toll pathway. genetic studies have shown that some gram-positive bacteria and the pgn from these same species, such as m. luteus, are sensed through pgrp-sa (michel et al., 2001). in fact, the structure of pgrp-sa bound to a lysine-containing muropeptide has been solved. pgrp-sa can also bind dap-type pgn, albeit to a lesser degree. however, pgrp-sa appears to be able to specifically cleave dap-type muropeptides, removing the final amino acid in the stem-peptide. it has been postulated that this carboxypeptidase activity prevents dap-type pgn from stimulating the toll pathway via pgrp-sa (chang et al., 2004). interestingly, not all lysine-type pgn requires pgrp-sa to trigger the toll pathway. in particular, staphylococcus aureus, enterococcus faecalis, streptococcus pyogenes, and staphylococcus saprophyticus infections still produce strong amp gene responses in pgrp-sa mutant (seml) flies. response to these bacteria or their pgns instead requires either pgrp-sa or pgrp-sd (bischoff et al., 2004). the mechanism of pgrp-sd-mediated recognition of some, but not all, lysine-type pgn producing bacteria remains unclear. one possibility is a structural difference common to those pgns sensed by pgrp-sd, which prevents detection by pgrp-sa, or vice versa. however, biochemical studies of pgrp-sd do not support the notion that it is involved in recognizing lysine-type pgn. crystallographic studies show that pgrp-sd has a deep pgn binding cleft, typical of all pgrps, and this binding cleft includes an arginine (arg90) in the key position typical of dap-pgn specific recognition. in fact, binding studies confirm that pgrp-sd binds dap-type pgn, from b. subtilis, but not lys-type from s. aureus (leone et al., 2008). in addition, the moderate induction of drosomycin observed following either b. subtilis or e. coli infection, which is toll dependent (leulier et al., 2003), required both pgrp-sa and pgrp-sd. so, somehow pgrp-sa and pgrp-sd function together in the recognition of dap-type pgn, for moderate toll induction, but function in a more redundant manner for the recognition of certain lystype pgn, for robust toll induction. however, the recognition of m. luteus pgn appears to more simply require only pgrp-sa. the molecular mechanisms involved in the recognition of various pgns by pgrp-sa and/or pgrp-sd remain to be determined. as mentioned above, gnbp1 also functions in pgn recognition and toll signaling. in fact, pgrpsa, -sd, and gnbp1 form a trimeric complex together in the presence of pgn fragments (wang et al., 2008). some groups have reported that gnbp1 provides a critical pgn processing activity to this complex, required to generate small pgn fragments which are bound by pgrp-sa and/or pgrp-sd for toll activation (filipe et al., 2005; wang et al., 2006). however, another group has reported that they do not observe a similar pgn digesting activity associated with gnbp1, in drosophila or tenebrio molitor (buchon et al., 2009). instead, this group proposes that gnbp1 serves to link the pgrps with the downstream serine protease cascade, described below. thus, it appears that gnbp1 functions in a complex with the pgn sensing receptors pgrp-sa and pgrp-sd, but the biochemical mechanism by which it contributes to immune recognition or toll signaling are not yet clear. both the gnbp3-mediated recognition of βglucans and the pgrp-sa/sd/gnbp1-mediated recognition of bacterial pgns trigger toll signaling through the same serine protease cascade. this cascade involves the modular serine protease (modsp), which is probably directly activated by these microbial sensing receptor complexes, and at least two downstream clip-domain serine proteases grass and spe. as mentioned above, spe cleaves and thereby activates spätzle, the ligand for toll. another protease, known as spirit may function between grass and spe, and other non-catalytic serine-protease homologs, sphinx 1/2 and spheroide, were also implicated this pathway by rnai based studies (kambris et al., 2006). however, the assignment of these factors to this pathway requires further genetic and biochemical characterization. the pathways presented in fig. 1 suggest a protease cascade that is consistent with the genetic analysis of mutants in drosophila and the biochemical analysis of the cascade from the hemolymph of tenebrio. however, biochemistry of the drosophila serine protease cascade still requires further study, as several issues remain unresolved, including the role of spirit. in addition, the predicted specificity of the drosophila modsp does not match the predicted cleavage site of the downstream serine protease grass, and modsp does not cleave grass in vitro (buchon et al., 2009). thus, it remains possible that other factors may be involved. currently, it is not clear how (or even if) pgn binding to pgrp-sa/sd/gnbp1 leads to the activation of modsp, and, as mentioned above the exact biochemical role of gnbp1 remains controversial. concluding remarks the goal of this review is to summarize recent work on the toll and imd pathways, two important innate immune signaling pathways in drosophila, with an emphasis on the molecular mechanisms involved in microbial recognition in this model system. this review is not meant to be a comprehensive analysis of all aspect of the insect immune response. notably, many very exciting studies have been published recently on phagocytosis, anti-viral immunity, melanization and clotting all topics not covered here. instead, significant detail is presented on the biochemistry and genetics of bacterial recognition by the pgrp family of innate immune receptors. over the past 5 years, much progress been made in this area, and the basic underpinnings of how bacteria are recognized by pgrgp receptors, which bind the bacterial cell-wall derived compound peptidoglycan, has been resolved. in addition, the preferential binding of dap-type pgn by some of these 170 receptors, notably pgrp-lc and pgrp-le, provides a firm explanation on the specific activation of the imd pathway by gram-negative and certain gram-positive bacteria. however, the specificity, or lack thereof, in activating the toll pathway by different types of peptidoglycans still requires further investigation. moreover, in both imd and toll signaling, it remains unclear how peptidoglycan binding by these pgrp receptors leads to the activation of downstream signaling events. these questions will be the focus of future study. references bischoff v, vignal c, boneca ig, michel t, hoffmann, ja, royet j. function of the drosophila pattern-recognition receptor pgrpsd in the detection of gram-positive bacteria. nat. immunol. 5: 1175-1180, 2004. bischoff v, vignal c, duvic b, boneca ig, hoffmann ja, royet j. downregulation of the drosophila immune response by peptidoglycan-recognition proteins sc1 and sc2. plos pathog 2, e14, 2006. buchon n, poidevin m, kwon hm, guillou a, sottas v, lee bl, et al. a single modular serine protease integrates signals from patternrecognition receptors upstream of the drosophila toll pathway. proc. natl. acad. sci. usa 106: 12442-12447, 2009. chang ci, ihara k, chelliah y, mengin-lecreulx d, wakatsuki s, deisenhofer j. structure of the ectodomain of drosophila peptidoglycanrecognition protein lca suggests a molecular mechanism for pattern recognition. proc. natl. acad. sci. usa 102: 10279-10284, 2005. chang ci, pili-floury ss, herve m, parquet c, chelliah y, lemaitre b, et al. a drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual l,dcarboxypeptidase activity. plos biol 2, e277, 2004. chang ci, chelliah y, borek d, mengin-lecreulx d, deisenhofer j. structure of tracheal cytotoxin in complex with a heterodimeric patternrecognition receptor. science 311: 1761-1764, 2006. charatsi i, luschnig s, bartoszewski s, nussleinvolhard c, moussian b. krapfen/dmyd88 is required for the establishment of dorsoventral pattern in the drosophila embryo. mech. dev. 120: 219-226, 2003. chen w, white ma, cobb mh. stimulus-specific requirements for map3 kinases in activating the jnk pathway. j. biol. chem. 277: 4910549110, 2002. choe km, lee h, anderson kv. drosophila peptidoglycan recognition protein lc (pgrplc) acts as a signal-transducing innate immune receptor. proc. natl. acad. sci. usa 102: 11221126, 2005. de gregorio e, spellman pt, rubin gm, lemaitre b. genome-wide analysis of the drosophila immune response by using oligonucleotide microarrays. proc. natl. acad. sci. usa 98: 12590-12595, 2001. de gregorio e, spellman pt, tzou p, rubin gm, lemaitre b. the toll and imd pathways are the major regulators of the immune response in drosophila. embo j. 21: 2568-2579, 2002. delaney jr, stoven s, uvell h, anderson kv, engstrom y, mlodzik m. cooperative control of drosophila immune responses by the jnk and nf-kappab signaling pathways. embo j. 25: 3068-3077, 2006. drier ea, govind s, steward r. cactus-independent regulation of dorsal nuclear import by the ventral signal. curr. biol. 10: 23-26, 2000. drier ea, huang lh, steward r. nuclear import of the drosophila rel protein dorsal is regulated by phosphorylation. genes dev. 13: 556-56, 1999. el chamy l, leclerc v, caldelari i, reichhart jm. sensing of 'danger signals' and pathogenassociated molecular patterns defines binary signaling pathways 'upstream' of toll. nat. immunol. 9: 1165-1170, 2008. erturk-hasdemir d, broemer m, leulier f, lane ws, paquette n, hwang d, et al. two roles for the drosophila ikk complex in the activation of relish and the induction of antimicrobial peptide genes. proc. natl. acad. sci. usa 106: 97799784, 2009. fernandez nq, grosshans j, goltz js, stein d. separable and redundant regulatory determinants in cactus mediate its dorsal group dependent degradation. development 128: 2963-2974, 2001. filipe sr, tomasz a, ligoxygakis p. requirements of peptidoglycan structure that allow detection by the drosophila toll pathway. embo rep. 6: 327-333, 2005. georgel p, naitza s, kappler c, ferrandon d, zachary d, swimmer c, et al. drosophila immune deficiency (imd) is a death domain protein that activates antibacterial defense and can promote apoptosis. dev. cell 1: 503-514, 2001. gobert v, gottar m, matskevich aa, rutschmann s, royet j, belvin m, et al. dual activation of the drosophila toll pathway by two pattern recognition receptors. science 302: 2126-2130, 2003. goto a, matsushita k, gesellchen v, el chamy l, kuttenkeuler d, takeuchi, et al. akirins are highly conserved nuclear proteins required for nf-kappab-dependent gene expression in drosophila and mice. nat. immunol. 9: 97-104, 2008. gottar m, gobert v, matskevich aa, reichhart jm, wang c, butt tm, et al. dual detection of fungal infections in drosophila via recognition of glucans and sensing of virulence factors. cell 127: 1425-1437, 2006. gottar m, gobert v, michel t, belvin m, duyk g, hoffmann ja, et al. the drosophila immune response against gram-negative bacteria is mediated by a peptidoglycan recognition protein. nature 416: 640-644, 2002. ha em, lee ka, seo yy, kim sh, lim jh, oh bh, et al. coordination of multiple dual oxidaseregulatory pathways in responses to commensal and infectious microbes in drosophila gut. nat. immunol. 10: 949-957, 2009. 171 hoffmann a, neumann p, schierhorn a, stubbs mt. crystallization of spatzle, a cystine-knot protein involved in embryonic development and innate immunity in drosophila melanogaster. acta crystallogr. sect. f struct. biol. cryst. commun. 64: 707-710, 2008. holland pm, suzanne m, campbell js, noselli s, cooper ja. mkk7 is a stress-activated mitogen-activated protein kinase kinase functionally related to hemipterous. j. biol. chem. 272: 24994-24998, 1997. hu x, yagi y, tanji t, zhou s, ip yt. multimerization and interaction of toll and spatzle in drosophila. proc. natl. acad. sci. usa 101: 9369-9374, 2004. huh jr, foe i, muro i, chen ch, seol jh, yoo sj, et al. the drosophila inhibitor of apoptosis (iap) diap2 is dispensable for cell survival, required for the innate immune response to gramnegative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of iap-binding apoptosis inducers. j. biol. chem. 282: 2056-2068, 2007. irving p, troxler l, heuer ts, belvin m, kopczynski c, reichhart jm, et al. a genome-wide analysis of immune responses in drosophila. proc. natl. acad. sci. usa 98: 15119-15124, 2001. kambris z, bilak h, d'alessandro r, belvin m, imler jl, capovilla m. dmmyd88 controls dorsoventral patterning of the drosophila embryo. embo rep. 4: 64-69, 2003. kambris z, brun s, jang ih, nam hj, romeo y, takahashi k, et al. drosophila immunity: a large-scale in vivo rnai screen identifies five serine proteases required for toll activation. curr. biol. 16: 808-813, 2006. kaneko t, goldman we, mellroth p, steiner h, fukase k, kusumoto s, et al. monomeric and polymeric gram-negative peptidoglycan but not purified lps stimulate the drosophila imd pathway. immunity 20: 637-649, 2004. kaneko t, yano t, aggarwal k, lim jh, ueda k, oshima, y, et al. pgrp-lc and pgrp-le have essential yet distinct functions in the drosophila immune response to monomeric dap-type peptidoglycan. nat. immunol. 7: 715-723, 2006. kim lk, choi uy, cho hs, lee js, lee wb, kim j, et al. down-regulation of nf-kappab target genes by the ap-1 and stat complex during the innate immune response in drosophila. plos biol 5, e238, 2007. kim t, yoon j, cho h, lee wb, kim j, song yh, et al. downregulation of lipopolysaccharide response in drosophila by negative crosstalk between the ap1 and nf-kappab signaling modules. nat. immunol. 6: 211-218, 2005. kleino a, valanne s, ulvila j, kallio j, myllymaki h, enwald h, et al. inhibitor of apoptosis 2 and tak1-binding protein are components of the drosophila imd pathway. embo j. 24: 34233434, 2005. lee jy, boman a, sun cx, andersson m, jornvall h, mutt v, et al. antibacterial peptides from pig intestine: isolation of a mammalian cecropin. proc. natl. acad. sci. usa 86: 9159-9162, 1989. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. lemaitre b, reichhart jm, hoffmann ja. drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. proc. natl. acad. sci. usa 94: 14614-14619, 1997. leone p, bischoff v, kellenberger c, hetru c, royet j, roussel a. crystal structure of drosophila pgrp-sd suggests binding to dap-type but not lysine-type peptidoglycan. mol. immunol. 45: 2521-2530, 2008. leulier f, lhocine n, lemaitre b, meier p. the drosophila inhibitor of apoptosis protein diap2 functions in innate immunity and is essential to resist gram-negative bacterial infection. mol. cell. biol. 26: 7821-7831, 2006. leulier f, parquet c, pili-floury s, ryu jh, caroff, m, lee wj, et al. the drosophila immune system detects bacteria through specific peptidoglycan recognition. nat. immunol. 4: 478-484, 2003. leulier f, rodriguez a, khush rs, abrams jm, lemaitre b. the drosophila caspase dredd is required to resist gram-negative bacterial infection. embo rep. 1: 353-358, 2000. leulier f, vidal s, saigo k, ueda r, lemaitre b. inducible expression of double-stranded rna reveals a role for dfadd in the regulation of the antibacterial response in drosophila adults. curr. biol. 12: 996-1000, 2002. lim jh, kim ms, kim he, yano t, oshima y, aggarwal k, et al. structural basis for preferential recognition of diaminopimelic acidtype peptidoglycan by a subset of peptidoglycan recognition proteins. j. biol. chem. 281: 8286-8295, 2006. lu y, wu lp, anderson kv. the antibacterial arm of the drosophila innate immune response requires an ikappab kinase. genes dev. 15: 104-110, 2001. maillet f, bischoff v, vignal c, hoffmann j, royet, j. the drosophila peptidoglycan recognition protein pgrp-lf blocks pgrp-lc and imd/jnk pathway activation. cell host microbe 3: 293-303, 2008. matova n, anderson kv. rel/nf-kappab double mutants reveal that cellular immunity is central to drosophila host defense. proc. natl. acad. sci. usa 103: 16424-16429, 2006. mellroth p, karlsson j, hakansson j, schultz n, goldman we, steiner h. ligand-induced dimerization of drosophila peptidoglycan recognition proteins in vitro. proc. natl. acad. sci. usa 102: 6455-6460, 2005. mellroth p, karlsson j, steiner h. a scavenger function for a drosophila peptidoglycan recognition protein. j. biol. chem. 278: 70597064, 2003. meng x, khanuja bs, ip yt. toll receptor-mediated drosophila immune response requires dif, an nf-kb factor. genes dev. 13: 792-797, 1999. mengin-lecreulx d, lemaitre b. structure and metabolism of peptidoglycan and molecular requirements allowing its detection by the 172 drosophila innate immune system. j. endotoxin res. 11: 105-111, 2005. meylan e, burns k, hofmann k, blancheteau v, martinon f, kelliher m, et al. rip1 is an essential mediator of toll-like receptor 3induced nf-kappa b activation. nat. immunol. 5: 503-507, 2004. michel t, reichhart jm, hoffmann, ja, royet j. drosophila toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. nature 414: 756-759, 2001. mishima y, quintin j, aimanianda, v, kellenberger c, coste f, clavaud c, et al. the n-terminal domain of drosophila gram-negative binding protein 3 (gnbp3) defines a novel family of fungal pattern recognition receptors. j. biol. chem. 284: 28687-28697, 2009. naitza s, rosse c, kappler c, georgel p, belvin m, gubb d, et al. the drosophila immune defense against gram-negative infection requires the death protein dfadd. immunity 17: 575-581, 2002. park jm, brady h, ruocco mg, sun h, williams d, lee sj, et al. targeting of tak1 by the nfkappa b protein relish regulates the jnkmediated immune response in drosophila. genes dev. 18: 584-594, 2004. persson c, oldenvi s, steiner h. peptidoglycan recognition protein lf: a negative regulator of drosophila immunity. insect biochem. mol. biol. 37: 1309-1316, 2007. qiu p, pan pc, govind s. a role for the drosophila toll/cactus pathway in larval hematopoiesis. development 125, 1909-1920, 1998. ramet m, manfruelli p, pearson a, mathey-prevot b, ezekowitz ra. functional genomic analysis of phagocytosis and identification of a drosophila receptor for e. coli. nature 416: 644648, 2002. royet j, dziarski r. peptidoglycan recognition proteins: pleiotropic sensors and effectors of antimicrobial defences. nat. rev. microbiol. 5: 264-277, 2007. rutschmann s, jung ac, hetru c, reichhart jm, hoffmann ja, ferrandon d. the rel protein dif mediates the antifungal but not the antibacterial host defense in drosophila. immunity 12: 569580, 2000a. rutschmann s, jung ac, zhou r, silverman n, hoffmann ja, ferrandon d. role of drosophila ikk gamma in a toll-independent antibacterial immune response. nat. immunol. 1: 342-347, 2000b. samakovlis c, kimbrell da, kylsten p, engstrom a, hultmark d. the immune response in drosophila: pattern of cecropin expression and biological activity. embo j. 9: 2969-2976, 1990. schleifer kh, kandler o. peptidoglycan types of bacterial cell walls and their taxonomic implications. bacteriol. rev. 36: 407-477, 1972. silverman n, zhou r, stöven s, pandey n, hultmark d, maniatis t. a drosophila ikappab kinase complex required for relish cleavage and antibacterial immunity. genes dev. 14: 2461-2471, 2000. simon a, kullberg bj, tripet b, boerman oc, zeeuwen p, van der ven-jongekrijg j, et al. drosomycin-like defensin, a human homologue of drosophila melanogaster drosomycin with antifungal activity. antimicrob. agents chemother. 52: 1407-1412, 2008. skaug b, jiang x, chen zj. the role of ubiquitin in nf-kappab regulatory pathways. annu. rev. biochem. 78: 769-796, 2009. sluss hk, han z, barrett t, davis rj, ip yt. a jnk signal transduction pathway that mediates morphogenesis and an immune response in drosophila. genes dev. 10: 27452758, 1996. stein d, goltz js, jurcsak j, stevens l. the dorsal-related immunity factor (dif) can define the dorsal-ventral axis of polarity in the drosophila embryo. development 125: 21592169, 1998. stenbak cr, ryu jh, leulier f, pili-floury s, parquet c, herve m, et al. peptidoglycan molecular requirements allowing detection by the drosophila immune deficiency pathway. j. immunol. 173: 7339-7348, 2004. stöven s, ando i, kadalayil l, engström y, hultmark d. activation of the drosophila nf-kb factor relish by rapid endoproteolytic cleavage. embo rep. 1: 347-352, 2000. stöven s, silverman n, junell a, hedengren-olcott m, erturk d, engstrom y, et al. caspasemediated processing of the drosophila nf{kappa}b factor relish. proc. natl. acad. sci. usa 100: 5991-5996, 2003. sun h, towb p, chiem dn, foster ba, wasserman sa. regulated assembly of the toll signaling complex drives drosophila dorsoventral patterning. embo j. 23: 100-110, 2004. sun h, bristow bn, qu g, wasserman sa. a heterotrimeric death domain complex in toll signaling. proc. natl. acad. sci. usa 99: 12871-12876, 2002. swaminathan cp, brown ph, roychowdhury a, wang q, guan r, silverman n, et al. dual strategies for peptidoglycan discrimination by peptidoglycan recognition proteins (pgrps). proc. natl. acad. sci. usa 103, 684-689, 2006. takehana a, katsuyama t, yano t, oshima y, takada h, aigaki t, et al. overexpression of a pattern-recognition receptor, peptidoglycanrecognition protein-le, activates imd/relishmediated antibacterial defense and the prophenoloxidase cascade in drosophila larvae. proc. natl. acad. sci. usa 99: 1370513710, 2002. takehana a, yano t, mita s, kotani a, oshima y, kurata s. peptidoglycan recognition protein (pgrp)-le and pgrp-lc act synergistically in drosophila immunity. embo j. 23: 4690-4700, 2004. tauszig-delamasure s, bilak h, capovilla m, hoffmann ja, imler jl. drosophila myd88 is required for the response to fungal and grampositive bacterial infections. nat. immunol. 3: 91-97, 2002. towb p, galindo rl, wasserman sa. recruitment of tube and pelle to signaling sites at the 173 surface of the drosophila embryo. development 125: 2443-2450, 1998. tsuda m, langmann c, harden n, aigaki t. the ring-finger scaffold protein plenty of sh3s targets tak1 to control immunity signalling in drosophila. embo rep. 6: 1082-1087, 2005. wang l, gilbert rj, atilano ml, filipe sr, gay nj, ligoxygakis p. peptidoglycan recognition protein-sd provides versatility of receptor formation in drosophila immunity. proc natl acad sci usa 105: 11881-11886, 2008. wang l, weber an, atilano ml, filipe sr, gay nj, ligoxygakis p. sensing of gram-positive bacteria in drosophila: gnbp1 is needed to process and present peptidoglycan to pgrpsa. embo j. 25: 5005-5014, 2006. weber an, tauszig-delamasure, s, hoffmann ja, lelievre e, gascan h, ray kp, et al. binding of the drosophila cytokine spatzle to toll is direct and establishes signaling. nat. immunol. 4: 794-800, 2003. werner t, borge-renberg k, mellroth p, steiner h, hultmark d. functional diversity of the drosophila pgrp-lc gene cluster in the response to lipopolysaccharide and peptidoglycan. j. biol. chem. 278: 2631926322, 2003. werner t, liu g, kang d, ekengren s, steiner h, hultmark d. a family of peptidoglycan recognition proteins in the fruit fly drosophila melanogaster. proc. natl. acad. sci. usa 97: 13772-13777, 2000. yano t, mita s, ohmori h, oshima y, fujimoto y, ueda r, et al. autophagic control of listeria through intracellular innate immune recognition in drosophila. nat. immunol. 9: 908-916, 2008. zaidman-remy a, herve m, poidevin m, pili-floury s, kim ms, blanot, d, et al. the drosophila amidase pgrp-lb modulates the immune response to bacterial infection. immunity 24: 463-473, 2006. zhou r, silverman n, hong m, liao ds, chung y, chen zj, et al. the role of ubiquitnation in drosophila innate immunity. j. biol. chem. 280: 34048-34055, 2005. zhuang zh, zhou y, yu mc, silverman n, ge bx. regulation of drosophila p38 activation by specific map2 kinase and map3 kinase in response to different stimuli. cell signal.18: 441-448, 2006. 174 isj 4: 101-xxx, 2007 isj 4: 101-111, 2007 issn 1824-307x review molluscan response to parasite: biomphalaria and schistosoma mansoni interaction d negrão-corrêa1, caj pereira1, fm rosa1, rl martins-souza1, za andrade2, pmz coelho3,4 1departamento de parasitologia, instituto de ciências biológicas (icb), universidade federal de minas gerais (ufmg), belo horizonte,minas gerais, brazil 2centro de pesquisas gonçalo muniz (cpgm-fiocruz), salvador, bahia, brazil 3centro de pesquisas rené rachou (cprr-fiocruz), belo horizonte, minas gerais, brazil 4santa casa de misericórida de belo horizonte, belo horizonte, minas gerais, brazil accepted november 7, 2007 abstract digenetic trematodes use molluscs, almost always a gastropoda, in their evolutive cycle, as intermediary hosts. the genus schistosoma, with three main species that infect humans s. mansoni, s. japonicum, and s. haematobium – shows a prevalence of 200 million patients in various countries worldwide, and 600 million people are still at risk of infection. s. mansoni is the most prevalent species, and biomphalaria snails are its intermediary hosts. although the campaigns of schistosomiasis control based on chemotherapy have reduced the morbidity and prevalence of this disease, transmission continues in almost all the areas submitted to intervention. one of the factors that has influence on the susceptibility of biomphalaria to s. mansoni infection is ability of the host internal defense system (ids) to recognize and destroy the parasite. in biomphalaria, the ids is composed of cellular elements named hemocytes that act jointly with soluble components present in hemolymph, which could affect directly the larvae, or act in the recognition of the parasite, and activation of hemocytes. the susceptibility level of the mollusc has been attributed to the hemocyte capacity of involving and destroying the parasite, and this will be the centre of interest of this review. the study of s. mansoni and biomphalaria interaction in resistant snail strains is important not only due to the academic-scientific value of this fascinating research area, but also to the potentially possible alternatives for the control of this endemia. key words: schistosoma mansoni sporocysts; biomphalaria glabrata; biomphalaria tenagophila; circulating hemocytes; soluble factors of hemolymph introduction although the great majority of the living beings is represented by invertebrates, up to now the publications in mass dealing with the defense mechanisms against pathogens is practically restricted to interactions between pathogens of vertebrate animals. the invertebrate animals must necessarily reckon upon their defense system to recognize and destroy infectious agents, although this system are not able to generate the diversity of ___________________________________________________________________________ corresponding author: deborah negrão-correa departamento de parasitologia, instituto de ciências biológicas universidade federal de minas gerais avenida antonio carlos 6627, belo horizonte, mg, brazil campus pampulha – zip code: 31270-901 e-mail: denegrao@icb.ufmg.br recognition observed during the adaptative immune response of vertebrates (van der knaap and loker, 1990; loker et al., 2004). recent studies have demonstrated many similarities between the innate defense response of vertebrates and the internal defense system of invertebrates (hoffman et al., 1999; hoffman, 2003), being identified in various invertebrates organisms production of complement-like proteins, antimicrobial peptides, pattern-recognition receptors (prrs) such as toll-like receptor and c-type lectins, phagocytic cells, production of highly toxic metabolites of oxygen and nitrogen (loker et al., 2004). although many similarities were identified in the defense system of several groups of invertebrates, it is important to note that genomic studies have indicated varied defense mechanisms in invertebrate groups phylogenetically associated, 101 mailto:denegrao@icb.ufmg.br but with very different alimentary habits and habitat (loker et al., 2004). several groups of invertebrates, such as insects and molluscs, are important intermediary hosts of parasites species that are transmitted to humans and domestic animals. as an example we can mention the necessity of diptera insects genus lutzomya in the development of different species of leishmania, the anophelins in the transmission of malaria or the anophelins and culicides for the development of lymphatic filariosis. among the molluscs, gastropoda are obligatorily intermediary hosts in the development of the majority of digenetic trematode species, such as s. mansoni and fasciola hepatica. thus, it is of the utmost importance to get a better understanding of the effector mechanisms used by the internal defense system (ids) of these invertebrates for the development of new strategies related to the control of these parasite infections. these studies have also contributed significantly to a better knowledge about the innate response of vertebrates. this review will focus primarily on the response of biomphalaria during infection with s. mansoni. schistosoma mansoni infection in biomphalaria schistosomiasis is an important health problem that affects over 200 million people worldwide. among the schistosomes species that infect human beings, s. mansoni is transmitted by biomphalaria snails and causes intestinal and hepatic schistosomiasis in africa, arabian peninsula, and south america (gryseels et al., 2006). recent estimative indicates that 6-7 million people are infected by s. mansoni only in brazil (katz and peixoto, 2000). s. mansoni infects biomphalaria by means of active penetration of the parasite´s ciliated larvae, named miracidia, at any site of the snail’s exposed parts, frequently the base of the antennae and cephalopodal mass. in the process of penetration, the parasite undergoes morphological and physiological changes, being transformed into primary sporocyst that remains in the fibro-muscular tissue of the host´s cephalopodal region near the penetration site. the primary sporocysts generate the secondary ones, which migrate from the cephalopodal musculature to the digestive glands or hepatopancreas of the mollusc, where they undergo profound anatomic changes and their germinative cells can generate the cercariae (maldonado and acosta-matienzo, 1947; pan, 1965; pereira et al., 1984). in brazil, out of ten species of molluscs genus biomphalaria described, only three were found naturally infected by s. mansoni: b. glabrata, b. tenagophila and b. straminea (paraense, 2001). the susceptibility level of these different species of biomphalaria to infection with the same lineage of s. mansoni is much diversified, and b. glabrata may present up to 75.3 % of susceptibility in experimental infections, b. tenagophila 32.6 %, and b. straminea 11.3 %, as demonstrated by souza et al. (1997). besides the difference in susceptibility observed among biomphalaria species compatible with the parasite, some lineages or geographic isolates of a same species of biomphalaria also present a great variation of susceptibility to the parasite. as far as b. tenagophila is concerned, the geographic lineage isolated at the biological reservoir in taim (rio grande do sul, brazil) was found to be completely resistant to the development of all s. mansoni isolates already tested. the character of resistance of this b. tenagophila lineage has been explored at our laboratory, aiming at studying the possible mechanisms of the parasite’s destruction, representing a potential model for the control of transmission in endemic areas, where b. tenagophila is the unique transmitter agent of the disease (coelho et al., 2004). genetic control of resistance of biomphalaria to s. mansoni infection the compatibility between s. mansoni and its intermediary host is influenced by behavioral and physiological factors of the mollusc. once found a suitable host, the susceptibility level of biomphalaria to s. mansoni can be determined by the genetic differences of the molluscs, as well as by the genetic constitution of schistosoma (basch, 1976). newton (1952, 1953) demonstrated for the first time that the susceptibility of b. glabrata snail to s. mansoni depends largely upon genetic factors. later, these results were corroborated by richards (1970), who demonstrated that the resistance character, acquired at the maturity phase, is determined by a single dominant gene, with mendelian inheritance. nevertheless, in b. glabrata, age is a determinant factor of the snail susceptibility; juvenile snails being more susceptible to infection even in lineages where the adult snail is resistant to s. mansoni infection. thus, the susceptibility of juvenile b. glabrata to s. mansoni infection is also regulated by genetic factors, being estimated that four or more genes affect this character (richards, 1977). b. tenagophila taim is a lineage that presents absolute resistance to infection by all s. mansoni strains tested at any phase of the mollusc development (santos et al., 1979; martins-souza et al., 2003; coelho et al., 2004). crossbreedings between b. tenagophila taim and b. tenagophila bh (susceptible lineage) showed that the character of resistance to s. mansoni infection is dominant (santos et al., 1979). the dominance of the resistance character of b. tenagophila taim was confirmed in crossbreedings with the susceptible joinville lineage (rosa et al., 2005), showing that 100 % of the f1 offspring were resistant and only 8 % of the f2 offspring were susceptible to infection by the parasite (table 1). this study suggests that at least two dominant genes would be responsible for the resistance to s. mansoni observed in b. tenagophila taim, one of them being the most important since it was expressed in all f1 offspring (rosa et al., 2005). the dominance of the resistance character in b. tenagophila taim added to the reproductive success of this lineage in the presence of susceptible snails of the same species (rosa et al., 2006), led us to suggest the use of this lineage as an alternative for biological control in 102 table 1 susceptibility rates of b. tenagophila joinville, b. tenagophila taim, f1 and f2 when submitted to infection with 25 miracidia of le strain of s. mansoni experiment group number of snails exposed to s. mansoni number of surviving snails number of infected snails eliminanting cercariae (%) 1 taim 64 60 0 (0) joinvillle 35 12 7 (58.3) f1 170 150 1 (0.6) f2 110 87 7 (8) 2 taim 30 25 0 (0) joinville 30 15 9 (60) f1 50 44 0 (0) f2 50 38 2 (5.3) some areas of schistosome transmission (coelho et al., 2004). one of the factors that influence the susceptibility and may be genetically determined is the activity of the snails ids. experimental infections in b. tenagophila taim have shown that s. mansoni miracidia are able to penetrate this snail lineage, however the parasites induce an intense cellular infiltration and are rapidly destroyed, suggesting an important participation of the ids on determination of resistance to s. mansoni in b. tenagophila taim. internal defense system (ids) of the mollusc the ids of snails is composed of cellular elements constituted by hemocytes, and by soluble factors present in hemolymph. the hemocytes may be circulating in hemolymph or fixed in tissues. in planorbids the hemolymph circulates in a semi-open system impelled by the heart. the hemoymph leaves the heart through the aorta reaching the tissues, draining in the venous sinus and returning to the heart via the pulmonary and renal veins, after being re-oxygenated in the pulmonar wall (baker, 1945). the heart, enclosed by the pericardium membrane, is divided into two chambers, the auricula, which receives hemolymph from the pulmonary cavity, and the ventricule that impels the hemolymph through the aorta. the aorta is divided into two arteries: the visceral artery, which irrigates the posterior part of the snail´s body, including the digestive and genital systems, and the cephalic artery, that reachs all the cephalopodal region. the arteries are exhausted in the pseudovascular spaces of the tissues, accumulating hemolymph in three venous sinuses: cephalopodal, visceral and sub-renal, returning to the heart after circulating through the kidney and lung (baker, 1945; paraense, 2001). in b. glabrata and bulinus sp a well defined region, located between the pericardium and the posterior epithelium of the mantle cavity (fig. 1a), also called amebocyte producing organ (apo), was identified as the main site for the production of hemocytes (lie, 1976). recent observations (sullivan et al., 2004; sullivan and castro, 2005) showed an increase of mitoses in the cells of this region, ranging from 24-72 h after inoculation of antigens of s. mansoni miracidia or cercariae, this being more evident in resistant lineage of b. glabrata. nevertheless, some authors (matricongondran, 1990; souza and andrade, 2006) demonstrated that b. glabrata hemocytes may present multi-centric origin, and sites with proliferation of hemocytes were detected also at the saccular portion of the renal tubules and in the ventricular cavity of the heart (fig. 1b). the circulating hemocytes of different species of molluscs present morphological and functional heterogeneity. according to ottaviani (1992; 2006), the population of circulating hemocytes of the majority of gastropod molluscs is constituted by two cellular types: the starry hemocytes that emit pseudopodes, and the roundish hemocytes. in planorbarius corneus, the starry hemocytes are cells that present phagocytic activity, adhere to glass and express proteins that are recognized by pro-inflammatory anti-cytokine antibodies of vertebrates. on the other hand, the roundish hemocytes are not endowed with phagocytic activity, they are not able to adhere to glass, and besides they proliferate in the presence of phytoagglutinin (ottaviani, 1992; ottaviani et al., 1993). similarly to p. corneus, the majority of the authors (harris, 1975; lie et al., 1987; barraco et al., 1993; borges and andrade, 2003) also distinguish two sub-populations of circulating hemocytes in hemolymph of b. glabrata. these subpopulations are called granulocytes, i.e., the hemocytes that emit pseudopodes and produce phagocytosis, and hyalinocytes that are the small and roundish hemocytes (fig. 2a). granulocytes can be easily identified by the uptake of neutral red stain into the cell vesicles (fig. 2b), showing that s. mansoni infection induce cellular proliferation (martins-souza et al., 2003). however, ultrastructural studies (matricon-gondran and letorcart, 1999 a,b), analyses of distribution and abundance of lysosomal enzymes (granath and yoshino, 1983), as well as of expression of lectin-ligants on the cellular surface (joky et al., 1983; martinssouza et al., 2006) suggest that the circulating hemocytes of biomphalaria constitute a cellular population significantly more heterogenous than that previously described (fig. 2b). the phenotypical and functional definition of biomphalaria hemocytes is of fundamental importance to understand the participation of these cells, or of any cellular subpopulation, in the destruction mechanism of s. mansoni larvae or other parasites. even though hemocytes are the main component of the mollusc ids, t here are some experimental 103 fig. 1 photomicrographs of biomphalaria glabrata heart region. (a) snail heart tissue in close contact with the pericardial membrane. the arrows indicate the amebocyte-forming organ (apo), a narrow and long band of epithelial-like cell along the pericardial membrane (100x). (b) heart tissue with a dense collection of hemocytes (arrow) (200x). hematoxylin-eosin stain evidences indicating that soluble elements of the hemolymph would participate in the protective mechanism against pathogens. soluble components of the hemolymph of molluscs can directly interact with pathogenic agents, by means of production of toxic substances or lytic peptides, or indirectly through mediator molecules for recognition of the pathogen or hemocyte activators. peptides with anti-microbial function, called mytilines, are produced and stored in hemocyte granules, and they are secreted in hemolymph of mytilus galloprovincialis (mitta et al., 2000) notwithstanding the participation of these peptides in the destruction of bacterial infections, there are no evidences of the participation of these peptides in the interaction of mollucs with metazoan parasites. fryer and bayne (1996) show that particles of polystireno treated with soluble factors of hemolymph of b. glabrata are significantly more phagocyted by hemocytes than the untreated particles, demonstrating that soluble factors of hemolymph may participate of the recognition mechanism and opsonization of particles by hemocytes. johnston and yoshino (1996) demonstrate that lectins similar to those of conavalia ensiformis (cona), erythrina corallodendrom (eca), glycine max (sba), tetragonolobus purpureas (tpa), and triticum vulgaris (wga) are present in hemolymph of b. glabrata. in molluscs, lectins are synthesized by hemocytes and released in hemolymph, where they immobilize the material particularized by agglutination, or are expressed at the surface of circulating hemocytes, where apparently they act as cytophylic receptors (richards and renwrantz, 1991; fryer and bayne, 1989). besides the lectins, other proteins with homologous function to cellular mediators, and already characterized in vertebrates, have been identified in hemolymph of molluscs and may be involved in the activation of hemocytes during infection by digenetic trematodes (ottaviani et al., 1993, 1995). ottaviani and co-workers (1993) reported the presence of a variety of proteins similar to pro-inflammatory cytokines of vertebrates, including interleukin-1 alpha (il-1α), interleukin-1 beta (il-1β), interleukin-2 (il-2), interleukin-6 (il-6), and alpha tumoral necrosis factor (tnf-α) in hemocytes of two species of molluscs, planorbarius corneus and viviparus ater, being present only in hemocytes with phagocytic activity. in further studies, ottaviani et al. (1995), relate the production of homologous proteins to cytokine with an increase of phagocytic activity and induction of nitric oxide synthase (nos) of mollusc hemocytes. these results suggest that cytokines-like may participate in the activation of hemocytes. functional mechanisms in parasite-mollusc interaction in the last years, many aspects of the interaction between the digenetic trematode larvae and the internal defense system of molluscs have been elucidated. nevertheless, the possible mechanisms responsible for destruction of the majority of larvae in resistant snails remain to be totally understood. the results reported up to now suggest that the hemocyte could be the effector element in the destruction mechanism of trematodes, being directly involved in the death of some encapsulated parasites (van der knaap and loker, 1990; bayne et al., 2001) or in the production of soluble factors which could be cytotoxic (connors et al., 1995). the majority of the authors (connors et al., 1995; bayne et al., 2001; martins-souza, 2003) agree that the snails´ defense generally occurs by means of destruction, total or partial, of the primary sporocyst at the first few hours following the penetration of the miracidium. the existence of a cellular defense mechanism deployed by molluscs against trematode infection was initially suggested by the finding of histological reactions around parasite sporocysts (newton, 1952). further studies have shown that hemocytes infiltration around parasite larvae in s. mansoni 104 fig. 2 morphological aspects of circulating hemocytes from biomphalaria tenagophila. (a) phase contrast photomicrography showing a granulocyte (g) and a hyalinocytes circulating in b. tenagophila hemolymph (400x). (b) bright field photomicrography of b. tenagophila circulating hemocytes after addition of neutral red staining, showing the heterogeneous granulocyte population stained in red (g) and the non-stained cells designated as hyalinocytes (h) (200x) infected biomphalaria was stronger in snail species that are more resistant to parasite infection, such as b. tenagophila and b. straminea (souza et al., 1997). in highly susceptible b. glabrata, confirmed by the great quantity of cercariae eliminated during a long period of time, sporocysts and cercariae at different developmental stages are found in abundance in the inner of the host, resulting in compression of the host´s structures, mainly in the interstice of the digestive glands, ovotestis and renal tubules. however, the presence of a great number of parasites did not induce cellular reaction in the parasite susceptible snail strains (godoy et al., 1997). on the opposite extreme appear the resistant snails, which shed a few cercariae and show an extensive infiltration in tissues with numerous hemocytes, frequently placed around the parasite structures in disintegration. the focal reactions frequently assumed a granuloma-like appearance (godoy et al., 1997). in our experimental model, s. mansoni infection in b. tenagophila of taim strain resulted in intense, diffuse or granuloma-like cellular infiltration in the infection site, mainly in the connective tissue of the snail cephalopodal region and antennae (fig. 3). the cellular infiltration was detected few hours after the parasite infection and no viable sporocysts were recovered from these infected snails. direct evidence of the hemocyte participation in s. mansoni infection control was obtained with experiments that transferred the apo from resistant to susceptible snail strains. in b. glabrata, the transplantation of apo from miracidiaexposed resistant strain to susceptible nih snails resulted in significantly more killed sporocysts than as observed during s. mansoni infection in nih snails (sullivan and spencer, 1994). recently, barbosa et al. (2006) showed in b. tenagophila that transplantation of the hematopoietic organ from taim lineage (totally resistant) to a susceptible s. mansoni strain resulted in an absolute resistance in the receptors whose transplant was successful. moreover, inoculation of silica particles in b. tenagophila cabo frio resulted in transitory reduction of a macrophage-like cell population of circulating hemocytes. the cellular depletion induced by silica-treatment in b. tenagophila cabo frio was accomplished by enhanced susceptibility to s. mansoni infection, shortening the intramolluscan phase of the parasite and increasing the number of sporocysts and cercariae produced (martins-souza et al., 2003). the process of destruction of s. mansoni larvae by hemocytes initiates with the recognition and encapsulation of the newly-penetrated sporocyst. bayne and co-workers (1980b) demonstrated that cell-free hemolymph obtained from susceptible and resistant b. glabrata strains are unable to change visibly the morphology of s. mansoni in vitro, the same occurring with hemolymph containing hemocytes of susceptible lineages. however, hemocytes of susceptible strains associated with soluble factors of hemolymph of resistant b. glabrata lineages acquire the ability to destroy s. mansoni sporocysts. the importance of the soluble fraction of biomphalaria hemolymph in the destruction mechanism of s. mansoni sporocysts was also confirmed in vivo in studies dealing with the transference of this fraction obtained from resistant b. glabrata snails to other ones susceptible to the parasite (granath and yoshino, 1984). recent results obtained with b. tenagophila taim also showed that addition of the cell-free hemolymph of this resistant snail strain significantly increased the ability of hemocytes from susceptible strain of b. tenagophila (cabo frio or joinville) to destroy s. mansoni sporocysts, in vitro. moreover, the increased mortality of sporocysts was associated with higher number of hemocytes on the parasite tegument (fig. 4), suggesting that cell-free hemolymph from resistant snail strain increased parasite recognition by hemocytes. however, in contrast with b. glabrata (bayne et al., 1980), cell-free 105 fig. 3 intense hemocyte infiltration in schistosoma mansoni infected biomphalaria tenagophila taim, a parasite-resistant snail strain. photomicrograph of a transversal section of antennae tissue from b. tenagophila taim 15 days after s. mansoni infection (20 miracidium/snail), showing a focal cellular reaction (arrow) with a granuloma-like appearance (200x). diffuse and focal cellular infiltration is observed in cephalopodal tissue of parasite infected resistant snails and has been associated with the parasite penetration and destruction. hematoxylineosin stain hemolymph from b. tenagophila taim was able to destroy a small, but statistically significant, percentage of s. mansoni sporocysts even in absence of hemocytes (pereira, 2005). the importance of b. tenagophila taim cell-free hemolymph in s. mansoni control was also confirmed in vivo, since susceptible snails treated with b. tenagophila taim cell-free hemolymph had lower percentage of infectivity (pereira, 2005; coelho and bezerra, 2006), and the snails that got infection produced lower number of sporocysts and cercariae (pereira, 2005). the main components of s. mansoni sporocysts tegument is glicoproteins and glicolipides (zelck and becker, 1990; uchikawa and loker, 1991). johnston and yoshino (1996) showed that lectins from b. glabrata cell-free hemolymph bind to glicoproteins extracted from the parasite tegument. more recently (adema et al., 1997a,b) a group of proteins, homologous to fibrogen and that has been associated with recognition, was identified in b. glabrata hemolymph, being its expression enhanced after infection of the mollusc with echinostoma paraensei, another digenetic trematode. these results suggested that lectins would serve as cell surface receptors for carbohydrate structures from trematode parasites. moreover, soluble lectins would also participate in the recognition mechanism by binding to carbohydrate structures from both hemocytes and parasite tegument (van der knapp et al., 1990). besides participating in the recognition of s. mansoni, lectins can also activate hemocytes. hemocytes of susceptible and resistant b. glabrata strains were stimulated with bovine albumin associated with one of the six carbohydrates: mannose, galactose, fucose, n-acetyl-glucosamine, n-acetyl-galactosamine and lactose, that are present in the tegument of s. mansoni sporocysts. hemocytes stimulated with bsa-galactose, bsamannose, and bsa-fructose were able to produce reactive oxygen-species (ros) (hahn et al., 2000). our results also confirmed the participation of soluble lectins in s. mansoni-sporocysts recognition mechanisms by b. tenagophila species. circulating hemocytes recovered from b. tenagophila both taim and cabo frio strains were intensively labelled by fitc-conjugated lectins, such as pna, sba, and wga. moreover, s. mansoni infection in resistant snail strain (taim) resulted in initial reduction in number of labelled-hemocytes in circulation (martins-souza et al., 2006). the reduction of circulating hemocytes during the first few hours after s. mansoni infection has been associated with the cell recruitment to the infection site (bezerra et al., 1997; martins-souza, 2003). other proteins similar to pro-inflammatory cytokines of vertebrates have been identified in hemolymph of molluscs and can participate in activation of hemocytes during the destruction process of parasites. specifically in b. glabrata was identified a protein with immunological and functional similarity to interleukin-1-like (il-1 like). in this snail, il1-like protein is induced by s. mansoni infection, and the level was significantly higher in resistant snail strains (granath et al., 1994). the inoculation of human recombinant il-1α in susceptible strain of b. glabrata resulted in increased production of ros by circulating hemocytes and reduced number of s. mansoni cercariae upon parasite infection (connors et al., 1995). these authors confirmed, in vitro, that cellfree hemolymph recovered from rhil-1α-treated snail, but not only rhil-1α, was capable of destroy s. mansoni sporocysts, suggesting that il-1 would activate b. glabrata-hemocytes to produce and secrete soluble cytotoxic mediators (connors et al., 1998). the effector mechanisms by which activated hemocytes are able to kill trematode larvae are not fully understood yet. dikheboom et al. (1988a,b) showed for the first time that gastropod hemocytes do produce reactive oxygen species (ros) in response to trematode infection. the initial reduction of o2 to superoxide anion (o2) is catalyzed by nadph-oxidase and o2 can be converted to other ros, including hydrogen peroxide (h202) and hypochlorous acid (hoci) (hampton et al., 1998; bayne et al., 2001). nadph oxidase-like activity has been identified in gastropod hemocytes (adema et al., 1993) and ros production 106 fig. 4 interaction of schistosoma mansoni sporocyst and hemocytes from biomphalaria tenagophila taim (b) or biomphalaria glabrata bh (c). (a) s. mansoni sporocyst in chernin’s balanced salt solution, showing an intact parasite larva (200x). (b) s. mansoni sporocyst completely encapsulated by hemocytes from b. tenagophila taim, a snail strain totally resistant to parasite infection (100x). (c) s. mansoni sporocyst incubated with hemocytes from b. glabrata bh, snail highly susceptible to parasite infection, showing an intact parasite with very few cell attached, x100 by b. glabrata hemocytes has been associated with s. mansoni resistance (adema et al., 1994). additionally, molluscan hemocytes also generate nitric oxide (no) from molecular oxygen and arginine (conte and ottaviani, 1995; arumugan et al., 2000). experimental evidences of ros and/or nos participation in killing of s. mansoni sporocysts by b. glabrata hemocytes were obtained using specific oxidant scavengers or enzyme inhibitors during the in vitro association. the results showed that inhibition of h2o2 and no production do favor sporocysts survival, indicating that this reactive species would be toxic to trematode larvae (hahn et al., 2001 a,b). however, attempts to associate h2o2 or no production with snail strain susceptibility to s. mansoni infection have not been conclusive (hahn et al., 2000; bayne et al., 2001). similarly, we were able to estimate no production by hemocytes isolated from b. glabrata or b. tenagophila at different time after s. mansoni infection and restimulated in vitro with s. mansoni egg antigen (sea). at each experimental point, as well as for each snail strain, analyses were performed in triplicate with total hemolymph collected and gathered together from 3 snails. after centrifugation, the hemocyte pellet was ressuspended in chernin´s balanced salt solution (5 x 105/ml of cbss) containing 100 μg/ml of sea, plated in 96-well tissue culture plates and incubated at 26 °c and 5 % co2. after 18 h of incubation, no presence was assayed directly in the cell supernatant by the greiss reaction (green et al., 1982) that quantifies the nitrite contents of the supernatants, as detailed by perreira et al. (2006). the no level increased in hemocytes recovered after the first few days of s. mansoni infection, however there were no detectable differences in no level between the snails, although each species or strain shows remarkable difference in parasite susceptibility (fig. 5). however, it is important to confirm if supernatant level do reflect the local production in hemocytesporocyts interaction. besides producing reactive species of oxygen and nitrogen, microscopy analyses indicate that hemocytes from parasiteresistant snails would phagocvte portions of sporocyst tegument leading to mechanical lesion that may be also lethal to the parasite (van der knaap and loker, 1990). finally, in order to evaluate the efficiency of biomphalaria spp defense system in the destruction of s. mansoni larvae, one must consider that there are many evidences indicating that the parasite is able to develop strategies to allow its evasion. thus, it has been described that the primary sporocyst of s. mansoni acquires quickly the antigens present in the host´s hemolymph (bayne et al., 1986), as well as express in the tegument antigens similar to those expressed by the host´s cells (yoshino and bayne, 1983) hindering the recognition process of the parasite by hemocytes. it has been also reported that components of the excreted/secreted material by the miracidium during the transformation process may reduce the motility of hemocytes, as well as their phagocytic capacity (connors and yoshino, 1990; lodes and yoshino, 1990), thus justifying the cellular reaction almost inexistent observed around s. mansoni sporocysts present in the tissue of b. glabrata susceptible strains. in addition to avoid the hemocytes´ approach, it has been also reported that sporocysts incubated in vitro with hemocytes of susceptible strains may be encapsulated, but not destroyed (boehmler et al., 1996; hahn et al., 2001a), suggesting the existence of anti-oxidant mechanisms. perspectives the most recent studies related to the internal defense system of invertebrates have shown new aspects of the invertebrate-pathogen relationship. these aspects afforded us a better understanding of the recognition and cellular activation mechanisms, which are also present in the innate defense response of vertebrates. biomphalaria-schistosoma mansoni interaction, besides being an important model in human health, constitutes an experimental approach that may add important information to the knowledge of the defense mechanism utilized by invertebrates, as well as of phylogenetic evolution of these mechanisms. in this context, our group has carried out researches dealing with the phenotypic and functional 107 fig. 5 nitric oxide (no) level in supernatant of circulanting hemocytes recovered from biomphalaria glabrata, b. tenagophila cabo frio e b. tenagophila taim during the s. mansoni infection. no levels was indirectly estimated by quantification of nitrite, using the greiss reaction, in hemocyte supernatant recovered after 18 h of in vitro restimulation with soluble egg antigen (sea 100 μg/ml). *** for p < 0.001 when comparing with nitrite level obtained after stimulation of non-infected hemocytes from the same snail strain. at each time point, there was no statistically significant difference in the nitrite level between the snail strains characterization of the hemocytes and hemolymph from different strains of b. glabrata and b. tenagophila. our goal is to identify possible mechanism of trematode recognition and hemocyte activation in b. tenagophila taim that would be responsible to the fast parasite destruction and consequent resistance against s. mansoni infection observed in this snail strain. in parallel, we are investigating the heritage of the resistance character from b. tenagophila taim to the offspring resulted of crossbreeding with the susceptible snail strains. a more comprehensive identification of these mechanisms would expand the theorical base that gives support to mass introduction of b. tenagophila resistant strain from taim in areas where transmission is maintained by this species (coelho et al., 2004). acknowledgement this work was supported by grants from fundação de amparo à pesquisa do estado de minas gerais (fapemig), and pronex (cnpq/fapemig). acknowledgement is also due to jeferson do carmo bernardes, josé carlos reis dos santos and selma fernandes for the technical support in the experiments. the authors are also grateful to mrs vera de paula ribeiro for reviewing the text and dr ary corrêa jr for the support in image production and documentation. references adema cm, arguello df, stricker sa, loker es. a time-lapse study of interactions between echinostoma paraensei intramolluscan larval stages and adherent hemocytes from biomphalaria glabrata and helix aspersa. j. parasitol. 80: 719-727, 1997a. adema cm, hertel la, miller rd, loker es. a family of fribrinogen related proteins that precipitates parasite derived molecules is produced by an invertebrate after infection. proc. natl. acad. sci. usa 94: 8691-8696, 1997b. adema cm, van deutekom-mulder ec, van der knaap wpw, sminia t. schistosomicidal activities of lymnaea stagnalis hemocytes: the role of oxygen radicals. parasitology. 109: 479485, 1994. adema cm, van deutekom-mulder ec, van der knaap wpw, sminia t. nadph-oxidase activity the probable source of reactive oxygen intermediate generation in hemocytes of the gastropod lymnaea stagnalis. j. leukocyte biol. 54: 379-383, 1993. arumugam m, romestand b, torreilles j, roch p. in vitro production of superoxide and no (as nitrite and nitrate) by mytilus galloprovincialis haemocytes upon incubation with pma or laminarin or during yeast phagocytosis. eur. j. cell biol. 79:513-519, 2000. baker fc. general morphology. in: the molluscan family planorbidae. university of illinois press, 1945. barbosa l, caldeira rl, carvalho os, vidigal thda, jannotti-passos l k, coelho p z. resistance to schistosoma mansoni by transplantation of apo biomphalaria tenagophila. parasite immunol. 28:209-212, 2006. barraco ma, steil aa, gargioni r. morphological characterization of the hemocytes of the pulmonate snail biomphalaria tenagophila. mem. inst. oswaldo cruz 88: 73-83,1993. basch pf. intermediate host specificity in schistosoma mansoni. exp. parasitol. 39: 150169, 1976. bayne cj, buckley pm, dewan pc. macrophagelike hemocytes of resistant biomphalaria glabrata are cytotoxic for sporocysts of schistosoma mansoni in vitro. j. parasitol. 66: 413-419, 1980a. bayne cj, buckley pm, dewan pc. schistosoma mansoni: cytotoxicity of hemocytes from susceptible snail hosts for sporocysts in plasma from resistant biomphalaria glabrata. exp. parasitol. 50: 409-416, 1980b. bayne cj, loker es, yui ma. interactions between plasma proteins of biomphalaria glabrata (gastropoda) and the sporocyst tegument of schistosoma mansoni (trematoda). parasitology 92: 653-664, 1986. bayne cj, hahn uk, bender rc. mechanisms of molluscan host resistance and of parasite strategies for survival. parasitology 123, s159s167, 2001. bezerra fsm, nogueira-machado ja, chaves mm, martins rl, coelho pmz. quantification of the number and phagocytary activity of hemocytes of resistant and susceptible strains of biomphalaria glabrata and biomphalaria tenagophila infected with schistosoma mansoni. rev. inst. med. trop. 39: 197-201, 1997. 108 boehmler am, fryer se, bayne cj. killing of schistosoma mansoni sporocysts by biomphalaria glabrata hemolymph in vitro: alteration of hemocyte behavior after poly-llysine treatment of plastic and the kinetics of killing by different host strains. j. parasitol. 82: 332-335, 1996. borges cmc, andrade za. extra-cellular matrix changes in schistosoma mansoni-infected biomphalaria glabrata. mem. inst. oswaldo cruz 98: 135-139, 2003. coelho jr and bezerra fs. compatibility of biomphalaria tenagophila with schistosoma mansoni: a study of homologous plasma transference. mem. inst. oswaldo cruz 101:111-112, 2006. connors va, de buron i, granath wo jr. schistosoma mansoni: interleukin-1 increases phagocytosis and superoxide production by hemocytes and decreases output of cercariae in schistosome-susceptible biomphalaria glabrata. exp. parasitol. 80: 139-148, 1995. connors va, de buron i, jourdane j, theron a, agner a, granath wojr. recombinant human interleukin-1-mediated killing of schistosoma mansoni primary sporocysts in biomphalaria glabrata. j. parasitol. 84: 920-926, 1998. connors va, yoshino tp. in vitro effect of larval schistosoma mansoni excretory-secretory products on phagocytosis-stimulated superoxide production in hemocytes from biomphalaria glabrata. j. parasitol. 76: 895-902, 1990. conte a, and ottaviani e. nitric oxide synthase activity in molluscan hemocytes. febs lett. 365:120-124, 1995. dikkeboom r, van der knaap wp, van der bovenkamp w, tijnagel jm, bayne cj. the production of toxic oxygen metabolites by hemocytes of different snail species. dev. comp. immunol.12: 509-520, 1988a. dikkeboom r, bayne cj, van der knaap wp, tijnagel jm. possible role of reactive forms of oxygen in in vitro killing of schistosoma mansoni sporocysts by hemocytes of lymnaea stagnalis. parasitol. res. 75:148-154, 1988b. fryer se, bayne cj. opsonization of yeast by the plasma of biomphalaria glabrata (gastropoda): a strain specific, time-depend process. parasite immunol. 11: 269-278, 1989. fryer se, bayne cj. phagocytosis of latex beads by biomphalaria glabrata hemocytes is modulated in a strain-specific manner by absorbed plasma components. dev. comp. immunol. 20: 323-337, 1996. godoy a, souza cp, guimarães ct, andrade za. unusual histological findings in biomphalaria glabrata with high degree of resistance to schistosoma mansoni miracidia. mem. inst. oswaldo cruz 92: 121-122, 1997. granath wo jr, connors va, tarleton rl. interleukin-1 activity in hemolymph from strains of the snail biomphalaria glabrata varying in susceptibility to the human blood fluke, schistosoma mansoni: presence, differential expression, and biological function. cytokine 6:21-27, 1994. granath wo jr, yoshino tp. characterization of molluscan phagocyte subpopulations based on lysosomal enzyme markers. j. exp. zool. 205226, 1983. granath wo jr, yoshino tp. schistosoma mansoni: passive transfer of resistance by serum in the vector snail, biomphalaria glabrata. exp. parasitol. 58:188-193, 1984. green lc, wagner da, glogowski j, skipper pl, wishnok jk, tannenbaum s. analysis of nitrate, nitrite, and ( 15n) nitrate in biological fluids. anal. biochem. 126: 131-138, 1982. gryseels b, polman k , clerinx j , kestens l. human schistosomiasis. lancet 368: 1106– 1118, 2006. hahn uk, bender rc, bayne cj. production of reactive oxygen species by hemocytes of biomphalaria glabrata: carbohydrate-specific stimulation. dev. comp. immunol. 24: 531541, 2000. hahn uk, bender rc, bayne cj. involvement of nitric oxide in killing of schistosoma mansoni sporocysts by hemocytes from resistant biomphalaria glabrata. j. parasitol. 87: 778785, 2001a. hahn uk, bender rc, bayne cj. killing of schistosoma mansoni sporocysts by hemocytes from resistant biomphalaria glabrata: role of reactive oxygen species. j. parasitol. 87: 292-299, 2001b. hampton mb, kettle aj, winterbourn cc. inside the neutrophil: oxidants myeloperoxidase, and bacterial killing. blood 92: 3007-3017, 1998. harris kr. the fine structure of encapsulation in biomphalaria glabrata. ann. n. y. acad. sci. 266: 446-464, 1975. hoffmann ja, kafatos fc, janeway ca, ezekowitz ra. phylogenetic perspectives in innate immunity. science 284:1313-1318, 1999. hoffmann ja. the immune response of drosophila. nature 426: 33-38, 2003. johnston la, yoshino tp. analysis of lectinand snail plasma-binding glycopeptides associated with the tegumental surface of the primary sporocysts of schistosoma mansoni. parasitology 112: 469-479, 1996. joky a, matricon-gondran m, benex j. fine structural differences in the amoebocytes of biomphalaria glabrata. dev. comp. immunol 7: 669-672, 1983. katz n, peixoto sv. critical analysis of the estimated number of schistosomiasis mansoni carriers in brazil. rev. soc. bras. med. trop. 33: 303-308, 2000. lie kj, jeong kh, heyneman d. molluscan host reactions to helminthic infection. in: soulsby ejl (ed), protozoa, arthropods and invertebrates, crc-prees inc., boca raton, florida, usa, pp 211-270, 1987. lie kj, heyneman d, jeong kh. studies on resistance in snails. 4. induction of ventricular capsules and changes in amebocyteproducing organ during sensitization of biomphalaria glabrata snails. j. parasitol. 62: 286-291, 1976. 109 lodes mj, yoshino tp. the effect of schistosome excretory-secretory products on biomphalaria glabrata hemocytes motility. j. invertebr. pathol. 56: 75-85, 1990. loker es, adema cm, zhangs-m, kepler tb. invertebrate immune systems not homogeneous, not simple, not well understood. immunol. rev. 198: 10-24, 2004. maldonado jf, acosta-matienzo j. evolution del schistosoma mansoni dentro de su hosped intermediario, el caracol australorbis glabratus. puerto rico j. pub. health trop. med. 22: 374404, 1947. martins-souza rl, pereira ca, coelho pm, negrão-corrêa d. silica treatment increases the susceptibility of the cabo frio strain of biomphalaria tenagophila to schistosoma mansoni infection but does not alter the natural resistance of the taim strain. parasitol. res. 91: 500-507, 2003. martins-souza r, pereira caj, martins-filho oa, coelho pmz, corrêa jra, negrão-corrêa d. differencial lectin labelling of circulating hemocytes from biomphalaria glabrata and biomphalaria tenagophila resistant or susceptible to schistosoma mansoni infection. mem. inst. oswaldo cruz 101 (suppl. 1): 185192, 2006. matricon-gondran m, letocart m. internal defenses of the snail biomphalaria glabrata i. caracterization of hemocytes and fixed phagocytes. j. invertebr. pathol. 74: 224-234, 1999a. matricon-gondran m, letocart m. internal defenses of the snail biomphalaria glabrata ii. defense cells have different phagocytic responses to various injected foreign materials. j. invertebr. pathol. 74: 235-247, 1999b. matricon-gondran m. the site of ultrafiltration in the kidney sac of the pulmonate gastropod biomphalaria glabrata. tissue cell. 22: 911923, 1990 mitta g, vandenbulcke f, hubert f, salzet m, roch p. involvement of mytilins in mussel antimicrobial defense. j. biol. chem. 275: 12954-12962, 2000. newton wl. the comparative tissue reaction of two strains of australorbis glabratus to infection with schistosoma mansoni. j. parasitol. 38: 362-366, 1952. newton wl. the inheritance of susceptibility to infection with schistosoma mansoni in autralorbis glabratus. exp. parasitol. 2: 242257, 1953. ottaviani e. immunorecognition in the gastropod molluscs with particular reference to the freshwater snail planorbius corneus (l.) (gastropoda, pulmonata). boll. zool. 59: 129139, 1992. ottaviani e. molluscan immunorecognition. inv. surv. j. 3: 50-63, 2006. ottaviani e, caselgrandi e, franceschi c. cytokines and evolution: in vitro effects of il-1 alpha, il-1 beta, tnf-alpha and tnf-beta on an ancestral type of stress response. biochem. biophys. res. commun. 207: 288292, 1995. ottaviani e, franchini a, franceschi c. presence of several cytokine-like molecules in molluscan hemocytes. biochem. biophys. res. comm. 195: 984-988, 1993. pan c. studies on the host-parasite relationship between schistosoma mansoni and the snail australorbis glabratus. am. j. trop. med. hyg. 14: 931-976, 1965. paraense wl. the schistosome vectors in the americas. mem. inst. oswaldo cruz 96: 7-16, 2001. pereira caj. influência de fatores solúveis da hemolinfa e de co-infecções com angiostrongylus vasorum na susceptibilidade de biomphalaria tenagophila à infecção por schistosoma mansoni. master thesis, 106 p, federal university of minas gerais, belo horizonte, mg, brazil, 2005. pereira ca, martins-souza rl, coelho pm, lima ws, negrao-correa d. effect of angiostrongylus vasorum infection on biomphalaria tenagophila susceptibility to schistosoma mansoni. acta trop. 98: 224-233, 2006. pereira lh, valadares te, da cunha ml, correa mc. recovery of young daughter sporocysts from snails infected with schistosoma mansoni. trans. r. soc. trop. med. hyg. 78: 563, 1984. richards cs. genetic studies of a molluscan vector of schistosomiasis. nature 227: 806-810, 1970. richards cs, renwrantz lr. two lectins on the surface of helix pomoatia haemocytes: a ca2+ dependent, galnac-specific lectin and a ca2+ independent, mannose 6-phosphate-specific lectin which recognizes activated homologous opsonins. j. comp. physiol. b 161: 43-54, 199. richards cs. schistosoma mansoni: susceptibility reversal with age in the snails host biomphalaria glabrata. exp. parasitol. 42: 165168, 1977. rosa fm, godard alb, azevedo v, coelho pmz. biomphalaria tenagophila: dominant character of the resistance to schistosoma mansoni and descendants of cross-breeding between resistant (taim, rs) and susceptible (joinville, sc) strains. mem. inst. oswaldo cruz 100: 1923, 2005. rosa fm, godard alb, rodrigues ha, carvalho os, caldeira rl, teles hms, maciel e, passos lj, negrão-corrêa d, coelho pmz. biomphalaria tenagophila: dynamics of populations of resistant and susceptible strains to schistosoma mansoni, with or without pressure of the parasite. mem. inst. oswaldo cruz 101 (suppl. 1): 247-251, 2006. santos mbl, freitas jr, correia mcr, coelho pmz. suscetibilidade ao schistosoma mansoni de híbridos de biomphalaria tenagophila do taim, rgs, cabo frio, rj, e belo horizonte, mg. rev. inst. med. trop. 21: 281-286, 1979. souza cp, borges cc, santana ag, andrade za. comparative histology of biomphalaria glabrata, b. tenagophila and b. straminea with variable degrees of resistance to schistosoma 110 mansoni miracidia. mem. inst. oswaldo cruz 92: 517-522, 1997. souza s and andrade za. on the origin of the biomphalaria glabrata hemocytes. mem. inst. oswaldo cruz 101 (suppl. 1): 213-218, 2006 sullivan jt, castro l.mitotic arrest and toxicity in biomphalaria glabrata (mollusca: pulmonata) exposed to colchicine. j invertebr. pathol. 90: 32-38, 2005. sullivan jt, pikios ss, alonzo aq. mitotic responses to extracts of miracidia and cercariae of schistosoma mansoni in the amebocyte-producing organ of the snail intermediate host biomphalaria glabrata. j. parasitol. 90: 92-96, 2004. sullivan jt, spence jv. transfer of resistance to schistosoma mansoni in biomphalaria glabrata by allografts of amoebocyte-producing organ. j. parasitol. 80: 449-453, 1994. uchikawa r, loker es. lectin-binding properties of the surfaces of in vitro-transformed schistosoma mansoni and echinostoma paraensei sporocysts. j. parasitol. 77: 742-748, 199. van der knaap wp, loker es. immune mechanisms in trematode-snail interactions. parasitol. today 6: 175-182, 1990. yoshino tp, bayne cj. mimicry of snail host antigens by miracidia and primary sporocysts of schistosoma mansoni. parasite immunol. 5: 317-328, 1983. zelck u, becker w. lectin binding to cells of schistosoma mansoni sporocysts and surrounding biomphalaria glabrata tissue. j. invertebr. pathol. 55: 93-99, 1990. 111 corresponding author: group << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj108.pdf 142 isj 2: 142-151, 2005 issn 1824-307x review insect immunorecognition e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted october 14, 2005 abstract the mechanisms of the innate immunity in the insects have been reviewed. in particular, the cellular component (phagocytosis, encapsulation, melanization, nodule formation, wound healing, hemolymph clotting and transplantation) and the humoral component (lectins, cytokine-like molecules and anti-microbial peptides) of the hemolymph have been investigated. key words: insects; immune and neuroendocrine responses introduction the first line of defence in insects is the cuticle. this external barrier of carbohydrate-protein material protects the animal from both physical damage and pathogen attack. the second and more important line of defence is the internal defence system, with a welldeveloped capacity to discriminate between self and non-self. as other coelomatic animals, insects implement this recognition through the cellular and humoral components of the hemolymph. cellular component with regard to the cellular component, there is a general problem in defining the number of immunocytes present in the invertebrate hemolymph and this is a subject of great debate. one reason for this situation is that in most cases there is no hemopoietic organ. when, however, the organ is present, as for example in insects, it supplies immunocytes during the animal’s development, and these continue to differentiate in the circulation (nappi and carton, 1986). however, the immunocyte proliferation occurs mainly in circulating hemolymph, thus containing different stages of maturation of the same cell. corresponding author: enzo ottaviani department of animal biology, university of modena and reggio emilia, via campi 213/d, 41100 modena, italy e-mail: ottaviani.enzo@unimore.it to avoid describing these different stages of maturation as specific cell types, morphological studies should be performed in parallel with functional analyses. this problem is also seen in insect immunocytes. these derive from the mitotic division of circulating blood cells and hemopoietic organs (jones, 1977). the hemopoietic organs have been described in different species and present peculiar locations and morphological characteristics. in particular, five pairs of lymph glands located along the anterior portion of the dorsal blood vessel are the sources for immunocytes in drosophila melanogaster (nappi and carton, 1986). hoffmann (1973) reported two possible organizations for hemopoietic organs. one is structured as dissociated accumulations of immunocytes that could be referred to as the “haemocyte reservoirs” described by jones (1977), while the other is considered a truly differentiated hemopoietic organ. in their elegant treatise, rowley and ratcliffe (1981) reported the presence in the insect hemolymph of the following cell types: prohaemocytes, plasmatocytes, granular cells, cystocytes, spherule cells and oenocytoids. subsequently brehélin and zachary (1986) proposed a new classification of blood cells, based mainly on the ultra-structural observations, and nine cell types are described: prohaemocytes, plasmatocytes, oenocytoids, spherule cells, thrombocytoids and four types of granular haemocytes. among the different types of cells described from different species, some have an immune function, while in others either the function is unknown or it is not directly involved in defence. prohaemocytes are present in all the species examined. they are small and rounded, with a large, round nucleus and seem to function as stem cells 143 (brehélin and zachary, 1986; franchini et al., 1996) (fig. 1). plasmatocytes share the typical functions of a macrophage, i.e. glass adhesion with the emission of pseudopodia allowing amoeboid movement, phagocytic capacity, encapsulation, nodule formation and wound repair (rowley and ratcliffe, 1981; franchini et al., 1996). despite the morphology, granular cells also present the immune functions described in plasmatocytes, even if to a lesser extent. spherule cells do not seem to be involved in immune functions, but the available data suggest a role in the synthesis of blood mucopolysaccharides (gupta and sutherland, 1967; akai and sato, 1973) and in silk production in bombix mori (nittono, 1960). the function of the oenocytoids is also as yet not established, even if the cytoplasmic presence of phenoloxidase (propo) activity may support an involvement in melanization processes. the crystal cells containing a crystal of tyrosine belong to this cell type and are found in some diptera (rizki and rizki, 1959; drif and brehélin, 1983). despite attempts to establish a correct, comparative classification of insect blood cell types, problems still remain. apart from the prohaemocytes, probably only two immunocytes, plasmatocyes and granular cells, appear to play a role in immune functions. however, some exceptions exist. d. melanogaster, for instance, only shows the plasmatocyte together with the specialized crystal cells (nappi and corton, 1986). this result reflects findings in other invertebrates such as molluscs and annelids (yoshino, 1976; cheng and guida, 1980; ottaviani, 1992; cooper et al., 1995). phagocytosis this phenomenon is of pivotal importance for nutrition and defence, and it is present throughout the animal kingdom. as mentioned before, both plasmatocytes (fig. 2) and granular cells are involved in phagocytosis. the process is characterized by various steps: recognition (chemotaxis and attachment), ingestion and killing. chemotaxis, i.e. non-random locomotion, allows cells to move towards a chemoattractant which has been recognized. the data on insect chemotaxis are controversial: some authors have demonstrated a chemotactic action of aspergillus flavus conidia towards immunocytes from galleria mellonella (vey et al., 1968; vey, 1969), while others failed to see such an action (salt, 1970; jones, 1956). furthermore, plasmatocyte chemotactic activity has been seen to be involved in encapsulation, nodule formation and wound repair (nappi, 1973; ratcliffe and gagen, 1977; rowley and ratcliffe, 1978). the study of the mechanism by which invertebrate immunocytes recognize non-self material is also still in its infancy. it should be remembered that the immune system involves two types of response: innate (or natural) and adaptive (or acquired). innate immunity is conserved from invertebrates to vertebrates, while adaptive immunity, based on antigen-specific t and b cells, only appears in vertebrates. in the latter, the old, innate immune system is able to discriminate between self and nonself by means of pattern-recognition receptors (prrs), able to recognize conserved pathogenassociated molecular patterns (pamps) produced by microorganisms (fearon, 1997; medzhitov and janeway, 2000). a similar scenario could also be present in invertebrates. mammalian immune cells express several toll-like receptors that are considered prrs (akira, 2001). in drosophila, the innate immune recognition of micro-organisms is mediated by signalling through toll receptors (hoffmann et al., 1999; takeda and akira, 2001). peptidoglycan recognition proteins (pgrfs) are able to recognize bacteria and their cell wall component, the peptidoglycan, and are well conserved from insects to mammals (dziarski, 2004). the mosquito anopheles gambiae secretes a thioester-containing protein (tep), αtep1, which is related to vertebrate complement factors and α2-macroglobulins (levashina et al., 2001). the reported studies are some examples of the extensive literature seeking to complete the mosaic of mechanisms involved in invertebrate innate immunity. immunocytes also recognize abiotic material suggesting that these cells exert their recognition not only on micro-organism pamps, but it is unknown how this happens. lavine and strand (2002) surmise the presence in invertebrate immunocytes of receptors with promiscuous capacities that can interact with a wide range of molecules normally not encountered in the hemocoel. in summary, as suggested by medzhitov and janeway (1997), innate immunity is based on the recognition of invariant modules in different bacterial and viral species. consequently, the limited number of recognition systems is mirrored by a limited number of molecular structures, which are recognized. paradoxically, from an invertebrate point of view, few are the microorganisms making the small number of recognition units (an extremely restricted repertoire) perfectly adequate for the small number of foreign modules to be recognized. the small number of recognition units induces a complex and efficient reaction by the ancestral defence based on the immune-neuroendocrine effector system present in invertebrates. fig. 1 cytospin preparation of immunocytes from calliphora vomitoria: a) plasmatocyte, b) granular cells (bar = 10 µm). 144 fig. 2 in vitro bacterial phagocytosis from calliphora vomitoria plasmatocyte (asterisk) (bar = 10 µm) (modified from franchini et al., 1996). innate immunity, stress and inflammation are interconnected in this system that has been conserved throughout evolution (ottaviani and franceschi, 1997; ottaviani et al., 1998). the last steps in phagocytosis are ingestion and killing. as in vertebrates, the ingested phase shows the formation of phagosomes and subsequent lysosomal fusion, as reported in the plasmatocytes of g. mellonella (rowley and ratcliffe, 1979). studies in the fruit fly ceratitis capitata suggest that signal transduction pathways that modulate phagocytosis are conserved in insects and mammals (foukas et al., 1998; metheniti et al., 2001). furthermore, teps seem to have an opsonic effect on the drosophila phagocytosis in a similar manner to mammalian complement factor c3 (lagueux et al., 2000). similar to mammals, the ingested phase needs energy that derives from the glycolitic pathway, as demonstrated by anderson et al. (1973) in the blaberus craniifer immunocytes. the authors also report an antimicrobial system. indeed, reactive oxygen intermediates (roi), reactive nitrogen intermediates (rni) and nitric oxide (no) are found in immunocytes of insects (whitten and ratcliffe, 1999; luckhart et al., 1998; nappi et al., 2000). in d. melanogaster and d. teissieri, no activates the gene encoding the anti-microbial peptide diptericin (nappi et al., 2000). encapsulation cellular encapsulation is a mechanism present in all the insect groups examined (rowley and ratcliffe, 1981). this means of defence involves immobilizing insect parasites, fungi and large protozoans that escape the phagocytic activity of the single immunocyte. the reaction is strong against living organisms and is followed by melanization, while it is weak against abiotic material and not always followed by melanization. the encapsulated material is surrounded by multi-cellular sheaths, hence the term capsule. götz (1986) defines ten steps in this cellular defence, with granular cells and plasmatocytes as the main actors. briefly, the first event in the encapsulation process is the contact of the immunocytes with foreign material and the degranulation of the granular cells. the material released from granules is sticky and adheres to foreign surfaces. the degranulation and disintegration of granular cells activate the plasmatocytes that participate in the formation of the capsule. subsequently, the process of melanization, originated in the granular cells, takes place. the approximate time of the encapsulation is about 1-3 days (fig. 3). role of melanization melanization is involved not only in encapsulation, but also in cuticular defenses, e.g. against bacteria, wounding and sclerotization, as well as in other defense responses such as the killing of bacteria or parasites and cytotoxicity (söderhräll and smith, 1986; ashida and brey, 1995; nappi et al., 1992; nayar et al., 1992; nappi and ottaviani, 2000). the melanin is synthesized by the activation of the propo-activating system (propo system) (söderhräll, 1982) comprising a complex cascade of serine proteases that allows the conversion of propo to phenoloxide (po), which, in turn, acts on substrates such as tyrosine and its derivatives (dopa and dopamine) to form melanin. this process was extensively studied for the first time in crustacean astacus astacus (söderhräll, 1982) and in the insect bombyx mori (ashida, 1971, ashida and ohnishi, 1967, ashida et al., 1983). the activation of propo has now been described in several invertebrate taxa (arizza et al., 1995; beschin et al., 1998; frizzo et al., 1999; tujula et al., 2001). the propo system has also been seen as a recognition system activated by different foreign materials, such as β-1,3-glucans from fungal and algal cell walls, and lipopolysaccharides and peptidoglycans from microbial cell walls (söderhräll and cerenius, 1998). the cdna encoding pgrps has been cloned from the silkworm b. mori fat body cdna library (ochiai and ashida, 1999). the specific binding of pgrp to the peptidoglycan induces the propo cascade. furthermore, these studies show that the means of extracellular recognition of microorganisms is conserved from insects to mammals. with regard the role of the propo system in the cuticular site, it has been demonstrated that propo originates from the circulating immunocytes in the silkworm b. mori and from immunocytes and epidermal cells in the caterpillar calpodes ethlius. subsequently, it is actively transported across the epidermis to the cuticular matrix (ashida and brey, 1995; sass et al., 1994). the propo cuticular silkworm is activated through a limited proteolysis by the serine proteinase, termed propo activating enzyme, which itself exists as a zimogen (ashida and brey, 1995). cuticular po is normally considered to be injury po, however, other two types of po are present in the cuticle of insects: granular po involved in the body colour and laccase-type po involved in sclerotization of a newly ecdysed cuticle (barrett, 1991). recently, 145 fig. 3 encapsulation of the egg of the wasp parasitoid leptopilina boulardi in drosophila melanogaster host (courtesy prof. nappi aj). asano and ashida (2001) studying cuticular po and its zymogen (propo) in b. mori purified two propo isoforms. in the same animal, the authors also found two propo isoforms in the hemolymph. these latter isoforms differ from the cuticular isoforms for the presence of five aminoacid residues. all the isoforms are activated by specific enzymes, and the hemolymph isofoms are transported to the cuticle. nodule formation this type of cellular defence takes place when phagocytosis is inadequate in the face of a large number of small non-self particles such as bacteria. rowley and ratcliffe (1981) divide nodule formation into two phases. in the first, the bacteria are entrapped in the material released by exocytosis from the granular cells and melanization then occurs externally. in the second, plasmotocytes, adhere and flatten the necrotic core forming the typical multicellular sheath. wound healing, hemolymph clotting and transplantation invertebrates respond to an external injury in order to avoid the loss of biological liquids. different mechanisms, such as fat or intestine extrusion, muscular contraction, hemolymph clot formation, cellular aggregation and melanin deposition, have been described (theopold et al., 2004, 2002; bidla et al., 2005). the hemolymph clot is well-known in the american cockroach, leucophaea maderae (bohn and barwig, 1984). the process involves clotting proteins present in the hemolymph plasma (plasma coagulogen) that are released from immunocytes (hemocyte coagulogen). the immunocytes migrate and aggregate around the altered region, and after their rupture the clotting starts. the process involves an enzymatic cascade which is activated by ca2+. the clotting reaction is very fast and in less than 3 minutes the clot is completely insoluble. a different clotting mechanism is described for locusta migratoria (brehélin, 1979). in this case, coagulation involves only the plasma (by means of the coagulogen), and this seems to be a functional equivalent in clotting of mammalian fibrinogen. lackie (1986) raised the question of how the damaged self is recognized, but following a study of the possible mechanisms involved, i.e. a non-specific response to the altered physiochemical surface proprieties of the connective tissue layer, a specific response via receptor-ligand interactions and the release of wounding factors by immunocytes, found no conclusive answer. tissue graft transplantation is a technique to test immunological specificity and memory in insects and, in general, in invertebrates, even if the mechanisms by which the host recognizes self and non-self implanted material have not yet been clarified. autoand allografts of integument in the insects b. craniifer and extatosoma tiaratum were accepted, while xenografts were rejected after melanization. all these phenomena showed an accumulation of immunocytes around the grafts which was more marked in the xenografts (thomas and ratcliffe, 1982). a different result was obtained with allogeneic cuticle and xenogeneic cuticle from blatta orientalis. both grafts were recognized as foreign by periplaneta americana, while implanted xenogeneic tissue from b. craniifer was not rejected by p. americana (lackie, 1983). the transplantation experiments by karp and meade (1993) using the same species, i.e. p. americana and b. orientalis, found that p. americana recognizes b. orientalis as foreign and mounts a rejection response against integumentary grafts. humoral component although lacking immunoglobulins, insects, as all other invertebrates, possess a variety of factors of varying degrees of specificity, including lectins, cytokine-like molecules and anti-microbial peptides. 146 lectins lectins are sugar-binding proteins or glycoproteins that agglutinate cells and/or precipitate glycoconjugates (goldstein et al., 1980). in insects and other invertebrates, the lectins are also called agglutinins and hemagglutinins. many insect species contain natural agglutinins (yeaton, 1981; ratcliffe and rowley, 1983; chen et al., 1993) that can be induced by antigenic stimulation (komano et al., 1980; ratcliffe and rowley, 1983). these are mainly produced by immunocytes (whitcomb et al., 1974) and fat body (komano et al., 1983). biomolecular studies have allowed the cdna from lectins of arachis hypogaea (shanker and das, 2001) and from pinellia ternata (yao et al., 2003) to be cloned. even if conflicting data have been reported, these substances appear able to agglutinate foreign material (komano et al., 1980; ratcliffe and rowley, 1983), they present opsonic properties (pendland et al., 1988; kawasaki et al., 1993), they increase phagocytic activity (wilson et al., 1999) and they have a toxic effect (powell et al., 1998). with regards this latter effect, galanthus nivalis agglutinin (gna) added to the diet of the insect pest, nilaparvata lugens, has been seen to cross the midgut epithelial barrier and pass into the circulatory system of the insect, so exerting its toxic effect. lectins from p. ternata and pinellia pedatisecta present insecticidal activity towards cotton aphids (aphis gossypii) and peach potato aphids (myzus persicae) when incorporated into artificial diets (huang et al., 1997; pan et al., 1998). cytokine-like molecules cytokines belong to the broad family of soluble factors that by communicating among different cell types induce the complex interactions that allow immune responses. these molecules have been called by different names according to their origin or function. nowadays, the general term cytokines is used for these signal molecules. interleukins (il), tumour necrosis factor (tnf), interferons (ifn), transforming growth factor (tgf)-β, platelet-derived growth factor (pdgf), etc. are the most common examples in mammals. cytokine-like molecules have been reported in various tissues of a number of invertebrates, including insects (table 1). the first findings on cytokine-like molecules in invertebrates reported a so-called "growth promoting substance" in insects and, in particular, in b. mori (aizawa and sato, 1963; vaugghn and louloudes, 1978) and samia cynthia (williams and kambysellis, 1969). cherbas (1973) indicated haemokinin as a fraction partially purified from both plasma and epidermal tissue of saturniid pupae and able to stimulate immunocyte activation. subsequently, the presence of epidermal growth factor (egf), fibroblast growth factor (fgf) and others has been demonstrated in different cells and tissues (table 1). particular attention has been paid to insulin-like growth factors, which have been described in different taxa, including insects (joosse et al., 1983; ebberink et al., 1989). moreover, genomic dna clones encoding brain secretory peptides that have been isolated from insects share substantial homologies with vertebrate preproinsulins. (adachi et al., 1989; iwami et al., 1989; lagueux et al., 1990). il-1α-, tnf-α-, pdgf-aband tgf-β1-like molecules have been detected in the plasmatocytes and granular cells of calliphora vomitoria and g. mellonella (franchini et al., 1996; wittwer et al., 1998). similar results have also been reported in the cell lines derived from the hemolymph of estigmene acraea and from the fat body cell of lymatria dispar (wittwer et al., 1998; ottaviani et al., 2000). the presence in insects of pro-inflammatory cytokines, i.e. il-1 and tnf-α, suggest an important role of these molecules in animal host defence responses. even if few functional data are present in the literature regarding insects, studies from our laboratory dealing with cytokines in invertebrates (mainly molluscs) have demonstrated that mammalian cytokines affect immune and neuroendocrine functions, wound repair and programmed cell death, while, at the same time, presenting the pleiotropicity, functional redundancy and receptor promiscuity seen in vertebrates (ottaviani et al., 2004). these findings suggest that the same frame may also exist in insects. furthermore, the insect cytoplasmic domain of the toll family proteins is homologous with the cytoplasmatic domain of the il-1 receptor family (kopp and medzhitov, 1999). given the lack of molecular biology data, it has been hypothesized in the literature that a correlation between invertebrate and vertebrate cytokine genes does not exist (beschin et al., 2001). however, to claim that mammalian cytokines intervene in the main invertebrate functions merely as a result of functional convergence does seem reductive. different research groups have found that the main molecules involved in the basic and fundamental functions of cell survival, for example adenocorticotropic hormone (acth), corticotrophin-releasing hormone (crh) and nitric oxide synthase (nos), show a gene homology with their vertebrate counterparts (duvaux-miret and capron, 1991; yuda et al., 1996; salzet et al., 1997). anti-microbial peptides anti-microbial peptides that are expressed constitutively or are readily inducible have been extensively studied in insects (boman, 1995; bulet et al., 1999). the main production site is the fat body, a functional equivalent of the mammalian liver (fehlbaum et al., 1994), but immunocytes (boman, 1991), the cuticular epithelial cells (brey et al., 1993) and the reproductive tract (rosetto et al., 1996) are also involved. bulet et al. (1999) classified the antimicrobial peptides into three classes on the basis of the sequence and structural characteristics: 1. linear peptides forming α-helices and devoid of cysteine residues; 2. cyclic peptides containing cysteine residues; 3. peptides with an over-representation in proline and/or glycine residues. the different anti-microbial peptides are named attacins, cecropins, defensins, drosomicins, etc. according to the bulet et al. (1999) classification, cecropins belong to the first class of anti-microbial 147 table 1 insecta cytokine-like molecules references ______________________________________________________________________ bombyx mori growth promoting factor aizawa and sato (1963); vaughn and louloudes(1978) samia cynthia growth promoting factor williams and hemokinin kambysellis (1969) cherbas (1973) antheraea polyphemus hemokinin cherbas (1973) hyalophora cecropea hemokinin cherbas (1973) drosophila melanogaster egf wharton et al. (1985); tgf-α, β kelley et al. (1987); neurotrophic factor padgett et al. (1987); imaginal disc gfs bryant (1988); kopczynski et al. (1988); hayashi et al. (1992); neuman-silberberg and schupback (1993); kutty et al. (1998); kawamura et al. (1999) manduca sexta hemolymph trophic factor wielgus et al. (1990) calliphora vomitoria tnf-α, pdgf-ab, tgf-β1 franchini et al. (1996) galleria mellonella il-1α, tnf-α wittwer et al. (1999) estigmene acraea il-1α, tnf-α wittwer et al. (1999) lymantria dispar pdgf-ab, tgf-β1 ottaviani et al. (2000) ______________________________________________________________________________ peptides, while defensins are ascribable to the second class. cecropins were the first anti-microbial peptides to be isolated and characterized (hultmark et al., 1980). they are small 4 kda peptides found in diptera and lepidotera and present an anti-bacterial activity against both gram positive and gram negative bacteria. cecropins share common features, i.e. molecular size (31-39 aminoacids), a strong basicity in the n-terminal region and a hydrophobic portion in cterminal part. they are induced by bacterial infection and are synthesized as preproproteins of 62-64 residues (boman, 1991). the precursors are first cleaved by a signal peptidase, and the pro-portion is then removed by a dipeptidyl aminopeptidase (boman et al., 1989). in contrast to cecropins, defensins mainly attack gram positive bacteria (boman, 1991). they are 4 kda cationic peptides with a characteristic six cysteine/three disulfide bridge pattern and three domains, a flexible amino-terminal loop, a central αhelix and a carboxy-terminal anti-parallel β-sheet (hanzawa et al., 1990; bonmatin et al., 1992; bulet et al., 1999). several reports have described the presence of defensins in different insect species (bulet et al., 1999), but not in lepidoptera. in our laboratory we recently identified for the first time the presence of a defensin active against gram positive bacteria in lepidoptera (izd-mb-0503 cell line derived from immunocytes of mamestra brassicae) (mandrioli et al., 2003). the biomolecular study revealed the presence of the defensin gene of 294 pb (fig. 4). alignment of m. brassicae defensin with homologous sequences from the insect defensin a genes revealed identity ranging from 43 % to 59 % (fig. 5). the analysis of the putative protein indicated the presence of 98 aminoacids, including 8 cysteine residues. in particular, cysteine residues 3-8 were highly conserved, suggesting their involvement in the formation of three disulfide bridges. northern blotting experiments showed a constitutive expression of the defensin gene in m. brassicae cells. its expression was increased by gram positive, but not by gram negative bacteria (fig. 6). fig. 4 complete sequence of m. brassicae defensin gene. upper lines show the nucleotide sequence, while the putative amino acidic sequence is indicated in the lower lines (modified from mandrioli et al., 2003). 148 fig. 5 defensin peptide alignment of mamestra brassicae (af465486) (a) with aedes aegypti (af156093) (b), apis mellifera (d17670) (c), phormia terranovae (af182164) (d), drosophila melanogaster (ac007414) (e) and anopheles gambiae (af063402) (f). highly conserved aminoacids are boxed (modified from mandrioli et al., 2003). fig. 6 northern blotting with the defensin probe on rna samples extracted at different times from m. brassicae cells after induction with heat-killed (a) and live bacteria (b). northern blotting indicates that defensin expression is increased by gram positive bacteria, in particular by live specimens, but not by candida albicans and saccharomyces cerevisiae (c) (modified from mandrioli et al., 2003). defensin induction was greater when using live bacteria rather than heat-killed specimens (fig. 6). no defensin gene induction was observed in the presence of candida albicans and saccharomyces cerevisiae (fig. 6). microbial killing by antimicrobial peptides involves both the charge and the structure of the latter. in cecropins and defensins, the mechanism is thought to relate to the peptides’ ability to assemble in the target membrane and form a pore (boman, 1991). the means by which cecropins associate with the plasma membrane is called “carpet-like” and involves: i) peptide arrangement parallel to membrane surface by binding to the phospholipid head groups; ii) rotation of peptides in order to re-orientate their hydrophobic residues toward the membrane hydrophobic core; iii) disruption of the membrane bilayer (shai, 1999). concluding remarks from an evolutionary point of view, insects, together with molluscs and annelids, are characterised by a body cavity, the coeloma, in which immunocytes appear with morpho-functional properties similar to mammalian blood cells. the new structure allows the immune system to develop more defined strategies to discriminate between self and non-self and to build up complex types of immune responses. acknowledgements this work was supported by a miur (italy) grant. references adachi t, takiya s, suzuki y, iwami m, kawakami a, takahashi sy, ishizaki h, suzuki a. cdna structure and expression of bombyxin, an insulin-like brain secretory peptide of the silkmoth bombyx mori. j. biol. chem. 264: 7681-7685, 1989. aizawa k, sato f. culture de tissus de ver a soie, bombyx mori, dans un milieu sans hemolymphe. ann. epiphyt. 14: 125, 1963. 149 akai h, sato s. ultrastructure of the larval haemocytes of the silkworm, bombyx mori (l) (lepidoptera, bombycidae). int. j. insect morphol. embryol. 2: 207-231,1973. akira s. toll-like receptors and innate immunity. adv. immunol. 78: 1-56, 2001. anderson rs, holmes b, good ra. comparative biochemistry of phagocytizing insect hemocytes. comp. biochem. physiol. 46b: 595-602, 1973. arizza v, cammarata m, tomasino mc, parrinello n. phenoloxidase characterization in vacuolar hemocytes from the solitary ascidian styela plicata. j. invertebr. path. 66: 297-302, 1995. asano t, ashida m. cuticular pro-phenoloxidase of the silkworm, bombyx mori. purification and demonstration of its transport from hemolymph. j. biol. chem. 276: 11100-11112, 2001. ashida m. purification and characterization of prophenoloxidase from the hemolymph of the silkworm bombyx mori. arch. biochem. biophys. 144: 749-762, 1971. ashida m, brey pt. role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. proc. natl. acad. sci. usa 92: 10698-10702, 1995. ashida m, ishizaki y, iwahana h. activation of prophenoloxidase by bacterial cell walls or β-1,3-glucans in plasma of the silkworm, bombyx mori. biochem. biophys. res. commun. 113: 562-568, 1983. ashida m, ohnishi e. activation of prophenoloxidase in the hemolymph of the silkworm bombyx mori. arch. biochem. biophys. 122: 411-416, 1967. barrett fm. phenoloxidases and the integument. in: binnington k, retnakaran a (eds), physiology of the insect epidermis, csiro publications, melbourne, australia, pp 195-212, 1991. beschin a, bilej m, hanssens f, raymakers j, vandyck e, revets h, brys l, gomez j, debaetselier p, timmermans m. identification and cloning of a glucan and liopoplysacchararide-binding protein from eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. j. biol. chem. 273: 2494824954, 1998. bidla g, lindgren m, theopold u, dushay ms. hemolymph coagulation and phenoloxidase in drosophila larvae. dev. comp. immunol. 29: 669-679, 2005. bohn h, barwig b. hemolymph clotting in the cockroach leucophaea maderae (blattaria). influence of ions and inhibitors; isolation of the plasma coagulogen. j. comp. physiol. b 154: 457-467, 1984. boman hg. antibacterial peptides: key components needed in immunity. cell 65: 295-207, 1991. boman hg, boman ia, andreu d, li z-q, merrifield rb, schlenstedt g, zimmermann r. chemical synthesis and enzymic processing of precursor forms of cecropins a and b. j. biol. chem. 264: 5852-5860, 1989. boman hg. peptide antibiotics and their role in innate immunity. ann. rev. immunol. 13: 61-92, 1995. bonmatin jm, bonnat jl, gallet x, vovelle f, ptak m, reichhart jm, hoffmann ja, ke legrain m, achstetter t. two-dimensional 1h-nmr study of recombinant insect defensin a in water: resonance assignments, secondary structure and global folding. j. biomol. nmr 2: 235-256, 1992. brehélin m. hemolymph coagulation in locusta migratoria: evidence of fibrinogen. comp. biochem. physiol. 62b: 329-334, 1979. brehélin m, zachary d. insect haemocytes: a new classification to rule out the controversy. in: brehélin m (ed), immunity in invertebrates, springer-verlag, berlin, pp 36-48,1986. brey pt, lee w-j, yamakawa m, koizumi y, perrot s, françois m, ashida m. role of the integument in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells. proc. natl. acad. sci. usa 90: 6275-6279, 1993. bryant pj. localized cell death by mutations in a drosophila gene coding for a transforming growth factor-β homolog. dev. biol. 128: 386-395, 1988. bulet p, hetru c, dimarcq jl, hoffmann d. antimicrobial peptides in insects; structure and function. dev. comp. immunol. 23: 329-344, 1999. chen c, ratcliffe na, rowley af. detection, isolation and characterization of mutiple lectins from the hemolymph of the cockroach, blaberus discoidalis. biochem. j. 294: 181-190, 1993. cheng tc, guida vg. hemocytes of bulinus truncatus rohlfsi (mollusca: gastropoda). invertebr. pathol. 35: 138-167, 1980. cherbas l. the induction of an injury reaction in cultured haemocytes from saturniid pupae. j. insect physiol. 19: 2011-2023, 1973. cooper el, franchini a, ottaviani e. earthworm coelomocytes possess immunoreactive cytokines and pomc-derived peptides. anim. biol. 4: 25-29, 1995. drif l, brehélin m. the circulating hemocytes of culex pipiens and aedes aegypti: cytplogy, histochemistry, hemograms and functions. dev. comp. immunol. 7: 687-690, 1983. duvaux-miret o, capron a. proopiomelanocortin gene and gene products: a comparative study. adv. neuroimmunol. 1: 41-57, 1991. dziarski r. peptidoglycan recognition proteins (pgrps). mol. immunol. 40: 877-886, 2004. ebberink rhm, smit ab, van minnen j. the insulin family: evolution of structure and function in vertebrates and invertebrates. biol. bull. 177: 176-82, 1989. fearon dt. seeking wisdom in innate immunity. nature, 388: 323-324, 1997. fehlbaum p, bulet p, michaut l, lagueux m, broekaert wf, hetru c, hoffmann ja. septic injury of drosophila induced the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. j. biol. chem. 269: 33159-33163, 1994. foukas lc, katsoulas hl, paraskevopoulou n, metheniti a, lambropoulou m, marmaras vj. phagocytosis of escherichia coli by insect hemocytes requires both activation of the ras/mitogen-activated protein kinase signal transduction pathway for attachment and β3 integrin for internalization. j. biol. chem. 273: 1481314818, 1998. franchini a, miyan ja, ottaviani e. induction of acthand tnf-α-like molecules in the hemocytes of calliphora vomitoria (insecta, diptera). tissue cell 28: 587-592, 1996. frizzo a, guidolin l, ballarin l, sabbadin a. purification and partial characterisation of phenoloxidase from the colonial ascidian botryllus schlosseri. marine biol. 135: 483-488, 1999. goldstein ij, hughes rc, monsigny m, osawa t, sharon n. what should be called a lectin ? nature 285: 66, 1980. götz p. encapsulation in arthropods. in: brehélin m (ed), immunity in invertebrates, springer-verlag, berlin, pp 153-170,1986. gupta ap, sutherland dj. phase contrast and histological studies of spherule cells in cockroaches. ann. entomol. soc. am. 60: 557-565, 1967. hanzawa h, shimada i, kuzuhara t, komano h, kohda d, inagaki f, natori s, arata y. 1h nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin. febs lett. 269 : 413420, 1990. hayashi i, perez-magallanes m, rossi jm. neurotrophic factor-like activity in drosophila. biochem. biophys. res. commun. 184: 73-79, 1992. hoffmann ja. les organes hématopoïétiques de deux insectes orthoptéres: locusta migratoria et gryllus bimaculatus. z. zellforsch. 106: 451-472, 1973. hoffmann ja, kafatos fc, janeway ca, ezekowitz ra. phylogenetic perspectives in innate immunity. science 284: 1313-1318, 1999. 150 huang df, pan yh, zhang sx, cao jp, yang xm, zhang j, yin wz. the discovery of insecticidal protein against aphids from pinellia pedatisecta and p. ternata. sci. agri. sin. 30: 94, 1997. hultmark d. immune reactions in drosophila and other insects: a model for innate immunity. trends genet. 9: 178-183, 1993. iwami m, kawakami a, ishizaki h, takahashi sy, adachi t, suzuki y, nagasawa h, suzuki a. cloning of a gene encoding bombyxin, an insulin-like brain secretory peptide of the silkmoth bombyx mori with prothoracicotropic activity. dev. growth diff. 31: 31-37, 1989. jones jc. the circulatory system of insects. thomas cc, springfield, illinois, 1977. jones jc. the hemocytes of sarcophaga bullata parker. j. morph. 99: 71-124, 1956. joosse j, geraerts wpm. endocrinology. in: saleuddin asm, wilbur km (eds), the mollusca. physiology, part 1, vol. 4, new york, academic press, pp 317-406, 1983. karp rd, meade cc. trasplantation immunity in the american cockroach, periplaneta americana: the rejection of integumentary grafts from blatta orientalis. dev. comp. immunol. 17: 301-307,1993. kawamura k, shibata t, saget o, peel d, bryant pj. a new family of growth factors produced by fat body and active on drosophila imaginal disc cells. development 126: 211-219, 1999. kawasaki k, kubo t, natori s. a novel role of periplaneta lectin as an opsonin to recognize 2-keto-3-deoxy octonate residues of bacterial lipopolysaccharides. comp. biochem. physiol. 106b: 675-680, 1993. kelley mr, kidd s, deutsch wa, young mw. mutations altering the structure of epidermal growth factor-like coding sequences at the drosophila notch locus. cell 51: 539-548, 1987. kopczynski cc, arlon ak, fechtel k, kooh pj, muskavitch ma. delta, a drosophila neurogenic gene, is transcriptionally complex and encodes a protein related to blood coagulation factors and epidermal growth factor of vertebrates. genes dev. 2: 1723-1735, 1988. komano h, mizuno d, natori s. purification of lectin induced in the hemolymph of sarcophaga peregrina larvae on injury. j. biol. chem. 255: 2919-2824, 1980. komano h, nozawa r, mizuno d, natori s. measurement of sarcophaga peregrina lectin under various physiological conditions by radioimmunoassay. j. biol. chem. 258: 2143-2147, 1983. kopp eb, medzhitov r. the toll-receptor family and control of innate immunity. curr. opin. immunol. 11: 13-18, 1999. kutty g, kutty k, samuel w, duncan t, jaworski c, wiggert b. identification of a new member of transforming growth factor-beta superfamily in drosophila: the first activin gene. biochem. biophys. res. commun. 246, 644-649, 1998. lackie am. immunological recognition of cuticular transplants in insects. dev. comp. immunol. 7: 41-50, 1983. lackie am. transplantation immunity in arthropods: is immunorecognition merely wound-healing? in: brehélin m (ed), immunity in invertebrates, springer-verlag, berlin, pp 125-138,1986. lagueux m, lwoff l, meister m, goltzene f, hoffmann ja. cdnas from neurosecretory cells of brains of locusta migratoria (insecta, orthoptera) encoding a novel member of the superfamily of insulins. eur. j. biochem. 187: 249-254, 1990. lagueux m, perrodou e, levashina ea, capovilla m, hoffmann ja. constitutive expression of a complement-like protein in toll and jak gain-offunction mutants of drosophila. proc. natl. acad. sci. usa 97: 11427-11432, 2000. lavine, m.d., strand, m.r. insect hemocytes and their role in immunity. insect biochem. mol. biol. 32: 1295-1309, 2002. levashina ea, moita lf, blandin s, vriend g, lagueux m, kafatos fc. conserved role of a complement-like protein in phagocytosis revealed by dsrna knockout in cultured cells of the mosquito, anopheles gambiae. cell 104: 709-718, 2001. luckhart s, vodovotz y, cui lw, rosenberg r. the mosquito anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. proc. natl. acad. sci, usa 95: 5700-5705, 1998. malagoli d, mandrioli m, ottaviani e. cloning and characterization of a procorticotrophin-releasing hormone in the izd-mb-0503 immunocyte line from the insect mamestra brassicae. peptides 23: 1829-1836, 2002. mandrioli m, bugli s, saltini s, genedani s, ottaviani e. molecular characterization of a defensin in the izd-mb0503 cell line derived from immunocytes of the insect mamestra brassicae (lepidoptera). biol. cell 95: 53-57, 2003. medzhitov r, janeway cajr. innate immune recognition: mechanisms and pathways. immunol. rev. 173: 89-97, 2000. medzhitov r, janeway cajr. innate immunity: the virtue of a nonclonal sysyem of recognition. cell 91: 295-298, 1997. metheniti a, paraskevopoulou n, lambropoulou m, marmaras vj. involvement of fak/src complex in the processes of escherichia coli phagocytosis by insect hemocytes. febs lett. 496: 55-59, 2001. nappi aj. hemocytic changes associated with the encapsulation and melanization of some insect parasites. exp. parasitol. 33: 287-302, 1973. nappi aj, ottaviani e. cytotoxicity and cytotoxic molecules in invertebrates. bioessays 22: 469-480, 2000. nappi, aj, carton y. cellular immune responses and their genetic aspects in drosophila. in: brehélin m (ed), immunity in invertebrates, springer-verlag, berlin, pp 171-187,1986. nappi aj, vass e, carton y, frey f. identification of 3,4dihydroxyphenylalanine, 5,6-dihydroxyindole, and nacetylarterenone during eumalin formation in the immune reactive larvae of drosophila melanogaster. arch. insect biochem. physiol. 20: 181-191, 1992. nappi aj, vass e, frey f, carton y. nitric oxide involvement in drosophila immunity. nitric oxide biol. chem. 4: 423430, 2000. nayar jk, mikarts ll, knight jw, bradley tj. characterization of the intracellular melanization response in anopheles quadrimaculatus against subperiodic brugia malayi larvae. j. parasitol. 78: 876880, 1992. neuman-silberberg fs, schupbach t. the drosophila dorsoventral patterning gene gurken produces a dorsally localized rna and encodes a tgf-alfa-like protein. cell 74: 165-174, 1993. nittono y. studies on the blood cells in the silkworm, bombyx mori (l). bull. sericult. exp. stn. japan 16: 171-266, 1960. ochiai m, ashida m. a pattern recognition protein for peptidoglycan. j. biol. chem. 274: 11854-11858, 1999. ottaviani e, franceschi f. the invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems. immunol. today 18: 169-174, 1997. ottaviani e. immunorecognition in the gastropod molluscs with particular reference to the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). boll. zool. 59: 129-139, 1992. ottaviani e, barbieri d, franchini a, kletsas d. pdgf and tgf-β partially .prevent 2-deoxy-d-ribose-induced apoptosis in the fat body cell line iplb-ldfb from the insect lymantria dispar. j. insect physiol. 46: 81-87, 2000. 151 ottaviani e, malagoli d, franchini e. invertebrate humoral factors: cytokines as mediators of cell survival. progr. mol. subcell. biol. 34: 1-23, 2004. ottaviani e, valensin s, franceschi c. tthe neuroimmunological interface in an evolutionary perspective: the dynamic relationship between effector and recognition systems. front. bioscience 3: d431-435, 1998. padgett rw, johnson rd, gelbart wm. a transcript a drosophila pattern gene predicts a protein homologous to the transforming growth factor-β family. nature 325: 81-84, 1987. pan yh, zhang sx, cao jp, haung df. the isolation, purification of pinellia pedatisecta lectin and its activity on aphid-resistance. progr. nat. sci. 8: 502-505, 1998. pendland jc, heath ma, boucias dg. function of a galactose-binding lectin from spodoptera exigua larval hemolymph: opsonization of blastospores from entomogenous hyphomycestes. j. insect physiol. 34: 533-540, 1988. powell ks, spence j, bharathi m, gatehouse ja, gatehouse amr. immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper, nilaparvata lugens (stal). j. insect physiol. 44: 529539, 1998. ratcliffe na, rowley af. recognition factors in insect hemolymph. dev. comp. immunol. 7: 653-656, 1983. ratcliffe na, gagen sj. studies on the in vivo cellular reactions of insects: an ultrastrural analysis of nodule formation in galleria mellonella. tissue cell 9: 73-85, 1977. rizki tm, rizki rm. functional significance of the crystal cells in the larvae of drosophila melanogaster. j. biophys. biochem. cytol. 5: 235-240, 1959. rosetto m, manetti ago, giordano pc, marri l, amons r, baldari ct, marchini d, dallai r. molecular characterization of ceratotoxin c, a novel antibacterial female-specific peptide of the ceratotoxin family from the medfly ceratitis capitata. eur. j. biochem. 241: 330-337, 1996. rowley af, ratcliffe na. an ultrastructural and cytochemical study of interactions between latex particles and haemocytes of the wax moth galleria mellonella. cell tissue res. 199: 127-137, 1979. rowley af, ratcliffe na. insects. in: ratcliffe na, rowley af (eds), invertebrate blood cells, vol. 2, academic press, london, pp 421-488, 1981. rowley af, ratcliffe na. a histological study of wound healing and hemocyte function in the wax-moth galleria mellonella. j. morphol. 157: 181-200, 1978. salt g. the cellular defence reactions of insects. cambridge university press, london,1970. salzet m, salzet-raveillon b, cocquerelle c, vergerbocquet m, pryor sc, rialas cm, laurent v, stefano gb. leech immunocytes contain proopiomelanocortin. nitric oxide mediates hemolymph proopiomelanocortin processing. j. immunol. 159: 5400-5411, 1997. sass m, kiss a, locke m. integument and hemocyte peptides. j. insect physiol. 40: 407-421, 1994 shai y. mechanism of binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides. biochim. biophys. acta 1462: 55-70, 1999. shanker s, das rh. identification of a cdna clone encoding for a galactose-binding lectin from peanut (arachis hypogaea) seedling roots. biochim. biophys. acta 1568: 105-110, 2001. söderhräll k. prophenoloxidase activating system and melanization – a recognition mechanism of artropods ? a review. dev. comp. immunol. 6: 601-611, 1982. söderhräll k, cerenius l. role of the prophenoloxidaseactivating system in invertebrate immunity. curr. opin. immunol. 10: 23-28, 1998. söderhräll k, smith vj. the prophenoloxidase activating system: the biochemical of its activation and role in arthropod cellular immunity, with special reference to crustaceans. in: brehélin m (ed), immunity in invertebrates, springer-verlag, berlin, pp 208233,1986. takeda k, akira s. roles of toll-like receptors in innate immune responses. genes cells 6: 733-742, 2001. theopold u, schmidt o, soderhall k, dushay ms. coagulation in arthropods: defence, wound closure and healing. trends immunol. 25: 289-294, 2004. theopold u, li d, fabbri m, scherfer c, schmidt o. the coagulation of insect hemolymph. cell. mol. life sci. 59: 363-372, 2002. thomas ig, ratcliffe na. integumental grafting and immunorecognition in insects. dev. comp. immunol. 6: 643-654, 1982. tujula n, radford j, nair sv, raftos da. effects of tributyltin and other metals on the phenoloxidase activating system of the tunicate, styela plicata. aquatic toxicol. 55: 191-201, 2001. vaughn jl, louloudes ej. isolation of two promoting fractions from insect hemolymph. in vitro 14: 351, 1978. vey a. etude in vitro des réactions hémocytaires anticryptogamiques des larves de lépidoptères. ann. zool. ecol. anim. 1: 93-100, 1969. vey a, quiot j-m, vago c. formation in vitro de réactions d'immunité cellulaire chez les insectes. in: barigazzi c (ed), proceeding 2nd international colloquium on invertebrate tissue culture, istituto lombardo, milan, pp 254-263, 1968. wharton ka, johansen km, xu t, artavanis-tsakonas s. nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing egf-like repeats. cell 43: 567-581, 1985. whitcomb rf, shapiro m, granados rr. insect defence mechanisms against microorganisms and parasitoids. in: rockstein m (ed), the physiology of insecta, vol. 5, academic press, new york, pp 447-536, 1974. whitten mma, ratcliffe na. in vitro superoxide activity in the haemolymph of the west indian leaf cockroach, blaberus discoidalis. j. insect physiol. 45: 667-675, 1999. wielgus jj, caldwell ga, nichols rl, white cf. purification, properties, and titer of a hemolymph trophic factor in larvae and pupae of manduca sexta. insect biochem. 20: 65-72, 1990. williams cm, kambysellis mp. in vitro action of ecdysone. proc. natl. acad. sci. usa 63: 231, 1969. wilson r, chen c, ratcliffe na. innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, blaberus discoidalis. j. immunol.162: 1590-1596, 1999. wittwer d, franchini a, ottaviani e, wiesner a. presence of il-1and tnf-like molecules in galleria mellonella (lepidoptera) haemocytes and in an insect cell line from estigmene acraea (lepidoptera). cytokine 11: 637-642, 1999. yao jh, zhao xy, liao zh, lin j, chen zh, chen f, song j, sun xf, tang kx. cloning and molecular characterization of a novel lectin gene from pinellia ternata. cell res. 13: 301-308, 2003. yeaton rw. invertebrate lectins: i. occurrence. dev. comp. immunol. 5: 391-402, 1981. yoshino tp. the ultrastructure of circulating hemolymph cells of the marine snail cerithidea californica (gastropoda: prosobranchiata). j. morphol. 150: 485-494, 1976. yuda m, hirai m, miura k, matsumura h, ando k, chinzei y. cdna cloning, expression and characterization of nitricoxide synthase from the salivary glands of the bloodsucking insect rhodnius prolixus. eur. j. biochem. 242: 807-812, 1996. title: isj 7: 32-44, 2010 issn 1824-307x review the complexity of drosophila innate immunity a reumer1, t van loy2, l schoofs1 1 department of biology, functional genomics and proteomics unit, k.u.leuven, naamsestraat 59, 3000 leuven, belgium 2 department of biology, molecular developmental physiology and signal transduction unit, k.u.leuven, leuven, belgium; current address: institut de recherche interdisciplinaire et biologie humaine et moléculaire (iribhm), faculty of medicine, université libre de bruxelles (belgium) accepted january 11, 2010 abstract metazoans rely on efficient mechanisms to oppose infections caused by pathogens. the immediate and first-line defense mechanism(s) in metazoans, referred to as the innate immune system, is initiated upon recognition of microbial intruders by germline encoded receptors and is executed by a set of rapid effector mechanisms. adaptive immunity is restricted to vertebrate species and it is controlled and assisted by the innate immune system. interestingly, most of the basic signaling cascades that regulate the primeval innate defense mechanism(s) have been well conserved during evolution, for instance between humans and the fruit fly, drosophila melanogaster. being devoid of adaptive signaling and effector systems, drosophila has become an established model system for studying pristine innate immune cascades and reactions. in general, an immune response is evoked when microorganisms pass the fruit fly’s physical barriers (e.g., cuticle, epithelial lining of gut and trachea), and it is mainly executed in the hemolymph, the equivalent of the mammalian blood. innate immunity in the fruit fly consists of a phenoloxidase (po) response, a cellular response (hemocytes), an antiviral response, and the nf-κb dependent production of antimicrobial peptides referred to as the humoral response. the jak/stat and jun kinase signaling cascades are also implicated in the defence against pathogens. key words: drosophila; innate immunity; signaling cascades introduction immune responses are typically distinguished in two main systems, the adaptive and the innate immune response. adaptive immunity is specific, has memory and is generally considered to be restricted to vertebrates. it relies on the generation of immune receptors, like immunoglobulins and tcell receptors, through somatic gene rearrangements in specified blood cells and on the clonal expansion of activated lymphocytes (b and t cells). innate immunity, on the other hand, refers to the evolutionary ancient and presumably conserved first-line host defence against the early phases of microbial infection, and it is believed to be naive in recognizing broadly conserved microbial moieties. the model organism drosophila melanogaster only seems to rely on this innate immune system for its ___________________________________________________________________________ corresponding author: ank reumer department of biology functional genomics and proteomics unit k.u.leuven, naamsestraat 59, 3000 leuven, belgium e-mail: ank.reumer@bio.kuleuven.be defence against pathogens. drosophila therefore has physical barriers (exoskeleton, chitinous epithelial lining of gut and trachea) to prevent microbial entrance into its body cavity. additionally, specialized blood cells, called hemocytes, floating in the hemolymph participate in phagocytosis and encapsulation of foreign invaders and some of their components are needed for their subsequent melanization. the larval fat body, a functional analog of the mammalian liver and the main larval energy reservoir, is the main site of the fruit fly larva’s humoral (or systemic) immune response, while in adults other tissues can take on major roles for immune surveillance (hoffmann, 2003). humoral response the humoral response is the most intensively studied part of drosophila innate immunity. it consists of recognition systems in the hemolymph which signal the presence of pathogens to the fat body, one of the main immune tissues. this leads to the activation of the toll and/or the immune deficiency 32 mailto:ank.reumer@bio.kuleuven.be fig. 1 the drosophila imd pathway. grambacterial cell wall components (dap type pgn) in the hemolymph are recognized by transmembrane or extracellular pgrp-le and pgrp-lc. imd pathway activation then is initiated by multimerization of transmembrane pgrp-lc and intracellular pgrp-le both containing a rhim-like [receptor interacting protein (rip) homotypic interaction motif] motif. pgrp-sc and pgrp-lb mediate the intensity of this activation. the adaptor protein that links this rhim-like motifs containing complex to imd is still unknown. downstream of imd, several processes can be distinguished of which some probably depend on k63 linked ubiquitin chains for the assembly of active protein complexes. 1) genetic evidence suggest the combined action of ubc-13 (ubiquitin conjugating enzyme), diap-2 (drosophila inhibitor of apoptosis protein 2) and uev1a (ubiquitin conjugating enzyme e2 variant 1) to be needed for the formation of these ubiquitin chains. 2) the complex formed between tak1 and tab2 mediates both jnk pathway and ikk signaling activation. the ikk complex composed of the regulatory subunit kenny and the catalytic subunit ird5 then phosphorylates relish. 3) the unit consisting of imd, fadd and dredd, which interact with each other through their death domains (dd), respectively death effector domains (ded), is supposed to be involved in the activation of tak1. dredd further is presumed to cleave phosphorylated relish resulting in the freeing of the rel transcription factor. further processing steps of the phosphorylated inhibitory part of relish are unknown. dnr-1 (defence repressor 1) probably is part of a negative regulatory loop that keeps the imd-fadd-dredd complex inactive until imd pathway activation upon infection while sickie positively regulates dredd mediated cleavage of relish (foley and o'farrell, 2004). the final result of all these processes is the translocation of the nf-κb transcription factor rel to the nucleus to start transcription of target genes. caspar and the ubiquitin-proteasome pathway further negatively regulate imd pathway signaling. 33 (imd) pathway resulting in the synthesis of amps as well as other effector molecules, and their subsequent release into the hemolymph to fight infection (reviewed in ferrandon et al., 2007). recognition of pathogens is mainly achieved by a diverse array of pattern recognition receptors (prrs) belonging to the peptidoglycan recognition protein (pgrp) and gram negative binding protein (gnbp) families which can discriminate between distinct classes of microorganisms (lemaitre et al., 1997). detection of the diaminopimelic (dap) type of peptidoglycan (pgn) constituting the inner membrane of gbacteria is mediated by pgrp-lc isoforms and pgrp-le resulting in the activation of the imd pathway (fig. 1) (wu et al., 2001; choe et al., 2002; gottar et al., 2002; ramet et al., 2002; takehana et al., 2004; kaneko et al., 2006). lysine (lys) type pgn of g+ bacteria on the other hand is recognized by pgrp-sa, pgrp-sd and gnbp1 leading to toll pathway activation (fig. 2) (michel et al., 2001; gobert et al., 2003; bischoff et al., 2004). sensing of fungal presence relies on the detection of glucan, a fungal cell wall constituent, by gnbp3 (gottar et al., 2006) and on the activation of the serine protease persephone (psh) in the hemolymph by virulence factors like fungal proteases (ligoxygakis et al., 2002). both fungal infection detection cascades converge on toll pathway activation (fig. 2). the ability to form several prr complexes furthermore expands the repertoire of microbial species of which the presence can be sensed (bischoff et al., 2004). recent findings also suggest that these recognition systems presumably detect proliferation of bacteria rather than their presence (ferrandon et al., 2007). furthermore, some microbial species can elicit both toll and imd signaling through a yet unknown process (de gregorio et al., 2002b). upon detection of gram negative bacterial infection, activation of the imd pathway (fig. 1) is probably achieved through cooperation of transmembrane pgrp-lc and intracellular pgrple (takehana et al., 2004; chang et al., 2005; mellroth et al., 2005; kaneko et al., 2006). pgrpsc and pgrp-lb, which reside in the hemolymph, further modulate the intensity of the imd pathway stimulation (mellroth et al., 2003; mellroth and steiner, 2006; zaidman-remy et al., 2006). this then results in the induction of several cascades downstream of the imd protein leading to activation of the relish derived nf-κb transcription factor rel (hedengren et al., 1999), as well as to jun nterminal kinase (jnk) pathway activation (boutros et al., 2002; park et al., 2004). both phosphorylation and cleavage of relish are needed to liberate the transcription factor fragment rel from its inhibitory part. phosphorylation is performed by the iκb kinase (ikk) signaling complex composed of kenny and ird5 (immune response deficient 5) whereas cleavage involves the coordinated action of at least three proteins, namely imd, fadd (fas-associated death domain) and dredd (death-related ced3/nedd2-like protein) (rutschmann et al., 2000; silverman et al., 2000; stoven et al., 2000; lu et al., 2001; leulier et al., 2002;). ikk and jnk activation furthermore is mediated by tak1 and tab2 (tak1binding protein 2) (vidal et al., 2001; boutros et al., 2002; silverman et al., 2003; gesellchen et al., 2005; geuking et al., 2005; kleino et al., 2005). rel-induced gene activation then results in the synthesis and release of amps and other effector proteins. among the drosophila amps, diptericin (dipt), attacin (att), drosocin (dros) and cecropin (cec) were identified as being imd pathway induced (hoffmann, 2003). negative regulation of the imd pathway furthermore involves caspar and the ubiquitin-proteasome pathway for controling relish and dredd activation levels (khush et al., 2002; kim et al., 2006). the physiological relevance of jnk signaling in the innate immune response remains elusive although it has been suggested to be involved in the control of the expression of some amps, and to regulate wound healing and melanization (boutros et al., 2002; igaki et al., 2002; silverman et al., 2003; kim et al., 2005) jnk signaling furthermore is shut off upon relish activation. the jnk-dependent response thus is transient and it precedes the sustained induction of relish-dependent innate immune loci (park et al., 2004). induction of the toll pathway (fig. 2) upon g+ and fungal infection is preceded by the cleavage of spätzle (spz) by the spätzle processing enzyme (spe) (jang et al., 2006). spz then binds to the membrane bound toll receptor thereby initiating the assembly of the toll induced signaling complex (tisc) which is composed of myeloid differentiation primary response gene 88 (myd88), tube and pelle (tauszig-delamasure et al., 2002; weber et al., 2003). tisc subsequently targets the cactus-dif (dorsal related immunity factor) complex in an unknown way to induce the degradation of the nfκb inhibitor cactus resulting in the release of the nf-κb transcription factor dif. gene activation is then accomplished upon translocation of dif to the nucleus (belvin et al., 1995; fernandez et al., 2001). drosomycin (drom), defensin (def) and metchnikowin (metch) are the amps of which the synthesis is induced by toll pathway activation (hoffmann, 2003). both the induction of the toll and the imd pathway thus leads to the activation of nf-κb transcription factors, dif and rel, which recognize distinct κb binding sites. many more putative toll and imd pathway components were discovered using several distinct assays, e.g., rnai on cultured s2 cells (avila et al., 2002) and loss-off-function screens (wu and anderson, 1998; wu et al., 2001), but their sites of action in toll and/or imd signaling still need to be explored. constitutive amp activation furthermore has been demonstrated in several epithelia in close contact with the environment. their expression however seems to be independent of both toll and imd signaling. upon infection, on the other hand, amp expression in these epithelia solely relies on imd pathway activation (ferrandon et al., 1998; tzou et al., 2000). cellular response the blood cells, or hemocytes, of drosophila participate in the immune response through the production of amps, the phagocytosis of bacteria, the 34 fig. 2 the drosophila toll pathway. structural components of the cell wall of g+ bacteria and fungi are detected by soluble pattern recognition receptors in the hemolymph. pgrp-sa, pgrp-sd and gnbp1 recognize lys type pgn probably upon formation of prr complexes, and gnbp3 binds to fungal derived β-glucans. virulence factors like fungal proteases [e.g., pr1 (gottar et al., 2006)] also can initiate an immune response through activation of the clip serine protease psh. the protease inhibitor necrotic (not shown) furthermore seems to be involved in sensing fungal infection in the same proteolitic pathway as psh (levashina et al., 1999). recently a second proteolitic cascade acting downstream of circulating prrs and including the activity of grass (gram positive specific serine protease) was uncovered (not shown) (el chamy et al., 2008). both the psh and the grass dependent pathways are required for full activation of toll upon fungal and g+ infection. all these diverse pathogen recognition systems converge in an largely unknown way to the cleavage of spätzle by spe resulting in the release of its 106 amino acid c-terminal fragment (c106). recently the modular serine protease (modsp) was shown to integrate signals originating from gnbp3 and pgrp-sa and to connect them to spe activity on spz (buchon et al., 2009b) spätzle c106 then binds to toll thereby inducing conformational changes in the receptor resulting in its activation. activated toll induces the assembly of the tisc composed of three members bearing dds. tube links pelle to myd88 through dd interactions, and myd88 probably interacts with toll through its tir (toll/il-1r) domain. tisc formation results in the activation of the kinase domain (kd) of pelle, and in an unknown way in the phosphorylation of the nf-κb inhibitor cactus thereby initiating its rapid polyubiquitylation and subsequent degradation. this allows the liberated nf-κb transcription factor dif to translocate to the nucleus and to induce gene activation upon binding to nf-κb elements. a cell culture study furthermore suggest the existence of a second branch downstream of toll activation consisting of atypical protein kinase c (apkc) and ref(2)p signaling components to aid in inducing dif activation (avila et al., 2002). 35 encapsulation of larger foreign particles such as parasitic eggs as well as the signalling of pathogen presence to the fat body (evans et al., 2003). at the larval stage, three types of circulating hemocytes can be distinguished originating from the embryonic head mesoderm (holz et al., 2003), the larval lymph glands (sorrentino et al., 2002) as well as from sessile hemocytes attached to larval epithelial tissues (markus et al., 2009). among these, the predominant type (90-95%) consists of small round plasmatocytes which essentially are phagocytic cells or macrophages. these engulf and degrade apoptotic and dead cells, and cellular detritus as well as microbial pathogens occurring in their hemolymph in a process known as phagocytosis. phagocytosis is initiated by the recognition of these targets mainly by four classes of molecules: the complement-like opsonin family of teps (thioestercontaining proteins), the class b scavenger receptors cd36, peste and croquemort, the egflike repeat containing receptors eater, nimrod c1 and draper, and the dscam (down syndrome cell adhesion molecule) isoforms (reviewed in stuart and ezekowitz, 2008). most of these components as well as a plethora of other intercellular phagocyte gene products were discovered with studies using a variant of drosophila embryonic s2 (schneider’s line 2) cells combined with rnai (pearson et al., 2003;ramet et al., 2001). additional studies are required to explore their function(s) in vivo. plasmatocytes also secrete amps as well as other immune signaling components. upd3 (unpaired 3), one of these molecules, has been shown to signal an infection induced by septic injury to the fat body thereby activating the jak/stat pathway in a cytokine-like manner (agaisse et al., 2003). a smaller proportion of circulating hemocytes is formed by the crystal cells which are recognized by pronounced crystal-like inclusions in their cytoplasm. these contain the enzymes necessary for humoral melanization which accompanies many immune reactions (see below). the third circulating hemocyte type, the lamellocyte, is normally not present in healthy larvae. lamellocytes are large flat cells of which the amount substantially increases after specific stimuli, e.g., presence of parasitic wasp eggs, through a small burst of mitosis and subsequent differentiation of sessile cells in the lymph glands (sorrentino et al., 2002) and through differentiation of a subepidermal lamellocyte precursor population (markus et al., 2009). the balance between the multipotent prohemocytes and the differentiating blood cells upon infection is controlled by a small cluster of signalling cells in the lymph glands, termed the posterior signalling centre (psc) (krzemien et al., 2007). lamellocytes mediate the encapsulation of invaders, e.g., parasitoids, that are often too large to be phagocytized. integrins seem to be involved in the strengthening of the capsule (irving et al., 2005). encapsulation furthermore is often accompanied by a localized melanization reaction and an augmented nitric oxide (cytotoxic) production, resulting in the killing of the parasite within the black capsule (carton and nappi, 1997, nappi et al., 2000). the hemocytes detected in drosophila adults originate from the embryonic and larval lineages that persist during metamorphosis to populate this developmental stage. no adult hematopoietic organ has been described so far (holz et al., 2003). recently, it was found that priming drosophila with sublethal doses of streptococcus pneumoniae or the natural fly pathogen beauveria bassiana has protective effects during subsequent challenges, and this persisted for the life of the fly. phagocytes as well as toll pathway activation, however independent of amp synthesis, were required for this presumably species specific but not generally observed effect. these findings raise questions about the absence of memory in drosophila’s innate immune responses (pham et al., 2007). for a more in depth description of the cellular immune response, see for review evans et al. (2003) and stuart and ezekowitz (2008). the phenoloxidase response the melanization reaction seems to be the most immediate immune response against invading pathogens in drosophila (cerenius and söderhäll, 2004). it frequently assists the encapsulation reaction in the killing of microbial pathogens (nappi and christensen, 2005). melanization is visible by the blackening of a wound site or of the surface of invaders, which results from the synthesis and deposition of melanin (tang et al., 2006). the melanization reaction starts off with the activation of prophenoloxidases (propo) to active phenoloxidase (po) enzyme in the hemolymph of arthropods, hence it is also referred to as po response. this system had been extensively studied in large insects such as manduca sexta (cerenius and söderhäll, 2004; söderhäll and cerenius, 1998). this has led to the present model (fig. 3) in which the recognition of microorganisms triggers a propoactivating enzyme (ppae) proteolytic cascade dedicated to the activation of po. the activated po then catalyses the oxidation of tyrosine-derived phenols to quinones. quinones then polymerize non-enzymatically to form insoluble melanin. quinones as well as melanin and its biosynthetic byproducts, hydrogen peroxide and nitric oxide amongst others, are directly toxic to microorganisms (nappi et al., 2000, nappi and ottaviani, 2000). melanin deposition is observed at all infection sites, where it possibly contributes to wound healing and to the control of microorganism growth as well as to their killing (leclerc et al., 2006, nappi et al., 1995, carton et al., 2009, nappi and christensen, 2005). of note, nearly all arthropod propos are devoid of a secretion signal sequence. there presence in the hemolymph is therefore assumed to result from hemocyte rupture, as reported for some drosophila propos (bidla et al., 2007). in drosophila, induction of the melanization response upon ginfection involves pgrp-le, which is also one of the key prrs of the imd pathway (fig. 1). the crystal cells furthermore are shown to express propo a1 (or 54) as well as propo45. the third drosophila propo is expressed exclusively in lamellocytes. the cells that mediate the encapsulation response thus also can provide 36 fig. 3 overview of the arthropod melanization cascade. the system is activated upon recognition of bacterial and fungal cell wall components by prrs as well as by some endogenous factors produced upon tissue damage, e.g., during wounding. a cascades of serine proteases presumably will result in the cleavage of proprophenoloxidase activating enzyme (pro-ppae) thereby activating it. cleavage of propo by active ppae results in the activation of po. next, po catalyzes the oxidation of phenols to quinones, which subsequently can polymerize to melanin. control of po activity is presumed to result from its synthesis as inactive precursor as well as from the presence of proteinase inhibitors which probably avoid excessive or premature activation (adapted from cerenius & söderhäll, 2004). one of the key enzymes for melanization (irving et al., 2005). the activation of propo in drosophila is partially controlled by the serine protease inhibitor serpin 27a (spn27a) (nappi and christensen, 2005). the target of spn27a is thought to be ppae since recombinant spn27a is able to inhibit beetle ppae (de gregorio et al., 2002). this however has not been demonstrated to occur in vitro or in vivo with drosophila ppae. two immune inducible serine proteases, mp1 (cg1102) and mp2 (cg3066) (melanization protease 1 and 2), furthermore were identified which act in the po cascade regulated by spn27a. mp1 seems to be required for activation of the melanization process upon bacterial and fungal infection whereas mp2 is only involved in fighting fungal infection and thereby acting upstream of mp1. mp2 is furthermore able to induce drosomycin expression independent of toll pathway activation (tang et al., 2006). mp2 (or ppae1) was furthermore reported to be a drosophila ppae as constitutive ppae mutants showed constitutive po activation (leclerc et al., 2006). further elucidation of the drosophila po response thus is required to explore whether it is consistent with the proposed model (fig. 3). recently, colinet et al. (2009) identified the first serpin used as a virulence factor from the parasitoid wasp leptopilina boulardi and they showed that it targets the drosophila phenoloxidase cascades. the jak/stat cascade in innate immunity the evolutionary conserved janus kinase (jak)/signal transducer and activator of transcription (stat) cascade plays a key role in a wide variety of biological processes (arbouzova and zeidler, 2006). in drosophila, only one stat protein, stat92, seems to exist and hopscotch (hop) is the homolog of vertebrate tyrosine kinase jak (binari and perrimon, 1994; hou et al., 1996; yan et al., 1996). loss-of-function mutants of both stat92e and hop show a severe decrease in immune response activation upon infection, implicating the drosophila jak/stat pathway in the innate immune defense (sorrentino et al., 2004). stat92e furthermore is activated in the fat body upon immunization (kwon et al., 2000). jak/stat pathway activation (fig. 4) requires binding of an extracellular ligand, unpaired (upd), to a transmembrane receptor, domeless/master of marelle (dome/mom). upd, upd2 or upd3 constitute the drosophila upd family of which only the latter is implicated in the fruit fly’s immune response (agaisse et al., 2003). ligand binding then results in the activation of receptor-associated jaks which recruit stats after their phosphorylation. next, the stats are phosphorylated and they will form the dimers that are responsible for gene activation upon translocation to the nucleus (see for review 37 fig. 4 the jak/stat pathway. upd binding onto the dome receptor dimer activates jak kinases. jaks phosphorylate (p) one another and subsequently attract stats. after jak dependent phosphorylation and subsequent dimerization, stat dimers are translocated to the nucleus through nuclear pore complexes to act as activators of gene transcription. arbouzova and zeidler, 2006). upon infection, turandot a (tota), totm and totc, which normally accumulate in the hemolymph in response to various stress conditions including immune challenge, are expressed in the fat body thereby requiring jak activity (agaisse et al., 2003). gene activation of vir1 (virus induced rna 1) furthermore is also attributed to jak/stat signaling since stat92e binds to its promoter (dostert et al., 2005). in drosophila, several negative regulators of jak/stat signalling were identified (fig. 4). among these, soc36e (suppressors of cytokine signaling 36e) and tyrosine phosphatase ptp61f (phosphotyr phosphatase 61f) both are a transcriptional target as well as a negative regulator, thereby forming a negative feedback loop to down-regulate pathway activity (baeg et al., 2005; muller et al., 2005). furthermore, the drosophila homologs of ranbp3 and ranbp10 control the nucleocytoplasmic transport of stat92e (baeg et al., 2005). ken and barbie (ken), a transcriptional repressor, selectively regulates stat92e activity (arbouzova et al., 2006). jak/stat pathway activity furthermore is also detected in hemocytes where it modulates hemocyte proliferation and differentiation, e.g., into lamellocytes in cooperation with the ras and toll pathway (evans et al., 2003). biological functions of jak/stat, besides its implication in the innate immune response, are summarized in agaisse and perrimon (2004) and in arbouzova and zeidler (2006). the antiviral response: implication of rna interference in the innate immune system rnai probably originated as an innate immune mechanism for fighting viral infections (fig. 5). viral dsrna thereby is used to trigger host-mediated degradation of viral rna. the identification of viral proteins, e.g., b2 of flock house virus and 1a from dcv (drosophila c virus), that are able to inhibit the rnai pathway (li et al., 2004) and the presence of viral small interfering rnas in infected cells (aliyari et al., 2008) support this rnai origin hypothesis. in drosophila, three rnai pathways are described of which at least two, an argonaute 2 (ago2) dependent and the piwi pathway, seem to be implicated in the anti-viral defence although both are elicited upon different viral infestations (van rij and berezikov, 2009). of note, dicer 2, argonaute 2 and r2d2 are encoded by the 3 % fastest evolving genes in drosophila. this probably is driven by the likewise fast evolving viral inhibitor proteins (obbard et al., 2006). in addition, studies using drosophila x virus (dxv, a dsrna virus) implied both toll and imd pathway signaling in the detection of viral infection, although only a toll induced nf-κb activation, dissimilar to the one described for amp production (fig. 2) seems to confer protective effects (zambon et al., 2005). dcv infection, adversely, results in jak/stat-induced immune signaling but not either tollnor imd-mediated immune responses (dostert et al., 2005). viruses thus evoke different defense 38 fig. 5 defense response against viral infection. left: viral infection (mainly through endocytosis) as well as viral replication in infected cells depend on endogenous cellular factors of the infected host (depicted in green). many viruses depend on dsrna production for replication. the hosts rnai machinery, composed of dicer-2 (dcr-2) and its cofactor r2d2, mediate cleavage of the viral dsrna into sirnas. these are subsequently incorporated into an ago2 containing risc complex which is devoted to the targeted destruction of analogical sequence specific viral rna (depicted in blue). viruses object this rnai mediated defence response by encoding rnai inhibitor components (depicted in red). some viral infections furthermore lead to the activation of a toll induced, yet unknown signaling cascade that results in nf-κb induced (dif) antiviral gene activation (depicted in blue). recently, the amino terminal dexd/h box helicase domain of dicer 2 was implicated in the inducible antiviral response thereby suggesting a connection between this and rnai (deddouche et al., 2008). right: uninfected cells may be induced to produce and release antiviral factors by an unknown mechanism probably involving recognition of viral products, cell debris or other host antiviral factors. both jak/stat and toll pathway activation have been implicated in the production of the proteins of which some are assumed to have antiviral proporties, e.g., vago (deddouche et al., 2008) and vir-1 (dostert et al., 2005). responses probably depending on differences in their pathogenesis and replication cycles. the exploration of the antiviral response in drosophila commenced about five years ago resulting in a yet limited understanding which is even more complicated by the different defense responses evoked by various viruses (see for reviews kemp and imler, 2009; van rij and berezikov, 2009). immune defence systems in epithelial tissues the innate immune responses described above were all explored in the two main immune tissues of drosophila, the fat body and the hemocytes. they focus on the recognition and signaling of a pathogenic invader in the body cavity, and the subsequent production and release of immune effectors into its main battlefield, the hemolymph. this is referred to as the systemic immune response. as is the case in mammals, also the epithelial linings of the digestive, respiratory and reproductive system that constitute the physical barrier for pathogen entrance into the body cavity do seem to rely on an effective immune defense system to try to prevent this invasion. epithelial expression of antimicrobial peptides, for example was explored using reporter flies in which the promoter sequence of each of the seven amps (att, cec, def, droc, dipt, drom and metch) was fused to the green fluorescent protein coding sequence. this way, the expression pattern of the amps was explored both in larvae and in adults (ferrandon et al., 1998; tzou et al., 2000). this led to the finding that amps can be induced in surface epithelia in a tissue-specific manner and that imd plays a critical role in the activation of this local response to infection (tzou et al., 2000). malphigian tubule epithelia, furthermore, shown expression of pgrps, e.g., pgrp-le when induced by tct (tracheal cytotoxin), disaccharide-tetrapeptide fragment of pgn (kaneko et al., 2006). furthermore, the drosophila gut lumen is considered to be hostile to transient microbial 39 colonization due to physical (acidity) and physiological (peristalsis of the gut) properties and the presence of lysozymes. and the gut epithelium further was shown to express amps and to catalyze the generation of ros that together most often provide an effective barrier against ingested microbes. next, global gene expression analysis of drosophila intestinal tissue, i.e., the gut minus the malpighian tubules and gastric caeca, to oral infection with the gbacterium erwinia carotovora recently revealed that immune responses in the gut are regulated by the imd and jak-stat pathways, but not the toll pathway (buchon et al., 2009a). in addition, the malpighian tubules also possess cellautonomous, immune-sensing capabilities. all components of both the imd and the toll pathway are expressed herein, and they are expected to mainly lead to the production of diptericin, cecropin and metchnikowin. the tubules furthermore produce nitric oxide (no) which was also shown to be extremely important for amp production as significant improvement in survival rates of the whole animal upon immune challenge was observed after forced no production (davies and dow, 2009). similarities between mammalian and insect immune responses insects and vertebrates display considerable overlap in the signalling pathways that regulate innate immunity and in some of the effector mechanisms used against microbes. the mode of detection of microbial patterns through activation of pattern recognition receptors and nf-κb signalling cascades have been conserved throughout evolution. the drosophila toll pathway, for example, has some parallels to the mammalian signalling systems downstream of the interleukin 1 receptor (il-1r) and the toll-like receptors (tlrs). the main difference seems to be the fact that the drosophila toll receptor does not sense microbial inducers directly, as most mammalian tlrs do, but instead relies on an upstream recognition system. furthermore, the mammalian system seems to be able to use the same nf-κb inducing cascade after recognition of both g+ and gbacterial infection, while drosophila uses two distinct systems, i.e., toll and imd signaling. the drosophila imd cascade furthermore is similar to the mammalian tumour necrosis factor receptor (tnfr) pathway (hoffmann, 2003; wang and ligoxygakis, 2006). a number of similarities between drosophila hemocytes and mammalian blood cells such as amp synthesis and cytokine production were also reported, as well as some specifics of invertebrate cellular immunity. the melanisation response, for example, has no clear counterpart in mammals but instead uses molecular building blocks (proteolytic cascades, integrins) that are conserved among phyla (irving et al., 2005). drosophila blood cells consist of only a few terminally differentiated types whose functions resemble those of the cells of the vertebrate myeloid lineage which gives rise to macrophages among others (evans et al., 2003). a proteomic analysis of the phagosome content of drosophila plasmatocytes, for example, has revealed that 70 % of its protein content (600 identified proteins) has a mammalian orthologue thereby validating fruit fly phagosomes as a model to study phagocytosis (stuart and ezekowitz, 2008). the encapsulation reaction furthermore has a similar function as the formation of granuloma in vertebrates (markus et al., 2009). in addition, the activation of tota in the drosophila fat body as a response to the release of upd3 by hemocytes, for example, is very similar to some aspects of the mammalian acute-phase response mediated by cytokines (agaisse and perrimon, 2004). next, the human genome encodes all major jak/stat pathway components. molecular and functional data clearly indicate that a high level of conservation exists between the structural components of both the insect/drosophila and the mammalian pathway (arbouzova and zeidler, 2006). for fighting off viral infections, the mammalian system seems to depend largely on recognition of dsrna, as does drosophila, but in mammals this mainly leads to the production of interferons while in drosophila rna interference is the main immune effector (cherry and silverman, 2006). the study of the drosophila antiviral immunity only just recently has gained serious interest. further similarities and differences thus are expected to be uncovered in the near future. overall, discoveries made through research in the fruit fly, drosophila melanogaster may be applicable to the study of innate immunity in humans. the exploration of innate immunity in insects has also garnered increasing attention because of the role of many insects in transmission of human disease agents. understanding how the insect immune system interacts with pathogens may contribute to the development of new strategies to block transmission of disease agents (shi and paskewitz, 2006). recently, questions were raised regarding the absence of memory and the absence of the diversity of immunoglobulin like recognition receptors in drosophila innate immunity as seen in mammals. induction of resistance to lethal doses of a pathogen by priming with sublethal doses was observed for s. pneumoniae and b. bassiana and required both the toll pathway and phagocytosis (pham et al., 2007). the discovery that the neuronal immunoglobulin superfamily member dscam, which is encoded by a gene that can potentially generate 18,000 splice isoforms, is expressed by hemocytes and by cells in the fat body has also raised considerable interest as to the possibility of the generation of a large receptor repertoire in d. melanogaster (watson et al., 2005). further research will probably shed light on these interesting findings. conclusion in drosophila, the epithelial defense system forms the first barrier to microorganismal infection. upon entry into the body cavity, pathogens subsequently are mainly countered in the hemolymph with humoral, cellular and phenoloxidase defence responses. both the jak/stat and the jnk pathways further seem to be implicated herein. drosophila further seems to 40 rely on rnai to combat viral infections. appropriate experimental studies to identify the actual killing elements, however, are still awaiting. interestingly, many drosophila immune components have a counterpart in mammals making the study of fruit fly immunity of great importance for the unraveling of the conserved innate immune cascades. references agaisse h, perrimon n. the roles of jak/stat signaling in drosophila immune responses. immunol. rev. 198: 72-82, 2004. agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell 5: 441-450, 2003. aliyari r, wu q, li hw, wang xh, li f, green ld, et al. mechanism of induction and suppression of antiviral immunity directed by virus-derived small rnas in drosophila. cell host microbe 4: 387-397, 2008. arbouzova ni, bach ea, zeidler mp. ken and barbie selectively regulates the expression of a subset of jak/stat pathway target genes. curr. biol. 16: 80-88, 2006. arbouzova ni, zeidler mp. jak/stat signalling in drosophila: insights into conserved regulatory and cellular functions. development 133: 26052616, 2006. avila a, silverman n, diaz-meco mt, moscat j. the drosophila atypical protein kinase c-ref(2)p complex constitutes a conserved module for signaling in the toll pathway. mol. cell. biol. 22: 8787-8795, 2002. baeg gh, zhou r, perrimon n. genome-wide rnai analysis of jak/stat signaling components in drosophila. genes dev. 19: 1861-1870, 2005. belvin mp, jin y, anderson kv. cactus protein degradation mediates drosophila dorsal-ventral signaling. genes dev. 9: 783-793, 1995. bidla g, dushay ms, theopold u. crystal cell rupture after injury in drosophila requires the jnk pathway, small gtpases and the tnf homolog eiger. j. cell. sci. 120: 1209-1215, 2007. binari r, perrimon n. stripe-specific regulation of pair-rule genes by hopscotch, a putative jak family tyrosine kinase in drosophila. genes dev. 8: 300-312, 1994. bischoff v, vignal c, boneca ig, michel t, hoffmann ja, royet j. function of the drosophila pattern-recognition receptor pgrpsd in the detection of gram-positive bacteria. nat immunol 5: 1175-1180, 2004. boutros m, agaisse h, perrimon n. sequential activation of signaling pathways during innate immune responses in drosophila. dev cell 3: 711-722, 2002. buchon n, broderick na, poidevin m, pradervand s, lemaitre b. drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. cell host microbe 5: 200-211, 2009a. buchon n, poidevin m, kwon hm, guillou a, sottas v, lee bl, et al. a single modular serine protease integrates signals from patternrecognition receptors upstream of the drosophila toll pathway. proc. natl. acad. sci. usa 106: 12442-12447, 2009b. carton y, frey f, nappi aj. parasite-induced changes in nitric oxide levels in drosophila paramelanica. j. parasitol. 95: 1134-1141, 2009. carton y, nappi aj. drosophila cellular immunity against parasitoids. parasitol. today 13: 218227, 1997. cerenius l, söderhäll k. the prophenoloxidaseactivating system in invertebrates. immunol. rev. 198: 116-126, 2004. chang ci, ihara k, chelliah y, mengin-lecreulx d, wakatsuki s, deisenhofer j. structure of the ectodomain of drosophila peptidoglycanrecognition protein lca suggests a molecular mechanism for pattern recognition. proc. natl. acad. sci. usa 102: 10279-10284, 2005. cherry s, silverman n. host-pathogen interactions in drosophila: new tricks from an old friend. nat. immunol. 7: 911-917, 2006. choe km, werner t, stoven s, hultmark d, anderson kv. requirement for a peptidoglycan recognition protein (pgrp) in relish activation and antibacterial immune responses in drosophila. science 296: 359-362, 2002. colinet d, dubuffet a, cazes d, moreau s, drezen jm, poirié m. a serpin from the parasitoid wasp leptopilina boulardi targets the drosophila phenoloxidase cascade. develop. comp. immunol. 33: 681-689, 2009. davies sa, dow ja. modulation of epithelial innate immunity by autocrine production of nitric oxide. gen. comp. endocrinol. 162: 113-121, 2009. de gregorio e, han sj, lee wj, baek mj, osaki t, kawabata s, et al. an immune-responsive serpin regulates the melanization cascade in drosophila. dev. cell 3: 581-592, 2002a. de gregorio e, spellman pt, tzou p, rubin gm, lemaitre b. the toll and imd pathways are the major regulators of the immune response in drosophila. embo j. 21: 2568-2579, 2002b. deddouche s, matt n, budd a, mueller s, kemp c, galiana-arnoux d, et al. the dexd/h-box helicase dicer-2 mediates the induction of antiviral activity in drosophila. nat. immunol. 9: 1425-1432, 2008. dostert c, jouanguy e, irving p, troxler l, galianaarnoux d, hetru c, et al. the jak-stat signaling pathway is required but not sufficient for the antiviral response of drosophila. nat. immunol. 6: 946-953, 2005. el chamy l, leclerc v, caldelari i, reichhart jm. sensing of 'danger signals' and pathogenassociated molecular patterns defines binary signaling pathways 'upstream' of toll. nat. immunol. 9: 1165-1170, 2008. evans cj, hartenstein v, banerjee u thicker than blood: conserved mechanisms in drosophila and vertebrate hematopoiesis. dev. cell 5: 673690, 2003. fernandez nq, grosshans j, goltz js, stein d. separable and redundant regulatory determinants in cactus mediate its dorsal group dependent degradation. development 128: 2963-2974, 2001. 41 ferrandon d, imler jl, hetru c, hoffmann ja. the drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. nat. rev. immunol. 7: 862-874, 2007. ferrandon d, jung ac, criqui m, lemaitre b, uttenweiler-joseph s, michaut l, et al. a drosomycin-gfp reporter transgene reveals a local immune response in drosophila that is not dependent on the toll pathway. embo j. 17: 1217-1227, 1998. foley e, o'farrell ph. functional dissection of an innate immune response by a genome-wide rnai screen. plos biol. 2: e203, 2004. gesellchen v, kuttenkeuler d, steckel m, pelte n, boutros m. an rna interference screen identifies inhibitor of apoptosis protein 2 as a regulator of innate immune signalling in drosophila. embo rep. 6: 979-984, 2005. geuking p, narasimamurthy r, basler k. a genetic screen targeting the tumor necrosis factor/eiger signaling pathway: identification of drosophila tab2 as a functionally conserved component. genetics 171: 1683-1694, 2005. gobert v, gottar m, matskevich aa, rutschmann s, royet j, belvin m, et al. dual activation of the drosophila toll pathway by two pattern recognition receptors. science 302: 2126-2130, 2003. gottar m, gobert v, matskevich aa, reichhart jm, wang c, butt tm, et al. dual detection of fungal infections in drosophila via recognition of glucans and sensing of virulence factors. cell 127: 1425-1437, 2006. gottar m, gobert v, michel t, belvin m, duyk g, hoffmann ja, et al. the drosophila immune response against gram-negative bacteria is mediated by a peptidoglycan recognition protein. nature 416: 640-644, 2002. hedengren m, asling b, dushay ms, ando i, ekengren s, wihlborg m, et al. relish, a central factor in the control of humoral but not cellular immunity in drosophila. mol. cell 4: 827-837, 1999. hoffmann ja. the immune response of drosophila. nature 426: 33-38, 2003. holz a, bossinger b, strasser t, janning w, klapper r. the two origins of hemocytes in drosophila. development 130: 4955-4962, 2003. hou xs, melnick mb, perrimon n. marelle acts downstream of the drosophila hop/jak kinase and encodes a protein similar to the mammalian stats. cell 84: 411-419, 1996. igaki t, kanda h, yamamoto-goto y, kanuka h, kuranaga e, aigaki t, et al. eiger, a tnf superfamily ligand that triggers the drosophila jnk pathway. embo j. 21: 3009-3018, 2002. irving p, ubeda jm, doucet d, troxler l, lagueux m, zachary d, et al. new insights into drosophila larval haemocyte functions through genome-wide analysis. cell microbiol. 7: 335350, 2005. jang ih, chosa n, kim sh, nam hj, lemaitre b, ochiai m, et al. a spätzle-processing enzyme required for toll signaling activation in drosophila innate immunity. dev. cell 10: 4555, 2006. kaneko t, yano t, aggarwal k, lim jh, ueda k, oshima y, et al. pgrp-lc and pgrp-le have essential yet distinct functions in the drosophila immune response to monomeric dap-type peptidoglycan. nat. immunol. 7: 715-723, 2006. kemp c, imler jl. antiviral immunity in drosophila. curr. opin. immunol. 21: 3-9, 2009. khush rs, cornwell wd, uram jn, lemaitre b. a ubiquitin-proteasome pathway represses the drosophila immune deficiency signaling cascade. curr. biol. 12: 1728-1737, 2002. kim m, lee jh, lee sy, kim e, chung j. caspar, a suppressor of antibacterial immunity in drosophila. proc. natl. acad. sci. usa 103: 16358-16363, 2006. kim t, yoon j, cho h, lee wb, kim j, song yh, et al. downregulation of lipopolysaccharide response in drosophila by negative crosstalk between the ap1 and nf-kappab signaling modules. nat. immunol. 6: 211-218, 2005. kleino a, valanne s, ulvila j, kallio j, myllymaki h, enwald h, et al. inhibitor of apoptosis 2 and tak1-binding protein are components of the drosophila imd pathway. embo j. 24: 34233434, 2005. krzemien j, dubois l, makki r, meister m, vincent a, crozatier m. control of blood cell homeostasis in drosophila larvae by the posterior signalling centre. nature 446: 325328, 2007. kwon ej, park hs, kim ys, oh ej, nishida y, matsukage a, et al. transcriptional regulation of the drosophila raf proto-oncogene by drosophila stat during development and in immune response. j. biol. chem. 275: 1982419830, 2000. leclerc v, pelte n, el cl, martinelli c, ligoxygakis p, hoffmann ja, et al. prophenoloxidase activation is not required for survival to microbial infections in drosophila. embo rep. 7: 231-235, 2006. lemaitre b, reichhart jm, hoffmann ja. drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. proc. natl. acad. sci. usa 94: 14614-14619, 1997. leulier f, vidal s, saigo k, ueda r, lemaitre b. inducible expression of double-stranded rna reveals a role for dfadd in the regulation of the antibacterial response in drosophila adults. curr. biol.12: 996-1000, 2002. levashina ea, langley e, green c, gubb d, ashburner m, hoffmann ja, et al. constitutive activation of toll-mediated antifungal defense in serpin-deficient drosophila. science 285: 19171919, 1999. li wx, li h, lu r, li f, dus m, atkinson p, et al. interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing. proc. natl. acad. sci. usa 101: 1350-1355, 2004. ligoxygakis p, pelte n, hoffmann ja, reichhart jm. activation of drosophila toll during fungal infection by a blood serine protease. science 297: 114-116, 2002. lu y, wu lp, anderson kv. the antibacterial arm of the drosophila innate immune response 42 requires an iκb kinase. genes dev. 15: 104110, 2001. markus r, laurinyecz b, kurucz e, honti v, bajusz i, sipos b, et al. sessile hemocytes as a hematopoietic compartment in drosophila melanogaster. proc. natl. acad. sci. usa 106: 4805-4809, 2009. mellroth p, karlsson j, håkansson j, schultz n, goldman we, steiner h. ligand-induced dimerization of drosophila peptidoglycan recognition proteins in vitro. proc. natl. acad. sci. usa 102: 6455-6460, 2005. mellroth p, karlsson j, steiner h. a scavenger function for a drosophila peptidoglycan recognition protein. j. biol. chem. 278: 70597064, 2003. mellroth p, steiner h. pgrp-sb1: an nacetylmuramoyl l-alanine amidase with antibacterial activity. biochem. biophys. res. commun. 350: 994-999, 2006. michel t, reichhart jm, hoffmann ja, royet j. drosophila toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. nature 414: 756-759, 2001. muller p, kuttenkeuler d, gesellchen v, zeidler mp, boutros m. identification of jak/stat signalling components by genome-wide rna interference. nature 436: 871-875, 2005. nappi aj, christensen bm. melanogenesis and associated cytotoxic reactions: applications to insect innate immunity. insect biochem. mol. biol. 35: 443-459, 2005. nappi aj, ottaviani e. cytotoxicity and cytotoxic molecules in invertebrates. bioessays 22: 469480, 2000. nappi aj, vass e, frey f, carton y. superoxide anion generation in drosophila during melanotic encapsulation of parasites. eur. j. cell biol. 68: 450-456, 1995. nappi aj, vass e, frey f, carton y. nitric oxide involvement in drosophila immunity. nitric oxide 4: 423-430, 2000. obbard dj, jiggins fm, halligan dl, little tj. natural selection drives extremely rapid evolution in antiviral rnai genes. curr. biol. 16: 580-585, 2006. park jm, brady h, ruocco mg, sun h, williams d, lee sj, et al. targeting of tak1 by the nfkappa b protein relish regulates the jnkmediated immune response in drosophila. genes dev. 18: 584-594, 2004. pearson am, baksa k, ramet m, protas m, mckee m, brown d, et al. identification of cytoskeletal regulatory proteins required for efficient phagocytosis in drosophila. microbes infect. 5: 815-824, 2003. pham ln, dionne ms, shirasu-hiza m, schneider ds. a specific primed immune response in drosophila is dependent on phagocytes. plos pathog. 3: e26, 2007. ramet m, manfruelli p, pearson a, mathey-prevot b, ezekowitz ra. functional genomic analysis of phagocytosis and identification of a drosophila receptor for e. coli. nature 416: 644648, 2002. ramet m, pearson a, manfruelli p, li x, koziel h, gobel v, et al. drosophila scavenger receptor ci is a pattern recognition receptor for bacteria. immunity 15: 1027-1038, 2001. rutschmann s, jung ac, zhou r, silverman n, hoffmann ja, ferrandon d. role of drosophila ikkγ in a toll-independent antibacterial immune response. nat. immunol. 1: 342-347, 2000. shi l, paskewitz sm. proteomics and insect immunity. inv. surv. j. 3: 4-17, 2006. silverman n, zhou r, erlich rl, hunter m, bernstein e, schneider d, et al. immune activation of nf-κb and jnk requires drosophila tak1. j. biol. chem. 278: 4892848934, 2003. silverman n, zhou r, stoven s, pandey n, hultmark d, maniatis t. a drosophila iκb kinase complex required for relish cleavage and antibacterial immunity. genes dev. 14: 2461-2471, 2000. söderhäll k, cerenius l. role of the prophenoloxidase-activating system in invertebrate immunity. curr. opin. immunol. 10: 23-28, 1998. sorrentino rp, carton y, govind s. cellular immune response to parasite infection in the drosophila lymph gland is developmentally regulated. dev. biol. 243: 65-80, 2002. sorrentino rp, melk jp, govind s. genetic analysis of contributions of dorsal group and jakstat92e pathway genes to larval hemocyte concentration and the egg encapsulation response in drosophila. genetics 166: 13431356, 2004. stöven s, ando i, kadalayil l, engström y, hultmark d. activation of the drosophila nf-κb factor relish by rapid endoproteolytic cleavage. embo rep. 1: 347-352, 2000. stuart lm, ezekowitz ra. phagocytosis and comparative innate immunity: learning on the fly. nat. rev. immunol. 8: 131-141, 2008. takehana a, yano t, mita s, kotani a, oshima y, kurata s. peptidoglycan recognition protein (pgrp)-le and pgrp-lc act synergistically in drosophila immunity. embo j. 23: 4690-4700, 2004. tang h, kambris z, lemaitre b, hashimoto c. two proteases defining a melanization cascade in the immune system of drosophila. j. biol. chem. 281: 28097-28104, 2006. tauszig-delamasure s, bilak h, capovilla m, hoffmann ja, imler jl. drosophila myd88 is required for the response to fungal and grampositive bacterial infections. nat. immunol. 3: 91-97, 2002. tzou p, ohresser s, ferrandon d, capovilla m, reichhart jm, lemaitre b, et al. tissue-specific inducible expression of antimicrobial peptide genes in drosophila surface epithelia. immunity 13: 737-748, 2000. van rij rp, berezikov e. small rnas and the control of transposons and viruses in drosophila. trends microbiol. 17: 163-171, 2009. vidal s, khush rs, leulier f, tzou p, nakamura m, lemaitre b. mutations in the drosophila dtak1 gene reveal a conserved function for mapkkks in the control of rel/nf-kappab-dependent innate immune responses. genes dev. 15: 1900-1912, 2001. 43 wang l, ligoxygakis p. pathogen recognition and signalling in the drosophila innate immune response. immunobiology 211: 251-261, 2006. wu lp, choe km, lu y, anderson kv. drosophila immunity: genes on the third chromosome required for the response to bacterial infection. genetics 159: 189-199, 2001. watson fl, puttmann-holgado r, thomas f, lamar dl, hughes m, kondo m, et al. extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 18741878, 2005. yan r, small s, desplan c, dearolf cr, darnell je jr. identification of a stat gene that functions in drosophila development. cell 84: 421-430, 1996. zaidman-remy a, herve m, poidevin m, pili-floury s, kim ms, blanot d, et al. the drosophila amidase pgrp-lb modulates the immune response to bacterial infection. immunity 24: 463-473, 2006. weber an, tauszig-delamasure s, hoffmann ja, lelievre e, gascan h, ray kp, et al. binding of the drosophila cytokine spätzle to toll is direct and establishes signaling. nat. immunol. 4: 794-800, 2003. zambon ra, nandakumar m, vakharia vn, wu lp. the toll pathway is important for an antiviral response in drosophila. proc. natl. acad. sci. usa 102: 7257-7262, 2005. wu lp, anderson kv. regulated nuclear import of rel proteins in the drosophila immune response. nature 392: 93-97, 1998. 44 conclusion 18 isj 14: 18-31, 2017 issn 1824-307x research report an integrated approach to study the biomarker responses in marine gastropod nerita chamaeleon environmentally exposed to polycyclic aromatic hydrocarbons j bhagat1, a sarkar2#, v deepti2, v singh2, l raiker2, bs ingole1 1biological oceanographic division, csir-national institute of oceanography, dona paula, goa 403004, india 2chemical oceanographic division, csir-national institute of oceanography, dona paula, goa 403004, india # present address: global enviro-care, kevnem, caranzalem, goa 403002, india accepted december 28, 2016 abstract ecological risk assessment using multiple biomarkers produce a large amount of data that is hard to interpret and the result are often contradictory. in this context, integrated biomarker response (ibr) index was used to integrate the biomarkers effects to assess the impact of environmental contaminants in marine gastropod nerita chamaeleon from goa, india. genotoxic (dna damage as measured by comet assay and alkaline unwinding assay) and biochemical [superoxide dismutase, catalase, glutathione s-transferase, lipid peroxidation and acetylcholinesterase] biomarkers were measured in snails collected from different sites (arambol, anjuna, sinquerim, dona paula, velsao, betul and palolem). total polycyclic aromatic hydrocarbons in snail tissue were in the range from 5.29 12.14 µg/g wet weight. standardized values of biomarker response were visualized using star plots, which show unique patterns for different biomarkers. the mean ibr value was found to be highest at dona paula (8.07 ± 0.91) followed by sinquerim (6.95 ± 0.91), velsao (4.48 ± 0.68), anjuna (3.28 ± 1.05), palolem (2.53 ± 0.73), arambol (1.81 ± 0.21) and betul (0.88 ± 0.77). additionally, the ibr values were found to be positively correlated with pah concentration in snail tissues. these results suggest that integration of biomarkers effects using ibr along with chemical analysis can be a useful tool for the assessment of environmental pollution and to identify spatial patterns of contamination in the aquatic ecosystem. key words: integrated biomarker response; oxidative stress; genotoxic damage; polycyclic aromatic hydrocarbon; comet assay introduction polycyclic aromatic hydrocarbons (pah) are persistent and ubiquitous environmental contaminants found in air, water, and soil. they are studied extensively due to their carcinogenic properties for human as well as animals. the international agency for research on cancer (iarc) has classified pahs as possible and probable carcinogen to human (iarc, 2010). the lipophilic and hydrophobic nature allows pahs to accumulate in the marine organism (mashroofeh et al., 2015). the accumulation of pahs in a marine organism can negatively affect their health (frouin et al., 2007; grintzalis et al., 2012). pahs and their metabolites interact with dna and form dna adducts. pah activation process also generates ___________________________________________________________________________ corresponding author: jacky bhagat biological oceanographic division csir-national institute of oceanography dona paula, goa-403004, india e-mail: bhagatjack@gmail.com reactive oxygen species (ros) which can induce genotoxic damage by modifying integrity of dna (mattson et al., 2009). biomarkers are an important tool to detect exposure and adverse effects of human-made or natural contaminants on aquatic organisms. some biomarkers are specific to chemicals or group of chemicals while other are non-specific and induces upon exposure to broad range of pollutants. due to the complexity of contaminants, use of multibiomarker has become an increasingly popular tool to study the environmental parameters as well as organism health. comet (or single cell gel electrophoresis) assay is the most commonly used as a biomarker of dna damage in various research areas because of it sensitive and reliable nature. dna damage as measured by comet assay has been reported in mussels, clams and several other aquatic organisms (martins et al., 2013; dailianis et al., 2014; sarkar et al., 2014). another technique is known as dna-alkaline unwinding assay (daua) is also widely used to detect dna damage in aquatic 19 fig. 1 sampling site located along goa, west coast of india. organisms (sarkar et al., 2008, 2013; oliveira et al., 2010). several antioxidant enzymes are induced to combat the excessive ros produced as a result of pah metabolism. overproduction of ros can cause oxidative damage in a cell leading to damage to proteins, molecules, and dna. studies on antioxidant enzymatic defenses as biomarkers of oxidative stress are well documented in marine organisms (niyogi et al., 2001a, b; pan et al., 2006). among the oxidative stress biomarkers superoxide dismutase (sod), catalase (cat), glutathione stransferase (gst), and lipid peroxidation (lpo) have been widely used as an environmental biomarker in gastropods (abdel-halim et al., 2013; zheng et al., 2013; wang et al., 2014). gst helps in detoxification of the reactive products produced as a result of lipid peroxidation (olsvik et al., 2010). correlation between gst activity and pah in the tissues has been reported in several studies on mussels mytilus edulis (gowland et al., 2002). pan et al. (2006) have studied lipid peroxidation in scallop chlamys ferreri exposed to pah, benzo(a)pyrene (bap). catalase activity was measured in clam (frouin et al., 2007) and fishes (nahrganga et al., 2009) exposed to pah. significant induction of lipid peroxidation was detected in mussels collected near oil spillage site (porte et al., 1991). the author also showed a strong correlation between lipid peroxidation and total body pah in mussels. acetylcholinesterase (ache) activity is another very useful biomarker of neurotoxic contaminants in marine organisms (gaitonde et al., 2006; sarkar et al., 2006). ache has been considered as specific biomarkers for organophosphate and carbamate pesticides. recent studies also report inhibition of ache in gastropods exposed to heavy metals and biocides (gaitonde et al., 2006; ma et al., 2014). the combination of biomarkers yields a complicated and vast data which is hard to interpret. integrated biomarker response (ibr) integrates results from individual biomarkers into a single index, called ibr index which provides a comparison between stations and also between biomarkers. it is widely used in aquatic organisms exposed to contaminants(barda et al., 2014; turja et al., 2014). ibr can also be summarized into a star plot where radius values are ibrs estimated at each station. star plots provide corresponding information regarding mechanisms of biological effects of contaminants (marigómez et al., 2013). beliaeff and burgeot (2002) were the first to construct star plot using ibr values in flounder platichthys flesus using erod, gst, cat, ache enzymatic activities and dna damage biomarkers along with pah and pcb concentrations in tissues. pah contamination in caged mussel mytilus trossulus and mytilus galloprovincialis was also studied using ibr index (tsangaris et al., 2011; dabrowska et al., 2013). molluscs have been widely used in environmental monitoring due to their economical and ecological importance. gastropods have received great attention in recent years thanks to the discovery of imposex (smith, 1981). environment contaminants such as organotin compounds are known to cause imposex in snails that lead to a decline in pollution because of sterility and reproduction abnormality (garaventa et al., 20 2008). nerita is among the oldest molluscan names, dating to linnaeus and is a potential biological monitor for environment monitoring (kumar and devi, 1997; kumar, 1990). in this study, a battery of biomarkers for genotoxic damage (comet assay and alkaline unwinding assay), oxidative stress (sod, cat, gst, lpo) and neurotoxicity (ache) were considered. ibr integrates the biological response of multiple biomarkers and provides a single value; hence, in this study, ibr was applied to study the spatial variation in biomarkers in n. chamaeleon. pah content in the tissues of snail was also determined to relate to the variability of the biomarker response. materials and methods chemicals 1-chloro-2, 4-dinitrobenzene (cdnb), acetylcholine bromide, bromothymol blue, calf thymus dna, epinephrine, ethidium bromide, ethylene glycol-bis (2-aminoethylether)-n,n,n',n'tetraacetic acid (egta), guaiacol glycerol ether, hydrogen peroxide (h2o2), l-glutathione reduced, trypan blue and sephadex g-50 were purchased from sigma-aldrichpvt. ltd, india. frosted slides and cover slips were supplied by himedia, goa, india, dimethyl sulfoxide was obtained from qualigens, goa. tris buffer and triton x-100 were obtained from merck, goa. sampling site and gastropods nerita chamaeleon (around 30 snails from each site) were collected during post-monsoon (2011) period from the intertidal rocks along the seven sites (anjuna, arambol, sinquerim, dona paula, velsao, betul and palolem) in goa, west coast of india (fig. 1). arambol is situated at the northern tip of goa near tiracol estuary and is chosen as reference site because of its comparatively uncontaminated environment (sarkar et al., 2014). anjuna and sinquerim are most popularly known for their touristic activities around the world and hold a large number of restaurants, resorts, and shacks in the close proximity of the beach. discharge of contaminated water from the shacks and restaurant might contribute largely towards the pollution at these sites. it should be noted that mv river princess has been grounded on sinquerimcandolim beach since 2000. the grounding of the ship for such a long time release of huge amount of petroleum hydrocarbon from the ship might also contribute to coastal pollution (ingole et al., 2006). dona paula is situated between mandovi and zuari estuaries, just opposite to mormugao harbor. excessive shipping activities such as tourist boats, cargo ships, fishing trawlers, casinos, and barges carrying iron ores from the mines and water scooters are major contributors to the rapid increasing water pollution at this site. velsao beach lies in the vicinity of one of the leading agrochemical industries (zuari agrochemicals) of india in verna industrial state region. during the sampling at velsao, the prevalence of pungent smell from the region of discharge outlet clearly indicated the prevailing state of coastal pollution. betul and palolem are situated in the southern part of goa. the collected snails were identified using the certified reference sample from zoological survey of india; kolkata, india (subba rao et al., 1992). snails of similar size (around 18 30 mm) irrespective of their sex were used in this study. snails were transported in a plastic container to the lab within 3 h of collection. the collected snails were acclimatized in 4 liters plastic aquaria for 48 h in aerated seawater at room temperature. the shells of the gastropods were gently broken and whole body tissue was carefully excised out. soft tissues from three to four snails were pooled together (1 gram) and used for biochemical, alkaline unwinding assay and comet assay. all the measurements were carried out in triplicate. measurement of physicochemical parameters physicochemical parameters such as ph of the water from the sampling site were measured using ph analyzer elico model li-612, whereas turbidity and conductivity were measured using turbidimeter (systronics type 132) and conductivity meter (systronics digital direct model: 304). nutrients (nitrate, nitrite, and phosphate) were measured spectrophotometrically using shimadzu uv 1800 spectrophotometer whereas dissolved oxygen (do) and biochemical oxygen demand (bod) were determined following the standard methods of grasshoff et al. (1983). measurement of dna damage dna damage in snails has been evaluated by comet assay and alkaline unwinding assay. comet assay was performed in n. chameleon using the methods as described in our previous studies (sarkar et al., 2015). the dna integrity was measured in n. chameleon using partial alkaline unwinding assay following the methods of sarkar et al. (2013). biochemical assays for extraction of sod, one gram of tissue was homogenized (using ultra-turrax t 25 basic 1ka werke homogenizer) in 1 ml of 0.1 m phosphate buffer (ph 7.4). following that 0.2 ml of ice cold chloroform, 0.15 ml of ice-cold ethanol, and 1 ml of distilled water was added and the whole solution was shaken thoroughly. the mixture was then centrifuged at 3000 rpm for 10 min at 4 °c (using eltek refrigerated centrifuge rc 4100d). sod activity was determined by the rate of auto-oxidation of epinephrine to adrenochrome (misra and fridovich, 1972). the reaction volume (1 ml) contained 10 mm epinephrine, 50 mm sodium carbonate buffer (ph 10.2) and 10 mm edta. sod activity is reported in per milligram of protein (u mg 1); where 1 u of sod is defined as the amount of sample causing 50 % of inhibition of epinephrine auto-oxidation. for catalase, one gram of tissue was homogenized with 4 ml of phosphate buffer (0.1m, ph7.4) using high-speed ultra turrax homogenizer for 1 min and centrifuged at 18,000 rpm, 1 h, 4 ºc. catalase activity was measured following the methods of sinha et al. (1972). 1 ml of sodium phosphate buffer (0.01 m, ph 7.0), 0.5 ml of 0.2 m hydrogen peroxide (h2o2), and 0.4 ml distilled water 21 were mixed to make the reaction mixture. the reaction was stopped by pouring 2 ml of dichromate-acetic acid reagent (containing potassium dichromate 1 part and glacial acetic acid 3 parts). it was then heated for 10 min and allowed to cool. after the mixture cools down, the absorbance was read at 583 nm against blank on a spectrophotometer. the activity of cat was expressed as mm of h2o2 consumed/min/mg protein. glutathione s-transferase (gst) activity was measured according to the method of habig et al. (1974). it is based on conjugation of 1-chloro-2,4dinitrobenzene (cdnb) solution (30 mm) and reduced glutathione (gsh) solution (30mm) in reaction buffer (0.1 m k2hpo4, edta-na2, ph 6.5). the change in absorbance was measured at 340 nm for every 30 seconds for 5 min using a uv-vis spectrophotometer. the activity of gst was determined using extinction coefficient of 9.6 mm-1 cm-1 for cdnb. gst activity was expressed as nm/min/mg of protein. lipid peroxidation was measured by the method adapted from ohkawa et al. (1979). briefly, 1 g of soft tissue was homogenized with 9 ml of 0.25 m sucrose using ultra turrax homogenizer for 1 min.0.2 ml of 8 % sds, 1.5 ml of 20 % acetic acid and 1.5 ml of 0.8 % tba was added to 0.2 ml of the tissue homogenate. it was then made up to 4 ml using distilled water and heated at 95 ˚c for 60 min. it was then cooled and centrifuged at 3,000 rpm for 10 min. following that, the absorbance was read at 532 nm. lpo value was measured as malondialdehyde (mda) equivalent and expressed as nm of mda min-1 mg-1. extraction of ache from tissue was performed in phosphate buffer (100 mm; ph 7.4) mixed with sucrose solution (250 mm) (1 ml of both per gram of tissue) spiked with 100μl tritonx-100 in order to break the tissue. the homogenate was centrifuged at 14,000 rpm at 4° c for 45 min. ache activity in snails was measured using the ∆-ph-metric method as described by sarkar et al. (1992) and gaitonde et al. (2006). briefly, 0.1 ml of sample enzyme was incubated with 0.2 ml of substrate (acetylcholine bromide) in phosphate buffer (0.01m, ph-8.0 ±0.10) with bromothymol blue as an indicator. the changes in ph (∆-ph) was measured at an interval of 10 min over a period of 1 h of incubation corresponding to the amount of acetic acid liberated due to interaction with the acetylcholinesterase enzyme. the unit of activity of ache was expressed as micromoles of acetic acid liberated per minute per mg of protein. proteins were determined according to the method of lowry et al. (1951), using bovine serum albumin as standard. estimation of polycyclic aromatic hydrocarbons (pah) pah were extracted by homogenization of tissues of snails with bi-distilled hexane using ultra turrax homogenizer. the moisture content in the solvent extracts was removed by anhydrous sodium sulphate and the solvent extracts were concentrated to 1 ml using kuderna danish evaporator followed by purification of aliquots through alumina (10 % deactivated) column using bi-distilled hexane (grasshoff et al., 1983). the final 10 ml of aliquots thus obtained were measured by a spectrofluorometer (shimadzu rf-5301 pc) with excitation at 310 nm and emission at 360 nm (burns, 1993; sarkar et al., 2008, 2014). kuwait oil was used as the standard. total pah in tissues was represented as µg/g wet weight. statistical analysis the data were expressed as mean ± standard deviation. results from biochemical assays and genotoxic damage were analyzed by analysis of variance (anova) followed by tukey hsd posttest. kolmogorov-smirnov test for normality of distribution was used prior to anova. spearman correlation matrix was also calculated to study the relationships between the different biomarkers measured. biomarker values were compared with the reference site, arambol. three levels were significance is reported: (a) p < 0.05, (b) p < 0.01, and (c) p < 0.001. all statistical comparisons were performed using originpro 8.5.0. principal component analysis (pca) was conducted to determine physicochemical water parameters association with biochemical using xlstat. integrative biomarker response (ibr) integrated biomarker response (ibr) was calculated as described by beliaeff and burgeot (2002) with modification by guerlet et al. (2010) and devin et al. (2014). briefly, the biomarker response data for each site was standardized using the formulae yi = (xi-m)/s where yi is the standardized biomarker response, xi is response value of each biomarker, m, and s are mean value and standard deviation for all sites respectively. the mean of standardized biomarker response (zi) was then calculated using the formulae as zi = yi or zi = –yi for biomarker responding to contamination by induction or inhibition, respectively. the minimum value for each biomarker at all station was also calculated from the standardized biomarker response. the scores for the biomarker was computed as si = zi+|mini|. individual areas ai connecting the ith and the (i + 1)th radius coordinates of the star plot were obtained according to the formulae ai = si * si+1 *sin (2π/k)/2, where si and si+1 represent the individual biomarker scores (calculated from standardized data) and their successive star plot radius coordinates and k represent the number of radii corresponding to the biomarkers used in the survey. biomarkers were ranged clockwise from sub-cellular level as follows: percentage tail dna (tdna), dna integrity value (dna-f), sod, cat, gst, lpo and ache (serafim et al., 2012).and the ibr value is calculated as follow: ibr= , where ai is the triangular area represented by two consecutive biomarker scores on the star plot and n is the number of biomarkers used in the ibr calculation. results water quality parameters physico-chemical water parameters varied significantly across all the sampling sites along the coast of goa (table 1). ph of the seawater was in the 22 table 1 physico-chemical properties of water sampled at different sites along the coast of goa, india sites ph temp. (˚c) turbidity (ntu) conductivity ms/cm nitrite µm/l nitrate µm/l phosphate µm/l do (mg/l) b.o.d (mg/l) arambol 7.97±0.01 31.75±0.35 5.60±0.14 40.50±0.71 0.47±0.12 1.66±0.06 0.54±0.14 4.29±0.00 1.19±0.08 anjuna 8.37±0.02*** 33.00±0.00*** 2.35±0.49*** 41.50±0.71 0.34±0.05 1.69±0.06 0.38±0.08 5.93±0.08*** 1.58±0.08** sinquerim 8.09±0.04* 30.00±0.35*** 5.15±0.49 36.50±0.71* 0.91±0.04** 9.14±0.20*** 0.73±0.15 5.87±0.32*** 1.69±0.08*** dona paula 7.75±0.01*** 30.00±0.35*** 10.05±0.35*** 40.00±1.41 1.11±0.01*** 11.08±0.02*** 0.80±0.02 4.40±0.16 0.56±0.02*** velsao 8.06±0.01 33.00±0.25*** 2.25±0.35*** 64.50±0.71*** 9.64±0.01*** 32.76±0.80*** 3.48±0.05*** 4.12±0.08 2.20±0.04*** betul 8.21±0.02** 32.00±0.25 1.24±0.02*** 39.50±0.71 0.23±0.03* 1.44±0.10 0.28±0.02 5.14±0.08** 4.01±0.02*** palolem 7.90±0.04 31.00±0.00** 4.15±0.07* 42.50±0.71 0.43±0.04 0.07±0.02 0.42±0.00 4.52±0.00 3.95±0.05*** values are represented as means± standard deviation. maximum values were shown in bold. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site arambol (anova, tukey hsd post-test). range from 7.75 to 8.37. maximum values for seawater temperature was observed at anjuna (33 ˚c), and minimum at palolem (31 ˚c). the highest turbidity was found at dona paula (10.05 ± 0.35 ntu) and the least at betul (1.24 ± 0.02 ntu). seawater from velsao showed significant variation in nutrients as compared to all other stations (p < 0.001). velsao also showed the maximum values for conductivity (64.50 ± 0.71 ms/cm). the values for nitrite, nitrate, and total phosphate at velsao were found to be 9.64 ± 0.01 µm/l, 32.76 ± 0.80 µm/l and 3.48 ± 0.05 µm/l respectively. dissolved oxygen (do) also varied significantly between the sites, with maximum values observed at anjuna (5.93 ± 0.08 mg/l). the highest biological oxygen demand (bod) was measured at betul (4.01 ± 0.02 mg/l) and the least at dona paula (0.56 ± 0.02 mg/l). biomarker responses dna damage the impairment of dna in whole body tissue of marine gastropods is clearly indicated by the decrease in the integrity of dna in n. chamaeleon exposed to various types of genotoxic contaminants prevalent at different sites along the coast of goa (fig. 2). the highest value of tdna was observed at sinquerim (55.86 ± 4.09). a significant difference in tdna was observed between sinquerim and arambol (p < 0.01) and sinquerim and anjuna (p < 0.01). the dna integrity at the reference site arambol was found to be relatively quite high (0.71 ± 0.03) as compared to the other sampling sites. except for betul and palolem, all the sampling sites showed a significant decrease in dna integrity as compared to arambol. the mean value of dna integrity in snails was found to be 0.48. fig. 2 (a) mean percentage dna in the tail (tdna) and (b) dna integrity (f value) in marine gastropod nerita chamaeleon collected from different sites along the coast of goa, india. values are means ± standard deviation (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site, arambol (anova, tukey hsd post-test). 23 fig. 3 (a) superoxide dismutase (sod), (b) catalase (cat), (c) glutathione s-transferase (gst), (d) acetylcholinesterase (ache) activities, and (e) lipid peroxidation (lpo) level in marine gastropod nerita chamaeleon collected from different sites along the coast of goa, india. values are means± standard deviation (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site, arambol (anova, tukey hsd post-test). biochemical assays all biomarkers varied across sites. sod activity in whole body tissue of snails was in the range from 1.57 to 17.27 u/mg of protein (fig. 3). significant differences in sod activity was observed for sinquerim, dona paula, velsao and palolem as compared to the reference site arambol. cat activity also showed significant variations among the sampling sites. the highest cat activity was observed at dona paula (0.39 ± 0.05 mm/min/mg) and the least at palolem (0.04 ± 0.01 mm/min/mg). all the other site except dona paula, showed significant decrease in cat activity as compared to arambol. cat activity between sinquerim and palolem were not significant, however, a significant change was observed between palolem and dona paula (p < 0.001). gst activity showed maximum value recorded at dona paula (191.54 ± 18.7 nm/min/mg) and minimum at sinquerim (28.11 ± 0.06 nm/min/mg). gst activity at dona paula was significantly (p < 0.001) higher than arambol (74.21 ± 1.47 nm/min/mg).the lowest lpo value (0.09 ± 24 0.01 mm mda/min/mg) was observed in snails collected at palolem whereas dona paula exhibited the highest value (1.18 ± 0.16 mm mda/min/mg). ache activity were significantly lower (p < 0.01) in snails from the sinquerim (10.44 ± 0.07 u/min/mg) in comparison to arambol (39.68 ± 0.18 u/min/mg). pca analysis showed that nutrients (phosphate, nitrate and nitrate) group together with conductivity. a strong positive correlation between lpo and turbidity (r = 0.84), and ache and turbidity (r = 0.801) was observed. dissolve oxygen showed negative correlation with dna integrity value, whereas ph showed negative correlation with all the biomarkers. measurement of pah pah concentrations in tissue of snail showed wide variation among the sites. sinquerim showed the highest value for total pah (12.14 ± 0.27 µg/g wet weight) in snail tissues whereas it was found to be least at arambol (5.29 ± 0.67 µg/g w.w.). the entire sampling site except anjuna showed significant variations in the pah content in snails (fig. 4). table 2 shows the pah content in molluscs from a different part of the world. relationship between biomarker response and pah content a moderate positive correlation was observed between tdna and pah (r = 0.626, p = 0.017) contents in snails (table 3). sod activity also showed positive correlation with pah (r = 0.604, p = 0.022) while ache activity was found to be negatively correlated with pah (r = 0.542, p = 0.045). strong positive correlation between gst activity and ache activity (r = 0.534, p = 0.049), gst and cat activity (r = 0.534, p = 0.047) were also observed. similar trend was observed between cat and sod activity (r = 0.613, p = 0.20,) and lpo value and sod activity (r = 0.942, p = 0.022). in contrast, dna-f was shown to be negatively correlated with sod activity (r = -0.617, p = 0.019) and lpo value (r = 0.679, p = 0.008). integrative biomarker response (ibr) ibr values for dona paula showed the maximum value for tdna (9.31 ± 0.91) as measured by comet assay and the minimum value was observed at palolem (0.25 ± 0.063) (fig. 5). the ibr values were significant for dna-f when sites arambol and dona paula were compared (p < 0.001). a very similar pattern was observed in star plots for all the oxidative stress biomarkers. the maximum ibr values for sod were measured at dona paula (7.17 ± 0.63) and the minimum values occurred at betul (0.91 ± 0.87). the ibr values for lpo at sinquerim and dona paula were significantly different from those of arambol (p < 0.001). however, no significant differences existed in any biomarker when ibr values for anjuna, betul and palolem were compared with arambol. sinquerim and dona paula showed significant differences in all the biomarkers studied with compared to arambol. the mean ibr values calculated from seven biomarkers was found to be highest at dona paula (8.07 ± 0.91) followed by sinquerim (6.95 ± 0.91), velsao (4.48 ± 0.68), anjuna (3.28 ± 1.05), palolem (2.53 ± 0.73), arambol (1.81 ± 0.21) and betul (0.88 ± 0.77). ibr values for genotoxic (tdna and dna-f), oxidative stress (sum of sod, cat, gst and sod) biomarker and total pah concentration in tissues were represented as star plot in figure 6. comparison of ibr for genotoxic, oxidative biomarkers and pah concentration shows that along with pah there might be other genotoxicants which are responsible for the impairment of dna in snails from different sites. fig. 4 polycyclic aromatic hydrocarbon (pah) content in marine gastropod nerita chamaeleon collected from different sites along the coast of goa, india. values are means± standard deviation (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site, arambol (anova, tukey hsd post-test). 25 table 2 polycyclic aromatic hydrocarbon (pah) concentrations in molluscs reported from different parts of the world organisms group location tissues no. of pah wet weight (µg/g) references nerita chamaeleon gastropod goa coast, india whole body tissues total pah 5.29-12.14 this study cronia contracta gastropod goa coast, india muscle tissue total pah 22.32–53.78 sarkar et al., 2008 turbo cornutus gastropod japan coast, japan soft tissue 8 0.044 koyama et al., 2004 sunetta scripta clam cochin harbor, india soft tissue total pah 13.35-21.49 menon & menon, 1999 donax trunculus clam comunidad valenciana coast soft tissue 8 0.43-10.09 bouzas et al., 2011 donax trunculus clam abu qir bay, egypt soft tissue 17 1137.07 el-deeb et al., 2007 saccostrea cucullata oyster hooghly estuary, india soft tissue total pah 0.8-12.5 niyogi et al., 2001a mytilus galloprovincialis mussel marmara sea, izmit bay soft tissue 16 5.67-14.81 telli-karakoç et al., 2002 mitylus galloprovincialis mussel adriatic sea, italy soft tissue 13 0.034 perugini et al., 2007 mytilus galloprovincialis mussel gulf of rijeka, croatia. soft tissue 10 0.049-0.134 bihari et al., 2007 mytilus galloprovincialis mussel gulf of trieste soft tissue 644-685 notar et al., 2001 mytilus galloprovincialis mussel comunidad valenciana coast soft tissue 8 0.21-8.95 bouzas et al., 2011 mytilus edulis mussel baltic sea, poland soft tissue 14 8.64–29.7 potrykus et al., 2003 palaeomonetes sp. shrimp norco, usa soft tissue total pah 7.18-10.86 oberdorster et al., 1999 penaeus japonicus shrimp gulf of suez muscle tissue 16 2.01 ali et al., 2014 sepia species cuttlefish gulf of suez muscle tissue 16 4.09 ali et al., 2014 portunus pelagicus crab gulf of suez muscle tissue 16 8.10 ali et al., 2014 nephrops norvegicus lobster adriatic sea, italy soft tissue 13 0.015 perugini et al., 2007 discussion in this study, seven biomarkers were measured in marine snail n. chamaeleon collected from goa coast and the obtained results show differences in individual biomarker responses. significant variations in genotoxic as well as biochemical biomarkers were observed and this can be associated with pollutant exposure. biomarker responses in snails showed clear spatial variations. the range of pah in snails in this study lies within the same range as reported in clams (menon and menon, 1999) and oysters (niyogi et al., 2001a) in indian coastal waters. however, higher values of pah were reported marine gastropods croniacontracta by sarkar et al. (2008) (table 2). total pah concentrations in snails from goa region range from the lowest value at arambol (5.29 ± 0.67 µg/g wet weight) to highest value at sinquerim (12.14 ± 0.27 µg/g wet weight). sinquerim also showed the highest values for tdna (55.86 ± 4.09) and lower dna-f (0.46 ± 0.04). accumulation of pah has been linked to dna damage through production of ros (jarvis et al., 2013). the ros produced as a result of pah exposure can cause single or double strand breakage in the dna (kaloyianni et al., 2009). sarkar et al. (2008) studied the seasonal variation of pah in cronia contracta collected from six sites along the goa coast and reported a positive correlation between the pah and dna damage. such a huge impairment in dna integrity in the gastropod at sinquerim can be attributed to genotoxic pollutants like polycyclic aromatic hydrocarbons being discharged extensively from various types shipping activities such as cargo ships, research vessel, tourist vessel, motor boats, fishing trawler, water scooters, barges sailing through this site as well as accidental oil 26 table 3 spearman’s correlation matrix on all the biomarkers and polycyclic aromatic hydrocarbon (pah) content in marine gastropod, nerita chamaeleon t-dna dna-f sod cat gst lpo ache dna-f -0.029 sod 0.332 -0.617* cat -0.143 -0.007 0.613* gst -0.398 -0.244 0.257 0.538* lpo 0.279 -0.679* 0.942* 0.495 0.182 ache -0.442 -0.152 0.099 0.327 0.534* 0.200 pah 0.626* -0.345 0.604* 0.095 -0.002 0.516 -0.542* (*) p < 0.05 significantly different(anova, tukey hsd post-test) spills, etc. (desai, et al, 2010). ingole et al. (2006) have reported a high level of total petroleum hydrocarbon at sinquerim-candolim beach due to the grounding of mv river princess. in velsao, the pah concentrations increase by two-fold as compared to arambol, such an increase in pah in snails at velsao revealed the severity of pollution in velsao. during sampling at velsao pungent smell from the discharge outlet from the industry has been observed, that indicates the severity of pollution at this site. there were no significant differences between pah concentration at velsao, sinquerim and dona paula. oxidative stress biomarker in n. chamaeleon showed spatial variability, with significant induction in sod activity at dona paula and sinquerim.sod activity at both of these sites is strongly correlated with the high values of pah measured in snails. numerous studies have also documented significant relationships between antioxidant activity and heavy metal/pah body burdens (giguere et al., 2003; manduzio et al., 2003). dona paula has been previously reported for heavy metal and pah contamination (sarkar et al., 2008). high values of tbt and other organotin compounds were also reported in dona paula (meena et al., 2009). catalase is well known to play an important role in scavenging h202 (di giulio and meyer, 2008), which is produced as a result of scavenging of superoxide radicals by sod. positive relationships between cat activity and pah levels were observed in oyster (niyogi et al., 2001a), barnacle (niyogi et al., 2001b), and in the gills of the mussel (cheung et al., 2001). in this study, a significant positive correlation was observed between sod and cat activity. the increase in sod and cat activity may be due to increasing in cellular ros produced due to exposure to pah (au et al., 1999). niyogi et al., (2001a) has also reported a positive correlation of cat and sod activity with pah tissue content in fig. 5 integrated biomarker response (ibr) represented by star plots for each sampling sites. 27 fig. 6 comparison of ibr index for genotoxic (dna damage as measured by comet assay and alkaline unwinding assay) and oxidative stress [superoxide dismutase (sod), catalase (cat), glutathione s-transferase (gst) and lipid peroxidation (lpo)] biomarkers with average polycyclic aromatic hydrocarbon (pah) value in snails from different sites of goa, india. s. cucullata. such a relationship has also been reported for different bivalve species exposed to hydrocarbons (richardson et al., 2008) and suggests that hydrocarbons induce oxidative stress by producing ros such as o2 -. the activity of cat is dependent on the level of h2o2 in the cell. another enzyme, gpx (glutathione peroxidase) eliminates h2o2 while carrying out gsh to gssg conversion. increase in gpx activity might lead to reduction of h2o2 that in turn can affect the activity of catalase. in our study high level of cat activity was observed at the reference site. in spite of high amount of pah in the other sites, a decreasing trend in cat activity was observed. wu et al. (2011) has also found that cat activity in eisenia fetida was unaltered suggesting that pah exposure does not induce increased cat activity. jifa et al. (2006) also reported unaltered changes in lateolabrax japonicus cat activity after (b[a]p) exposure. gst is a phase ii metabolizing enzyme and catalyzes the conjugation of reduced glutathione (gsh) with pah derivatives. the activity of gst depends on the availability of gsh. contrarily to other antioxidant enzymes, gst showed a negative correlation with pah in this study. decrease in gst activity after long exposure to pah can be due to the reduction of gsh levels. gsh can also be converted to its oxidized state (gssg) by glutathione peroxidase (gpx). decrease in gsh has been reported in molluscs exposed to pahs (grintzalis et al., 2012). studies with gst in aquatic organisms exposed to environmental or anthropogenic contaminants has shown increase (zheng et al., 2013; cabecinhas et al., 2014), unaltered (bianco et al., 2013; rivadeneira et al., 2013) or decrease (ma et al., 2014; ali et al., 2015) enzyme activities. free radicals produced as a result of metabolic activities can react with polyunsaturated fatty acids in the cell membrane, resulting in an increase of lipid peroxidation (livingstone et al., 2001). lpo is well known oxidative stress biomarkers in bivalves exposed to pah (kaloyianni et al., 2009). lpo results in production of mda which can react with dna and form dna adduct. increased formation of dna adduct in gill cells of mytilus galloprovincialis after exposure to pah, benzo(a)pyrene have been reported by venier and canova (1996). enhancement of lipid peroxidation and inhibition of ache were detected in gills of mussels m.galloprovincialis exposed to phenanthrene (grintzalis et al., 2012). elevated levels of cat, lpo and sod were observed in mussels (mytilus galloprovincialis) collected from sites affected by the oil spill (sureda et al., 2011). in this study significant decrease in ache activity was observed in sinquerim. this may be due to the prevalence of hydrocarbon pollution as observed by high amount of pah reported in the gastropods. presence of high amount of pah in snails from sinquerim may be due to extensive shipping activities as well as accidental oil spills (desai et al., 2010). enhancement of lipid peroxidation by-products and inhibition of ache in mussels exposed to phenanthrene and/or anthracene were reported by grintzalis et al. (2012). pca indicated that biochemical biomarkers tend to be increased under condition of higher turbidity and lower ph. pca analysis provided an important association between the physicochemical parameters and biomarker response. the fact that the biomarkers assessed were moderately associated with the environmental variables suggest that other contaminants, besides those measured here, were also contributing to the lower health status of the snails. ibr with star plots showed higher biomarker response in snails from dona paula, sinquerim, and velsao, and moderate at anjuna and palolem. thus higher values of pah and oxidative stress biomarkers at velsao, sinquerim and dona paula shows critical unbalance ros formation and the severity of pollution at these sites. several authors have reported higher values of ibr in contaminated sites as compared to the reference site (tankoua et al., 2013; turja et al., 2014). in this study elevated the level of pah as well as oxidative stress biomarkers response was observed in snails from sinquerim. vega-lópez et al. (2013) has investigated t h e r el a t i o n b et w e e n o xi d a t i v e s t r e s s a n d 28 fig. 7 principal component analysis (pca) on the data set with physicochemical water parameters [ph, temperature (temp.), turbidity (turb.), conductivity (cond.), nitrite (no2 -), nitrate (no3 -), phosphate (po4 3-), dissolve oxygen (do), biological oxygen demand (bod)] and biomarkers response [dna integrity (dna-f), percentage tail dna (tdna), superoxide dismutase (sod), catalase (cat), glutathione s-transferase (gst), acetylcholinesterase (ache) activities, and lipid peroxidation (lpo)] in nerita chamaeleon. antioxidant defenses in phytoplankton with heavy metal and pah. the author stated that oxidative damage was related with pah (benzo[b]fluoranthene) using ibr. pah are suspected of having an oxidative damage leading to damage in the genetic material and some of the pah such as benzo(a)pyrene, indeno[1,2,3c,d]pyrene, benzo[g,h, i]perylene etc. are well known for such actions (perez-cadahia et al., 2004; woo et al., 2006). there is strong evidence that some of them are carcinogenic (diguilio et al., 1995) with the capacity to cause various types of oxidative stress and dna damage. these results suggest that integration of genotoxic and biochemical biomarker can serve as a useful tool in environmental monitoring programs. conclusion the present study showed that tdna, dna-f, sod, cat, gst, lpo and ache levels in snails varied along different sites. integrated biomarker response index based on a battery of biomarkers proved a useful tool for visualization of biological responses in snails, facilitating comparisons between different sites. increased value of ibr index at sinquerim and dona paula can be attributed to exposure of snails to contaminants prevalent at these sites. our study demonstrated, the sensitivity of marine snail n. chamaeleon as a good candidate species for pah contamination. this study demonstrates the usefulness of multibiomarker approach in the coastal bio-monitoring program. acknowledgements authors would like to pay their deep sense of gratitude to the director, csir-nio for his wholehearted cooperation and keen interest to carry out the work in this institute. they are indebted to department of biotechnology, new delhi for providing fellowship to mr. jacky bhagat. they also extend their thanks to the council of scientific and industrial research (csir) for providing the financial support and infrastructural facilities. references abdel-halim k, el-saad aa, talha m, hussein a, bakry n. oxidative stress on land snail helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals. chemosphere 93: 1131-1138, 2013. ali d, ali h, alarifi s, kumar s, serajuddin m, mashih ap, et al. impairment of dna in a freshwater gastropod (lymnea luteola l.) after exposure to titanium dioxide nanoparticles. arch. environ. contam. toxicol. 68: 543-552, 2015. au d, wu r, zhou b, lam p. relationship between ultrastructural changes and erod activities in liver of fish exposed to benzo [a] pyrene. 29 environ. pollut. 104: 235-247, 1999. barda i, purina i, rimsa e, balode m. seasonal dynamics of biomarkers in infaunal clam macoma balthica from the gulf of riga (baltic sea). j. mar. syst. 129: 150-156, 2014. beliaeff b, burgeot t. integrated biomarker response: a useful tool for ecological risk assessment. environ. toxicol. chem. 21: 13161322, 2002. bianco k, yusseppone ms, otero s, luquet c, de molina mdcr, kristoff g. cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (chilina gibbosa) with low sensitivity carboxylesterases. aquat. toxicol. 144: 26-35, 2013. burns ka. evidence for the importance of including hydrocarbon oxidation products in environmental assessment studies. mar. pollut. bull. 26: 77-85, 1993. cabecinhas as, novais sc, santos sc, rodrigues ac, pestana jl, soares am, et al. sensitivity of the sea snail gibbula umbilicalis to mercury exposure-linking endpoints from different biological organization levels. chemosphere 119: 490-497, 2015. cheung c, zheng g, li a, richardson b, lam p. relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, perna viridis. aquat. toxicol. 52: 189-203, 2001. dabrowska h, kopko o, turja r, lehtonen kk, góra a, polak-juszczak l, et al. sediment contaminants and contaminant levels and biomarkers in caged mussels (mytilus trossulus) in the southern baltic sea. mar. environ. res. 84: 1-9, 2013. dailianis s, tsarpali v, melas k, karapanagioti hk, manariotis id. aqueous phenanthrene toxicity after high-frequency ultrasound degradation. aquat. toxicol. 147: 32-40, 2014. deasi s, verlecar x, ansari z, jagtap t, sarkar a, vashistha d, et al. evaluation of genotoxic responses of chaetoceros tenuissimus and skeletonema costatum to water accommodated fraction of petroleum hydrocarbons as biomarker of exposure. water res. 44: 22352244, 2010. devin s, burgeot t, giambérini l, minguez l, paindevin s. the integrated biomarker response revisited: optimization to avoid misuse. environ. sci. pollut. res. 21: 2448-2454, 2014. di giulio rt, hinton de. the toxicology of fishes: crc press, 2008. frouin h, pellerin j, fournier m, pelletier e, richard p, pichaud n, et al. physiological effects of polycyclic aromatic hydrocarbons on soft-shell clam mya arenaria. aquat. toxicol. 82: 120134, 2007. gaitonde d, sarkar a, kaisary s, silva c, dias c, rao d, et al. acetylcholinesterase activities in marine snail (cronia contracta) as a biomarker of neurotoxic contaminants along the goa coast, west coast of india. ecotoxicology 15: 353-358, 2006. giguère a, couillard y, campbell pg, perceval o, hare l, pinel-alloul b, et al. steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient. aquat. toxicol. 64: 185200, 2003. gowland b, mcintosh a, davies i, moffat c, webster l. implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione stransferase activity in mussels. mar. environ. res. 54: 231-235, 2002. grasshoff k, ehrhardt m, kremling m. 1983. methods of sea water analyses analysis, verlag chemie: weinheim, germany. grintzalis k, georgiou cd, dailianis s. total thiol redox status as a potent biomarker of pahmediated effects on mussels. mar. environ. res. 81: 26-34. 2012. guerlet e, vasseur p, giambérini l. spatial and temporal variations of biological responses to environmental pollution in the freshwater zebra mussel. ecotoxicol. environ. saf. 73: 11701181, 2010. habig wh, pabst mj, jakoby wb. glutathione stransferases the first enzymatic step in mercapturic acid formation. j. biol. chem. 249: 7130-7139, 1974. humans iwgoteocrt. some non-heterocyclic polycyclic aromatic hydrocarbons and some related exposures. iarc monographs on the evaluation of carcinogenic risks to humans/world health organization, international agency for research on cancer 92: 1, 2010. ingole b, sivadas s, goltekar r, clemente s, nanajkar m, sawant r, et al. ecotoxicological effect of grounded mv river princess on the intertidal benthic organisms off goa. environ. int. 32: 284-291, 2006. ismert m, oster t, bagrel d. effects of atmospheric exposure to naphthalene on xenobioticmetabolising enzymes in the snail helix aspersa. chemosphere 46: 273-280, 2002. jarvis iw, bergvall c, bottai m, westerholm r, stenius u, dreij k. persistent activation of dna damage signaling in response to complex mixtures of pahs in air particulate matter. toxicol. appl. pharmacol. 266: 408-418, 2013. jifa w, yu z, xiuxian s, you w. response of integrated biomarkers of fish (lateolabrax japonicus) exposed to benzo[a]pyrene and sodium dodecylbenzene sulfonate. ecotox. environ. safe. 65:230-236, 2006. kaloyianni m, dailianis s, chrisikopoulou e, zannou a, koutsogiannaki s, alamdari d, et al. oxidative effects of inorganic and organic contaminants on haemolymph of mussels. comp. biochem. physiol. 149c: 631-639, 2009. kumar k, devi v. effect of heavy metals on toxicity and oxygen consumption of intertidal gastropods nerita albicilla and nerita chamaeleon. j. ecotoxicol. environ. monitoring 5: 1-5, 1995. kumar kp, devi vu. accumulation of copper and zinc by two intertidal gastropods and their potential as biological monitors. toxicol. 30 environ. chem. 67: 71-81, 1998. livingstone d. contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. mar. pollut. bull. 42: 656-666, 2001. lowry oh, rosebrough nj, farr al, randall rj. protein measurement with the folin phenol reagent. j. biol. chem. 193: 265-275, 1951. ma j, zhou c, li y, li x. biochemical responses to the toxicity of the biocide abamectin on the freshwater snail physa acuta. ecotoxicol. environ. saf. 101: 31-35, 2014. manduzio h, monsinjon t, rocher b, leboulenger f, galap c. characterization of an inducible isoform of the cu/zn superoxide dismutase in the blue mussel mytilus edulis. aquat. toxicol. 64: 73-83, 2003. marigómez i, garmendia l, soto m, orbea a, izagirre u, cajaraville mp. marine ecosystem health status assessment through integrative biomarker indices: a comparative study after the prestige oil spill “mussel watch”. ecotoxicology 22: 486-505, 2013. martins m, costa pm, ferreira am, costa mh. comparative dna damage and oxidative effects of carcinogenic and non-carcinogenic sediment-bound pahs in the gills of a bivalve. aquat. toxicol. 142: 85-95, 2013. mattsson å, lundstedt s, stenius u. exposure of hepg2 cells to low levels of pah‐containing extracts from contaminated soils results in unpredictable genotoxic stress responses. environ. mol. mutag. 50: 337-348, 2009. menon nn, menon n. uptake of polycyclic aromatic hydrocarbons from suspended oil borne sediments by the marine bivalve sunetta scripta. aquat. toxicol. 45: 63-69, 1999. misra hp, fridovich i. the role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. j. biol. chem. 247: 3170-3175, 1972. mashroofeh a, bakhtiari ar, pourkazemi m. distribution and composition pattern of polycyclic aromatic hydrocarbons in different tissues of sturgeons collected from iranian coastline of the caspian sea. chemosphere 120: 575-83, 2015. nahrgang j, camus l, gonzalez p, goksøyr a, christiansen js, hop h. pah biomarker responses in polar cod (boreogadus saida) exposed to benzo (a) pyrene. aquat. toxicol. 94: 309-319, 2009. niyogi s, biswas s, sarker s, datta a. antioxidant enzymes in brackishwater oyster, saccostrea cucullata as potential biomarkers of polyaromatic hydrocarbon pollution in hooghly estuary (india): seasonality and its consequences. sci. total environ. 281: 237246, 2001. niyogi s, biswas s, sarker s, datta ag. seasonal variation of antioxidant and biotransformation enzymes in barnacle, balanus balanoides, and their relation with polyaromatic hydrocarbons. mar. environ. res. 52: 13-26, 2001. ohkawa h, ohishi n, yagi k. assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. anal. biochem. 95: 351-358, 1979. oliveira m, maria v, ahmad i, pacheco m, santos m. seasonal liza aurata tissue-specific dna integrity in a multi-contaminated coastal lagoon (ria de aveiro, portugal). mar. pollut. bull. 60: 1755-1761, 2010. olsvik p, nordtug t, altin d, lie k, overrein i, hansen b. transcriptional effects on glutathione s-transferases in first feeding atlantic cod (gadus morhua) larvae exposed to crude oil. chemosphere 79: 905-913, 2010. pan l, ren j, liu j. responses of antioxidant systems and lpo level to benzo (a) pyrene and benzo (k) fluoranthene in the haemolymph of the scallop chlamys ferrari. environ. pollut. 141: 443-451, 2006. pérez-cadahía b, laffon b, pásaro e, méndez j. evaluation of pah bioaccumulation and dna damage in mussels (mytilus galloprovincialis) exposed to spilled prestige crude oil. comp. biochem. physiol. 138c: 453-460, 2004. porte c, sole m, albaiges j, livingstone d. responses of mixed-function oxygenase and antioxidase enzyme system of mytilus sp. to organic pollution. comp. biochem. physiol. 100c: 183-186, 1991. potrykus j, amaya a, pempkowiak j, porte visa c. content and pattern of organic pollutants (pahs, pcbs and ddt) in blue mussels mytilus trossulus from the southern baltic sea, 2003. richardson bj, mak e, de luca-abbott sb, martin m, mcclellan k, lam pk. antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (perna viridis): do mussels “integrate” biomarker responses? mar. pollut. bull. 57: 503-514, 2008. rivadeneira pr, agrelo m, otero s, kristoff g. different effects of subchronic exposure to low concentrations of the organophosphate insecticide chlorpyrifos in a freshwater gastropod. ecotoxicol. environ. saf. 90: 82-88, 2013. sarkar a, bhagat j, ingole bs, rao dp, markad vl. genotoxicity of cadmium chloride in the marine gastropod nerita chamaeleon using comet assay and alkaline unwinding assay. environ. toxicol. 30: 177-187, 2015. sarkar a, bhagat j, sarker s. evaluation of impairment of dna in marine gastropod, morula granulata as a biomarker of marine pollution. ecotoxicol. environ. saf. 106: 253-261, 2014. sarkar a, gaitonde dc, sarkar a, vashistha d, d’silva c, dalal s. evaluation of impairment of dna integrity in marine gastropods (cronia contracta) as a biomarker of genotoxic contaminants in coastal water around goa, west coast of india. ecotoxicol. environ. saf. 71: 473-482, 2008. sarkar a, ray d, shrivastava, amulya n, sarker s. molecular biomarkers: their significance and application in mar. pollut. monit. ecotoxicol. 15: 333-340, 2006. sarkar a. evaluation of the toxicity of organic matter in marine sediments. water sci. tech. 25: 255 257, 1992. serafim a, company r, lopes b, fonseca v, 31 frança s, vasconcelos r, et al. application of an integrated biomarker response index (ibr) to assess temporal variation of environmental quality in two portuguese aquatic systems. ecol. indicators 19: 215-225, 2012. sinha ak. colorimetric assay of catalase. anal. biochem. 47: 389-394, 1972. subba rao n, dey a, barua s. estuarine and marine molluscs. fauna of west bengal part 9 (state fauna series 3): calcutta, zoological survey of india, 1992. tankoua of, buffet p, amiard j, berthet b, mouneyrac c, amiard-triquet c. integrated assessment of estuarine sediment quality based on a multi-biomarker approach in the bivalve scrobicularia plana. ecotoxicol. environ. saf. 88: 117-125, 2013. tsangaris c, hatzianestis i, catsiki v-a, kormas ka, strogyloudi e, neofitou c, et al. active biomonitoring in greek coastal waters: application of the integrated biomarker response index in relation to contaminant levels in caged mussels. sci. total environ. 412: 359-365, 2011. turja r, höher n, snoeijs p, baršienė j, butrimavičienė l, kuznetsova t, et al. a multibiomarker approach to the assessment of pollution impacts in two baltic sea coastal areas in sweden using caged mussels (mytilus trossulus). sci. total environ. 473: 398-409, 2014. vega-lópez a, ayala-lópez g, posadas-espadas bp, olivares-rubio hf, dzul-caamal r. relations of oxidative stress in freshwater phytoplankton with heavy metals and polycyclic aromatic hydrocarbons. comp. biochem. physiol. 165a: 498-507, 2013. venier p, canova s. formation of dna adducts in the gill tissue of mytilus galloprovincialis treated with benzo [a] pyrene. aquat. toxicol. 34: 119133, 1996. wang x, liu z, wang w, yan z, zhang c, wang w, et al. assessment of toxic effects of triclosan on the terrestrial snail (achatina fulica). chemosphere 108: 225-230, 2014. woo s, kim s, yum s, yim uh, lee tk. comet assay for the detection of genotoxicity in blood cells of flounder (paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons. mar. pollut. bull. 52: 1768-1775, 2006. wu s, wu e, qiu l, zhong w, chen j. effects of phenanthrene on the mortality, growth, and anti-oxidant system of earthworms (eisenia fetida) under laboratory conditions. chemosphere 83: 429-434, 2011. zheng s, wang y, zhou q, chen c. responses of oxidative stress biomarkers and dna damage on a freshwater snail (bellamya aeruginosa) stressed by ethylbenzene. arch. environ. contam. toxicol. 65: 251-259, 2013. societ italiana di immunobiologia comparata isj 5: 30-40, 2008 issn 1824-307x report of meeting ixth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 27 29 february 2008, biological departments, university of insubria, varese, italy organizers: m de eguileor, a grimaldi, g tettamanti, r valvassori department of structural and functional biology, university of insubria, varese, italy session 1. chairman: m cammarata, university of palermo, italy the immune system of compound ascidians l ballarin department of biology, university of padua, padua, italy differently from vertebrates, having an adaptive immunity, ascidians are chordates with a germ-line based innate immunity in which phagocytosis and cytotoxicity are the main effector systems. in recent years, the comprehension of the immune system of compound ascidian has greatly improved. most of the research was carried out in species of the genus botryllus and botrylloides. in these organisms, immunocytes constitute a considerable fraction of circulating haemocytes and are represented by phagocytes and phenoloxidase (po)-containing, cytotoxic cells. phagocytes include hyaline amoebocytes (ha) and macrophage-like cells (mlc). the former are wandering cells which quickly recognise and engulf foreign particles or cells: after the ingestion, they withdraw their cytoplasmic projections and acquire a globular shape, turning to mlc. phagocytosis requires the recognition of molecular patterns on the surface of target particles by receptors on phagocytes and is greatly influenced by the nature of the particle. the recognition triggers signal transduction pathways which lead to the activation of the map-kinase cascade and of nf-kb. soluble lectins can increase the phagocytosis of foreign particles acting as opsonins. phagocytes play an important role during the colonial generation change (take-over), when the old adult zooids stop their filtering activity and are progressively resorbed. during this period, tissues of adult zooids undergo diffuse apoptosis and are rapidly infiltrated by blood phagocytes which rapidly and massively engulf senescent cells. cytotoxic morula cells are characterised by the presence of vacuoles containing inactive pro-po and its polyphenol substrata. they are the effectors of the rejection reaction between contacting, genetically incompatible colonies. these cells crowd inside the ampullae contacting the alien colony, cross the epithelium of the ampullar tips and enter the tunic where they degranulate and release the content of their vacuoles. the activated po acts on substrata and induce necrotic death through the induction of an oxidative stress. there is an interesting cross-talk between cytotoxic an phagocytic cells: i) phagocytosis can be modulated by soluble molecules (cytokines) released upon the recognition of foreign particles (yeast cells and bacterial spores) by the cytotoxic cells; ii) some ha are exposed to seawater, on the internal side of the siphons, where they act as guard cells. once recognised foreign materials, they activate morula cells in the tentacular lacunae which, in turn alert the whole immune system towards the potentially dangerous particles that can be phagocytosed or degraded. further insights on siphonal guard cells of ascidians f cima department of biology, university of padua, padua, italy in the oral siphon of the colonial ascidian botryllus schlosseri, hyaline amoebocytes directly exposed to the sea-water flow entering into the pharynx have been recently observed and described. these cells, named “siphonal guard cells” (sgc), are free of moving on the surface of the tunic that internally covers the siphons. our previous observations by means of histochemical, histoenzymatic and immunohistochemical techniques showed that they share many morphofunctional characteristics with the phagocytic blood cell line, from which probably they originate, and are able to recognise and phagocytise various foreign particles. after exposure of colonies to bacterial spores, the observations at both light and electronic microscope revealed that these cells are involved in a complex and unusual series of local and 30 systemic immune events. already after 5 min, the sgc showed bacteria inside their heterophagic vacuoles. after 10-15 min, as a transitory plug of floccular and colloidal material formed in the lumen of the siphon by exocytosis of some sgc, other ones with engulfed bacteria crossed the epidermis of the siphon reaching the siphonal sinus; cells of the cytotoxic blood cell line (morula cells) were drawn and crowded into the siphonal sinus, where most of them were positive to antitnf-α and anti-cd57 antibodies and degranulated stimulating, after this time and until 12 h, large scavenger phagocytes. the latter showed bacteria engulfed in their large phagosomes, increased in number in the blood circulation and were continuously eliminated through the peribranchial chamber with a mechanism which was never previously described. as regards the ability to transfer an alert signal, the role of sgc appears important as regards the immunosurveillance of the opening of the alimentary canal, similarly to what occurs in the vertebrate oropharyngeal lymphatic tissues. apoptosis signalling pathways in the compoud ascidian botryllus schlosseri during the colonial blastogenetic cycle a menin, l comini, l ballarin department of biology, university of padua, padua, italy in the colony of the ascidian botryllus schlosseri, three blastogenetic generations are usually present: adult zooids, primary buds on zooids and secondary buds on primary buds. colonies undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenetic generations. it is possibile, therefore, to define a colonial blastogenetic cycle that begins with the appearance of a new generation, and ends with the generation change, during the take-over phase, in which programmed cell death by apoptosis is largely diffuse. using the haemocytes as reference tissue we investigate the extent of cell death during the colonial blastogenetic cycle . our results confirm the expression, on cells surfaces, of fas receptors and theirs ligands fasl. moreover, we showed the presence of members of the caspase family: the initiator caspases 8 and 9 and the executioner caspases 3 and 7. the activated executioner caspases can subsequently cleave distinct cellular proteins such as parp: using immunoblot assay we observed the cleaveage of proteins recognised by anti-parp. in vertebrates, intrinsically mediated initiation begins with mitochondrial membrane disruption resulting in cytochrome c (cyt c) release. we observed an encrease of h2o2 in cytoplasmatic contents and a different expression of cyt c during the take-over. these results confirm botryllus an interesting model organism for the study of apoptosis. a novel rhamnose-binding lectin from the colonial ascidian botryllus schlosseri n franchi, f gasparini, b spolaore1, l ballarin department of biology, university of padua, padua, italy 1cribi, university of padua, padua, italy lectins are carbohydrate-binding proteins which agglutinate cells and/or precipitate glycoconjugates. the family of rhamnose binding lectins (rbls) includes various proteins, previously classified as galectins, with common sugar specificity and one to three homolougous carbohydrate-recognition domains (crds), about 100-aminoacids long and characterised by eight highly conserved cysteine residues. from a fulllength cdna library from the compound ascidian botryllus schlosseri we identified five complete transcripts homologous to known rbls. comparisons of the predicted amino acid sequences (118 aa) suggest that they represent different isoforms of a novel rbl, called bsrbl-1-5 with only one crd. reverse-phase hplc and mass spectrometry of the affinity-purified material confirmed the presence of four of these isolectins in botryllus homogenate. analysis of both molecular masses and tryptic digests of bsrbls indicated that the n-terminal sequence of the purified proteins starts from residue 22 of the putative amino acid sequence, so that residues 1-21 represent a signal peptide. analysis by mass spectrometry of v8protease digests confirmed the presence and alignments of the eight cysteines involved in the disulphide bridges characterising rbls. functional studies confirmed the enhancing effect on phagocytosis of the affinity-purified material. the phylogenetic relationship of bs-rbls with orthologous molecules from protostomes and deuterostomes was also studied. enhanced expression of a cintnf gene in the lps challenged inflammatory responses of the ascidian ciona intestinalis n parrinello, a vizzini, v arizza, m cammarata, d parrinello, f giaramita, g salerno, m pergolizzi, ma sanfratello, m vazzana marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy in invertebrates immune system, cell proliferation, phagocytosis and chemotaxis are regulated by cytophilic humoral molecules with functional similarities to vertebrate cytokines. these molecules modulate defense responses to exogenous and endogenous insults, tissue repair and recovery of homeostasis by ligand-specific receptor interactions required to initiate and regulate immune responses. tumor necrosis factor (tnf) is a pro-inflammatory cytokine produced as part of the innate response. in invertebrates tnf-like molecules have been identified by using various methods. since in ciona intestinalis genome (ensembl) tnf gene has been identified (cintnf), 31 real-time pcr analysis was carried out. results showed a prompt (2-4 hrs) enhanced cintnf gene expression in the inflamed body wall after intratunic lps injection. in situ hybridization assays supported the involvement of pharynx hemocytes in the inflammatory response, and transcript was mainly found in morula cells and in unidentified cells associated with epidermis. similar results were found by examining hemocytes from the hemolymph. immunoblotting assay with anti-cintnf specific antibodies revealed that a 17 kda cintnf is released in the hemolymph. sphingomyelin as well as carbohydrates are involved in the mechanism of cytotoxic molecules contained and released in vitro by ciona intestinalis granulocytes v arizza, d parrinello, f giaramita, a vizzini, m cammarata, m vazzana, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy the immune-system of invertebrates recognizes and then reacts to foreign particles potentially pathogen by means of cytotoxicity, phagocytosis, encapsulation, and humoral effector mechanisms (agglutination, lysis). recent studies have shown the presence in invertebrates, including tunicates, of hemocyte/coelomocyte cytolytic molecules (lysins) that can be released into the hemolymph or coelomic fluid, and their lytic activity depends on their integration inside target cell membranes. in some cases membrane lesions appear as circular pores as shown by electron microscopy observations. there are few data on ascidian lysins and their lytic mechanisms. in ciona intestinalis a cytotoxic activity towards mammalian erythrocytes has been reported. the lytic activity, examined using a tris-buffered saline containing calcium ions and isosmotic to the hemolymph, was inhibited by sphingomyelin (25 µg/ml). to identify the lysin-releasing cells, hemolymph was separated through a discontinuous percoll gradient. the hemocyte populations were separated in 6 bands, each of them appeared to be enriched with a particular hemocyte population. the hemocytes from band 5 (b5) mainly composed with unilocular refractile granulocytes were responsible for the lysis of rabbit erythrocytes (re). to characterize the activity, the supernatant of the b5 hemocyte lysate supernatant (b5hls) or b5 hemocyte culture supernatant (b5hcs) was assayed with different mammal targets. both b5hls and b5hcs contain at various extent lytic activity against re and k562 tumor cell line. a lower level of cytotoxicity was found against sheep erythrocytes. such an activity was calcium-dependent, thermo-stable (56 °c), inhibited by sphingomyelin (25 µg/ml), phospholypase a2 inhibitors e.g. dibucain and quinacrine, as well as by d-galactose and cell-free hemolymph. present results suggest that a complex lytic mechanism dependent on sugar-crd interaction may be involved in innate immune response of ciona intestinalis. session 2. chairman: l abelli, university of ferrara, italy leech neuroimmunity: a crossing point between injury and nerve repair j vizioli, pe sautière, c lefebvre, f croq, m tahtouh, m salzet, j pestel laboratoire de neuroimmunologie des annélides fre 2933 cnrs. university of lille 1-59655 villeneuve d’ascq, france lophotrochozoans (annelids and molluscs) are increasingly contributing, along with vertebrates and ecdysozoans, to a deeper understanding of important biological processes like innate immunity or neurogenesis. among the annelids, the medicinal leech hirudo medicinalis, is one of the most extensively used model organism since the xix century for neurobiology studies. indeed, the development, the anatomy and the physiology of many nerve cells are now well characterized. in addition, the medicinal leech is a recognized model in central nervous system (cns) regeneration studies because of its capacity to get in a few weeks a complete morphological and functional repair of the injured nerve cord. recent results showed that leech cns is able to activate an innate immune response upon bacterial challenge and that several induced molecules are also known to be involved in regeneration events. early alert signals, glial cells recruitment, neuronal growth and axonal guidance are the major events occurring during cns regeneration in hirudo, but the basic molecular mechanisms of these processes are little known. we presently develop a research project aimed to the comprehension of such mechanisms and the characterization of some protagonist of this complex phenomenon. the main points of interest and the guidelines of our research are here briefly exposed to discuss the role of neuroimmunity during leech nerve repair. leech hematopoietic cells involved in muscle regeneration a grimaldi, g tettamanti, s banfi, ml guidali, g greco, c bianchi, r valvassori, m de eguileor department of structural and functional biology, university of insubria, varese, italy recently, myogenic and endothelial (myoendothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle. these are primitive cells, distinct from satellite cells (potent myogenic stem cell population residing in the muscle of vertebrates and responsible for postnatal muscle regeneration and growth), they are located outside from the basal lamina and expressing the hematopoietic precursor marker cd34. these cd34+ precursors cells play a fundamental role during muscle regeneration, differentiating into myogenic cells and contributing to new fibres formation. hematopoietic and endothelial precursors cells of leeches show an impressive conservation in 32 morphofunctional and molecular mechanisms compared to vertebrates. for this reason leeches can be considered a simple model to better understand several mechanism regulating musclederived hematopoietic stem cell biology. we have previously demonstrated that in leeches endothelial and circulating precursor cells, express cd34 and the vegf receptor flk1 that in vertebrate are specific for both the endothelial and the myo-endothelial cells involved in muscle regeneration. initially, leeches show in the area of lesion a plug that is in a short time replaced by a pseudoblastema only made of fibroblasts and macrophages without any type of muscle fibres. after 2 months from injury, new muscle are present in the cicatricial area and the damaged body sac is regenerated. we focus on the origin of the new muscle fibres since, unlike from vertebrates, no satellite cells are present in the adult muscle body. our findings suggest that cd34+ circulating precursors cells in leeches, as muscle-derived hematopoietic vertebrate stem cell, can be a source of myogenic precursors, directly recruited from vegf in the regenerative area. self/non-self recognition in the ciliate euplotes raikovi: characterization of er-mapk1, a downstream component of the autocrine signal transduction pathway a vallesi, b di pretoro, p luporini department of biology, university of camerino, camerino, italy in the ciliate euplotes raikovi, cell typespecific, water-borne signal proteins (pheromones) control self/non-self recognition phenomena by binding their target cell-surface receptors and activating downstream signal transduction pathways. immunorecognition analyses of e. raikovi cell extracts revealed that at least three distinct protein kinases are activated (phosphorylated) in functional association with the autocrine pheromone-receptor loop that promotes the vegetative (mitogenic) cell growth. one of these kinases, designated as er-mapk1 (from mitogen-activated protein kinases), was structurally characterized by molecular cloning of the relevant gene. the er-mapk1 n-terminal half of 300 amino acids bears unmistakable structural homology with the “intestinal cell kinase” and the “male-germ cell associated kinase”, that are involved in the regulation of proliferation and differentiation of specialized animal cells. it contains all the basic structural features that are required for a mapk catalytic activity, in particular the dual phosphorylation site represented by the thr-asp-tyr motif in the activation loop. in contrast, the er-mapk1 c-terminal half of 331 amino acids appears to be structurally unique. it is particularly rich in glycine residues and potential sites of regulatory activities, and shows sequence motifs that clearly predicts a nuclear localization of er-mapk1. old and new immunomarkers to assess health status of clams (tapes philippinarum) from the lagoon of venice v matozzo, mg marin department of biology, university of padua, padua, italy the aim of the present study was to examine the health status of clams (tapes philippinarum) from the lagoon of venice by means of immunomarkers. bivalves were collected in june 2007 in 8 sites of the lagoon characterised by differing contamination levels. immunomarkers included total haemocyte count (thc), lysozymelike activity in cell-free haemolymph (as measure of cell membrane stability), and vitellogenin (vg)-like protein levels in both digestive gland and cell-free haemolymph. the results showed that clams sampled at marghera and fusina (highly polluted sites) had significantly reduced thc values with respect to those of animals from the other sites. conversely, significantly increased thc was observed in clams from valle di brenta and cà roman, influenced by wastewater from agricultural areas and intense fishing and passage of ships, respectively. significantly increased lysozyme-like activity was also recorded in cell-free haemolymph of clams collected at the most polluted sites (campalto, marghera, cà roman), suggesting that destabilisation of cell membranes occurred in haemocytes. interestingly, altered vg-like protein levels were observed in digestive gland and haemolymph of both male and female clams from contaminated sites. vg induction is generally recognised as a biomarker of exposure to estrogenic compounds. however, in the light of recent findings concerning involvement of vg in immune responses (zhang et al. fish shellfish immunol. 19: 93-95, 2005), application of vg as potential immunomarker in future laboratory and field studies is suggested. on the basis of the immune responses analysed in the present study, we can conclude that the health status of t. philippinarum at contaminated sites is poor. however, influence of both exogenous (i.e., water temperature and salinity, food availability) and endogenous factors (i.e., reproductive cycle) on cell functional responses investigated have to be taken into proper account. a new hemagglutinin from the coelomic fluid of the sea urchin paracentrotus lividus (lamarck, 1816) (echinodermata) f drago department of animal biology, “ marcello la greca”, university of catania, catania, italy agglutinins or lectins are glycoproteins that play a fundamental role in the innate immune responses in invertebrates. they are usually detected in cellfree coelomic fluid or hemolymph by the ability to agglutinate particles such as vertebrate erythrocytes (hemagglutinin), bacteria, protozoa or fungal cells. hemolymph lectins have been isolated from several 33 species of invertebrate taxa, while few data are available in echinodermata. in the present study a hemagglutinin was purified from the coelomic fluid of the sea urchin paracentrotus lividus, by ionexchange chromatography on deae-sephadex. a fraction, corresponding to a band at 11 kda in sds page, was found to have hemagglutinating activity. the amino acid composition of the lectin is in progress by the use of a 2d electrophoresis followed by maldi tof analysis. session 3. chairman: r valvassori, university of insubria, varese, italy immunosuppression and host regulation by parasitic hymenoptera f pennacchio department of entomology “filippo silvestri”, university of naples “federico ii”, portici, naples, italy the success of parasitism in hymenoptera is largely influenced by molecules and genes that the ovipositing female injects along with the egg into the host’s body or that offspring produces during the course of development. here we analyze how the basal “toolkit” of gene products in ancestral ectoparasitic idiobionts has changed with the evolution of more intimate developmental strategies, which require very efficient mechanisms of evasion and/or suppression of host immune response. the functional and molecular bases of the major alterations of host immune system, endocrine balance, development and reproduction induced by regulation factors, both of maternal and embryonic/larval origin, are presented and the impact of these changes on host suitability and parasitoid fitness analyzed. hril-8 stimulates unpaired (upd)-3 expression and other immune-related activities in drosophila melanogaster sl2 cell line d malagoli, s sacchi, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy invertebrate innate immunity relies on both cellular and humoral components. among humoral factors, there is less information on soluble molecules able to act as signals during the immune response (i.e. cytokines). to date, only a few cytokines have been observed in different arthropod taxa such as insects (spätzle and unpaired [upd]-3) and crustaceans (astakine). as it has been proposed that a possible involvement of chemotactic factors may increase the expression of upd-3, we have studied the effects of human recombinant ilnterleukin-8 (hril-8) on the immune functions of drosophila melanogaster sl2 macrophage-like cells. we have found that hril-8 enhances the expression of upd-3 and of the putative cytokine, drosophila helical factor (dhf). furthermore, hril-8 promotes the transcription of the antimicrobial peptide genes defensin and cecropin a1. beside these evidences on humoral factors, hril-8 also increases the phagocytic activity of sl2 cells. our data suggest the existence in d. melanogaster of one or more soluble factors that possibly share some structural similarity with il-8, eliciting an immune response involving simultaneously cellular and humoral components. il-1 system in reproduction of invertebrates a pellegrini, s jantra, f ietta, n bechi, e bigliardi1, l paulesu department of physiology, university of siena, siena, italy 1department of evolutionary biology, university of siena, siena, italy interleukin-1 (il-1) is a key regulator of the inflammatory response and an important mediator of materno-fetal immunotolerance in mice. we recently showed that il1-β and its functional membrane receptor type i (il-1r ti) are also expressed in the female reproductive tract of vertebrate species with different reproductive strategies, i.e. viviparity and oviparity. in both cases, there are mechanisms of materno-fetal immunotollerance since the sperm enter and cross the female genital tract and then, the fertilized egg/zygote/embryo is transported or retained in the oviduct. to date, research available is limited to vertebrates while no data are reported in invertebrates. since the cytoplasmic domain of il1r ti shares high homology with the cytoplasmic region of toll protein that recognize many pathogen associated molecular patterns in drosophila, we investigated the expression of toll-il1r ti (tir) domain in reproductive tissues of this invertebrate. by using a specific anti-human antibody against the tir domain, western blot analysis revealed a band of 117 kda in membrane lysates corresponding to drosophila toll protein. immunohistochemistry showed protein expression in the epithelial cells of oviductal tissues and in the spermathecae. these findings suggest a potential ubiquitous role of il-1 system in reproductive tissues of vertebrates and invertebrates. differential responses of mussel hemocytes to bacterial challenge c ciacci 1, m betti1, p roch2, l canesi3 1institute of physiological science, university of urbino “carlo bo”, loc. crocicchia, urbino, italy 2umr ecosystèmes lagunaires, universitè de montpellier 2-34099 montpellier, cedex 5, france 3department of biology, university of genoa, genoa, italy marine bivalves are widespread molluscs in coastal waters at different latitudes. due to their filter-feeding habits, they accumulate large number of both autochthonous and allochtonous bacteria from the harvesting waters. in this work the functional responses of mytilus hemocytes to in vivo challenge with different 34 bacteria were evaluated. two gram-negative bacteria, vibrio splendidus and vibrio anguillarum, and one gram-positive bacterium, micrococcus lysodeikticus, were used. mussels have been injected with heat-killed bacteria and hemocytes sampled at different time post-injection (from 3 to 24 h). the results demonstrated that different bacteria elicited significant functional responses in mussel hemocytes. all bacteria lead to a significant lysosome membrane destabilisation (v. splendidus > v. anguillarum > m. lysodeikticus) at short time post-injection followed by recovery at longer times. similar effects have been observed in mussels collected from two different populations (adriatic sea and ligurian sea). bacterial challenge also induced a significant increase (about 100 %) in serum lysozyme activity. moreover, challenge with v. anguillarum significantly stimulated the bactericidal activity of hemocytes towards e. coli. the results indicated differential functional responses of mussel hemocytes to different bacteria. the application of this knowledge to the understanding of the actual adaptive responses of bivalve when exposed to micro-organisms in their natural environment can represent significant ecological, economical and public health-related interest. this work was partially supported by the eu program imaquanim (food-ct-2005-007103). morphological changes of mytilus galloprovincialis hemocytes after bacterial interaction p pagliara, p roch1 department of biological and environmental sciences and technologies, university of salento, lecce, italy 1umr ecosystèmes lagunaires, université de montpellier 2-34099 montpellier, cedex 5, france apoptosis represents a physiological process used by multicellular organisms to regulate the cell number (tissue homeostasis), to remove damaged or unwanted cells or to eliminate different pathogens. protozoan parasites can induce or inhibit apoptosis in host cells, whereas bacteria increased apoptosis in human neutrophils and macrophages, interfering with such essential components of the innate immunity. so, upregulation of apoptosis due to infection may result in immune suppression. mainly studied in vertebrates, only few data are available on the relationships between apoptosis and immunity in invertebrates. in the present study, we investigated the response of the marine bivalve mytilus galloprovincialis hemocytes to different bacteria species, as well as the morphological modifications of their nucleus using microscopic observations.. the numbers of hemocytes that detached from the substrate and died by necrosis increased after bacteria contact. the detached hemocytes became round losing their typical cytoplasm extensions. they were also evidences for morphological changes of nucleus with chromatin condensation and bordering at the nucleus periphery, and sometimes its fragmentation. control of apoptotic induction in mussel has been done on adhering hemocytes treated with h2o2 and puromicin. in conclusion, the changes in nucleus morphology and the rounding of cells suggested apoptosis activation, being more evident with vibrio splendidus than with vibrio anguillarum. session 4. chairman u oreste, cnr, naples, italy immune defences and interactions with the environment g scapigliati, e randelli, d casani, f buonocore department of enviromental sciences, university of tuscia, viterbo, italy animal species live in a condition of homeostasis with the microbiome present in their body and in the environment. this microbiome is constituted by a great number of microorganism species, many of which are potential pathogens. the homeostatic condition describes a continuous and dynamic equilibrium present between aggression strategies developed by microorganisms, and defence strategies invented by animal eukaryiotes. during millenia, the continuous development of aggression and defence strategies became an evolutionary machine that produced a great number of orthologous and paralogous genes. also, the evolution of animal behaviours has been possible by the evolutive capabilities offered by the immune system, that must guarantee full immune defence in every environmental condition. the research performed in our lab considered the morphological and functional organisation of immune defences initially in insects, subsequently in teleost fish and amphibians. teleost fish represent an experimental model widely employed, since are oldest living vertebrates with a functional platform of immune system conserved in all its components until mammals, they are present in every aquatic environment of the planet, and are of interest for animal and environmental biotechnolgy. in addition, teleosts have a free-swimming larval stage, and it’s possible to divide vital stages when only innate defences are present, and when both innate and acquired defences are functional. employing as main investiagted species the sea bass dicentrarchus labrax, we defined in a teleost species the panel of distribution and the ontogenesis of lymphocytes, the involvement of lymphocytes during antigenic stimulation “in vivo” and “in vitro”, the cloning of genes coding for immunoregulatory molecules, and the expression of these genes during immune responses. in the imaquanim consortium, we are developing systems of transcriptomic analysis by using pcrarrays, to evaluate immune responses through the simultaneous expression of groups of genes coding i) for the whole sea bass immunome (ca 50 genes), ii) for the “innate” immunome (ca 15 genes), and iii) for the “acquired” immunome (ca 35 genes). of great interest for immune studies is gut-associated 35 immune system, whose functional organisation could be also related to the intestinal microbiome and food habits. many t cells are present in the intestinal epithelium of predatory teleosts, and current research is in progress to extend these observations to herbivorous and plancton-eating fish species. research funded by european union (6fp, n° 000173). thymocyte decisions in sea bass, dicentrarchus labrax (l.): cd4 or cd8? gene expression profiling and in situ hybridisation studies l guerra, f buonocore, e randelli, am fausto, l abelli1, s picchietti department of environmental sciences, university of tuscia, viterbo, italy 1department of biology and evolution, university of ferrara, ferrara, italy in teleosts no precise information is still available on differentiation and selection of t cells, crucial steps to establish a functional immune system, although the role of thymic microenvironment in t-cell development has long been recognized in mammals. this work first defines the expression levels of cd4, cd8-α and tcr-β, three important genes in t cell function, in the thymus of the marine teleost dicentrarchus labrax (l.). specific primers were used to analyse by real time pcr gene expression in the thymus of one-year-old specimens. the results reported high tcr-β and cd8-α transcripts, while cd4 transcripts were lower (p<0.05 vs. tcrβ). the in situ hybridization with rna probes identified cd4 and cd8-α expressing cells in each thymic lobe, cd8α+ and cd4+ thymocytes almost filled the cortex drawing a cortex-medulla demarcation and extended in large cords in the medulla. in sea bass thymus, the expression pattern of cd4 and cd8-α largely overlapped that of tcrβ, except in the subcapsular zone where tcrβ+ double-negative thymocytes were detected. these results provide new information about the thymic compartmentalization in teleosts and leads to new hypotheses about thymocyte differentiation pathways. gene expression and functional studies on gut immune system of the sea bass, dicentrarchus labrax (l.) s picchietti, l guerra, e randelli, f bertoni1, am fausto, f buonocore, l abelli1 department of environmental sciences, university of tuscia, viterbo, italy 1department of biology and evolution, university of ferrara, ferrara, italy morphological and molecular data have evidenced in a few teleost species a gut-associated lymphoid tissue (galt), consisting mainly of t cells, whose origin, selection and functions are still unclear. this work reports about tcr-β, cd8-α and cd4 transcription in the intestine of the marine teleost dicentrarchus labrax (l.) and localization of t cells expressing such genes. anterior, middle and posterior intestine segments of one-year-old specimens were sampled for rq-pcr and in situ hybridization (ish) studies. tcr-β and cd4 transcription did not differ along the intestine, while cd8-α transcripts rose in the posterior segment (p<0.05 vs. middle). in the whole intestine tcr-β and cd8-α transcripts were higher than cd4 ones (p<0.001). in anterior and middle segments tcr-β expression was higher than cd8-α (p<0.001). the ish with rna probes identified numerous tcr-β+ and cd8-α+ cells intraepithelially and in the lamina propria of mucosa, the latter forming aggregates, and rare cd4+ cells. percoll-purified leucocytes from the whole intestine showed innate cytotoxic activity against a xenogenic tumour cell line. there results confirm that the sea bass galt consists mainly of t cells with significant cytotoxic activity. early administration of probiotic strains stimulates the gut immune system of the marine teleosts dicentrarchus labrax and sparus aurata l abelli, s picchietti1, e randelli1, o carnevali2 department of biology and evolution, university of ferrara, italy 1department of environmental sciences, tuscia university, viterbo, italy 2department of marine sciences, marche polytechnic university, ancona, italy early feeding (started during gut metamorphosis) with probiotic-supplemented diets, besides modifying the intestinal microflora, provoked profound effects on physiology of fish larvae. using rotifers and artemia as living vectors, the autochthonous bacterium lactobacillus delbrueckii or a multispecies probiotic formulation (autochthonous lactobacillus fructivorans + lactobacillus plantarum from human faeces) were orally administered to sea bass and gilthead seabream larvae, respectively. the treatments stimulated the larval rearing (significantly increased body weight, decreased cortisol levels and improved stress response) and the immune system. in sea bass, the probiotic raised intestinal tcells (+105 %), in keeping with increased total body tcr-β transcript (+41 %), and acidophilic granulocytes (+118 %) concomitant to lower transcription of pro-inflammatory genes (il-1β, tgfβ, il-10, cox-2). in seabream, the multispecies formulation raised intestinal ig+ cells (+51 %) and acidophilic granulocytes (+284 %), mainly belonging to the mab g7+ phagocytic population (+536 % vs. control). 36 these results point to stimulatory effects of probiotics on the gut immune system that correlates with improvement of fry survival. a cd4 homologue in sea bass (dicentrarchus labrax): molecular characterisation and structural considerations e randelli, f buonocore, d casani, s costantini1, am facchiano1, jj zou2, cj secombes2, g scapigliati department of environmental sciences, tuscia university, viterbo, italy 1laboratorio di bioinformatica e biologia computazionale; institute of food sciences, cnr, avellino, italy 2scottish fish immunology research centre, aberdeen university, aberdeen, uk the cd4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. its action as a t cell co-receptor increases the avidity of association between a t cell and an antigenpresenting cell by interacting with portions of the complex between mhc class ii and tr molecules. the sea bass cd4 cdna consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. the analysis of the sequence shows the presence of four putative ig-like domains and that some fundamental structural features are conserved from sea bass to mammals. by real time pcr analyses very high levels of cd4 mrna basal transcripts have been evidenced in thymus, followed by gut and gills. in vitro stimulation of head kidney leukocites with lps and pha-l gave an increase of cd4 mrna levels after 4 h and a decrease after 24 h. homology modelling has been used to create a 3d model of sea bass cd4 and to investigate its interaction with sea bass mhc-ii. our results will add new insights in sea bass t cell immune response and will help to identify t cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity. this work was supported by the european commission within the project imaquanim (ec contract number food-ct-2005-007103). effects of exogenous cortisol on the expression of glucocorticoid receptor (dlgr1) and hsp70 in head-kidney and peritoneal cavity cells from dicentrarchus labrax a vizzini, m vazzana, g salerno, m celi, ma sanfratello, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy fish are constantly exposed to stressful conditions, and have evolved responses including the prompt release of corticosteroids hormones. the physiological effects of corticosteroids are regulated by cellular glucocorticoid receptors (gr), that mediate gene expression influencing a variety of physiological function related to metabolism, immunity, beahaviour, osmoregulation and cardiovascular transport. the assembly, functionality and transport of grs are dependent upon the activity of heat shock proteins (hsp) including hsp 90, hsp 70 and hsp 56. in previous papers we showed that high plasma cortisol level due to fish confinement affects sea bass innate immunity, then we cloned and sequenced a gr from sea bass peritoneal cavity leucocytes (dlgr1). in this study we examined the expression of dlgr1 as related with hsp 70 tissue content in the presence of high exogenous plasma cortisol level. cortisol (20mg/kg body mass) was injected into fish peritoneal cavity, and, at various times after the injection, dlgr1 gene expression was evaluated by real time pcr. hsp 70 was identified by specific antibodies an densitometry analysis of western blot pattern was carried out. results showed that, despite a drop in dlgr1 protein content, there was a significantly enhanced dlgr1 mrna expression in head kidney cells due to stress for manipulation procedures, whereas in leucocytes from peritoneal cavity the enhanced expression of dlgr1 could be related to a cortisol effect. an increased level of hsp 70 protein was also shown. research are in progress to evaluate hsp 70 gene expression. session 5. chairman: n parrinello, university of palermo, italy longevity genes across species: conservation versus evolvability s salvioli1,2,3, ptieri1,2, g castellani2,4, m capri1, c barbi1, a santoro1,2, s altilia1,2, l invidia1,2, m pierini1,2, e bellavista1,2, d monti5, c franceschi1,2,3,6 1 department of experimental pathology, university of bologna, 40126 bologna, italy 2interdepartmental center “l. galvani” (c.i.g.), university of bologna, 40126 bologna, italy 3er-gentech laboratory, 44100 ferrara, italy 4dimorfipa, university of bologna, 40064 ozzano dell'emilia, italy 5department of experimental pathology and oncology, university of florence, 50134 florence, italy 6department of gerontological science, italian national research centre on aging (inrca), 60131 ancona, italy the search for longevity genes has greatly developed in recent years basing on the idea that a consistent part of longevity is determined by genetics. the ultimate goal of this research is to identify possible genetic determinants of human aging and longevity, but studies on humans are limited by a series of critical restrictions. for this reason, most of the studies in this field have been, and still are, performed on animal models, basing on the assumption that fundamental biological mechanisms are highly conserved throughout evolution and that, accordingly, extrapolation from model systems to humans is quite reasonable. 37 indeed, many comparative data obtained on single genes or gene families fit with this assumption. however, it is also clear that, despite such a basic conservative scenario, major changes also occurred in evolution, particularly regarding biological regulatory processes and integration between and among pathways. this consideration raises the fundamental question of the transferability of the results obtained from model systems to humans. indeed, many data obtained on animal models were not confirmed on humans or, on the contrary, some contributions to the genetics of longevity were discovered in humans and not always have been replicated in animals. recent conceptualizations stressed the importance of robustness as a fundamental property of living organisms, a characteristic acquired during evolution that allow them to be error-tolerant and to easily respond to external perturbations. a “bow-tie” organizational architecture is likely to be a common feature of highly organized, robust systems. a bow-tie model is present when many inputs converge on, and are integrated by, few elements, and many different outputs come out as the product of the integration. the elements composing the core (“knot”) of the bow-tie in a biological system are “hub proteins” and the genes that encode them are likely to be in many cases robustness genes. much of the core of a bowtie is often conserved throughout evolution, but this conservation does not prevent, but rather facilitates the variability of the possible outputs. thus, a bowtie structure allows both robustness and evolvability. we propose that longevity genes should participate with a core position to a bow-tie metabolic structure and that a new way to identify putative genetic determinants of longevity could be thus the conservation of their products in bow-tie structures all along evolution. thymus development is influeced by hypophyseal and thymic allograft after hypophysectomy: expression of pcna and cd3 markers m aita, e caccia1, n romano1 department of human physiology and pharmacology, university “la sapienza”, rome, italy 1department of environmental sciences, university of tuscia, viterbo, italy experiments of hypophysectomy, followed by hypophyseal or thymic allograft were performed in chicken embryos in order to study the influence of thymus or hypophysis on the thymus development. experimental and control thymuses, collected at the 18th day of incubation, were tested for antipcna (proliferating cell nuclear antigen) and anticd3 immune reaction. the immunoreactive cells were evaluated by computer-assisted statistical analysis. in the first series of experiments, hypophysectomy was performed on chick embryos at 36-40 hr of incubation. the thymuses collected evidenced in the cortex, a significant reduction of pcna immunereactive thymocytes (p<0,05). in the second series of experiments hypophysectomized embryos received at the 12th day of incubation or a hypophyseal allograft or a thymic allograft onto the chorio-allantoic membrane from 18 day-old donor embryos. the hypophyseal allograft allowed an increase of the cd3 expression of the cortical and medullary thymocytes but no improvement of pcna expression in cortical thymocytes. the thymic allograft let a very good recovery (p<0,001) both in pcna and cd3 expressions in the cortical thymocytes and a good recovery, of cd3 similar to that found in hypophyseal allograft, in the medullary thymocytes. these findings confirm that the lack of hypophysis decreases the possibility of cortical thymocytes to proliferate, but not to differentiate further on and that a thymic allograft may influence the recovery of the thymus of a hypophysectomized embryo, probably also by an emigration of thymocytes from the thymic graft. comparative analysis of fucose binding lectins isolated and characterized from different teleost species, and distribution of a f-lectin during dicentrarchus labrax ontogenesis m cammarata, mg parisi, g benenati, v mangano, a vizzini, g salerno, d parrinello, m vazzana, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy fucose-binding lectins (fbl) are contained in tissues and fluids from invertebrates and vertebrates. the lectin repertoire in teleost fish is highly diversified and, recently, the fbl structure with a novel lectin fold of f-type carbohydrate recognition sequence (the eel “f-type”) has been described (bianchet et al. nat. struct. biol. 8: 628634, 2002). such a lectin shared an unique fucosebinding sequence motif contained in carbohydratebinding domain of lectins and unrelated proteins. recently, we have shown that a serum fbl from sea bass dicentrarchus labrax (dlfbp) and sea bream sparus aurata (safbp) are involved in innate immunity. both lectins were composed of two tandem domains that exhibit the eel “f-type” motif. these lectins were purified, characterized, cloned and sequenced, and biological activity and tissue distribution were examined. a complete coding of teleost f-type lectin crds showed that dlfbp and safbp are members of the f-lectin family. the phylogenetic sequence analysis from bacteria to vertebrates, showed that the f-lectin motif is broadly distributed, and significantly diversified in terms of molecular organization and number of domains. preliminary results on comparative analysis of purified and characterized lectins from different teleost species are also reported. these lectins, that showed calcium independent hemoagglutinating activity against rabbit erythrocytes, have been purified using a fucose-agarose affinity chromatography. serum fucose binding lectins are present in all the studied species with a molecular size range from 32 to 41 38 kda, and share shrinkage property in sds-page. the relationship with the f-lectin family has been supported with western blot analysis using dlfbp antibody. finally, we examined distribution and expression of dlfbp during fish ontogeny and showed lectins are also present in eggs. cloning and expression analyses of an interferon (ifn) in sea bass (dicentrarchus labrax l.) after poly i:c induction d casani, f buonocore, e randelli, v scala, jj zou1, cj secombes1, g scapigliati department of environmental sciences, tuscia university, viterbo, italy 1scottish fish immunology research centre, aberdeen university, aberdeen, uk interferons (ifns) are a multigene family of inducible cytokines and posses antiviral activity through a jak/stat signalling pathway. ifn action induces the synthesis of ifn-regulated proteins, as mx, that collectively constitute the antiviral response responsible for the inhibition of virus multiplication. ifn cdna cloning was possible after in vitro stimulation of head kidney sea bass (dicentrarchus labrax l.) leukocytes with polyinosinic: polycytidilic acid (poly i:c). poly i:c is a synthetic ribonucleotide that acts as a viral rna to induce the ifn system. we used the “homology cloning” strategy: with degenerate primers we obtained an initial fragment of 189 bp. from this fragment we designed specific primers for 3’ and 5’ race pcr to complete the cdna sequence. an alignment was performed using available ifn amino acid sequences to study the conservation of characteristic features and a phylogenetic tree was generated using the same sequences. finally, specific primers were used to analyse by real time-pcr the ifn and mx expression in head kidney leukocytes stimulated with poly i:c for 6 and 24 h. the ifn expression after 6 h is higher with respect to 24 h expression, whereas for mx it is the contrary. this is an agreement with the teory that ifn is produced after viral infection and, successively, it induce mx synthesis. the same cloning procedure will be used on genomic dna, as it is important to determine the number of introns in the ifn sequence to investigate its evolution from fish to mammals. this work was supported by the european commission within the project imaquanim (ec contract number food-ct-2005-007103). phylogenetic analysis of teleost light chain immunoglobulin u oreste, s varriale, m r coscia cnr, institute of protein biochemistry, napoli , italy in the last decade a large number of cdna igl sequences have been obtained from teleosts. because of the extensive igl diversity within the genome of each species and the very wide phylogenetic distance among teleost orders, the classification of igl isotypes from teleost species remains questionable. at present, because the number of igl isotypes identified so far varies in different teleost species, it remains unclear whether all possess the same number of igl isotypes. the discrepancy in the number of igl isotypes identified in each species may be explained by incomplete screening of differently expressed isotype specific mrnas and by a different degree of diversity between sequences identified in each species. to date, igl genes have been sequenced from 27 different species, two of them being antarctic species investigated by us: trematomus bernacchii and gymnodraco acuticeps. in the present work we compared all the available teleost immunoglobulin light chain sequences in order to evaluate the evolutionary relationships among them. we searched the data banks for all teleost immunoglobulin light chain, either genomic or cdna, sequences. for each species we aligned the sequences by clustalx, distinguished different isotypes and constructed a consensus sequence for each isotype. then we calculated the distances from the consensus of individual sequences and used the closest one in a data set. the sequences of the data set were aligned and phylogenetic trees were constructed by neighbor-joining and maximum-likelihood methods. our results indicated that three different isotypes can be identified in teleosts. in the acanthopterygean species one isotype can be distinguished into two subisotypes one of them carrying a dna microsatellite insertion. specific sequential features of each group of isotypes have also been observed. work supported by the pnra project 2005.1.2 screening for antimicrobial peptides in zebrafish (danio rerio) el caccia, g vasta1, ag ficca, p strickler1, n romano department of environmental sciences, university of tuscia, viterbo, italy 1comb, university of maryland, baltimore, maryland, usa antimicrobial peptides (amps) are small-sized (from 12 to 45 amino acids in length), cationic and amphipathic molecules able to neutralize pathogenic microrganisms. the amp are very ancient in evolution life and wide expressed in methazoan species. they were described as being inducible and exert the killing or slow growth of bacteria, thus representing an old immune system that share adaptive and natural characteristics. probably, the amps are early expressed during ontogeny of vertebrate as “evolution memory” before more complicate immune system has to differentiate. with the emergence of antibiotic resistant bacteria the amps represent good candidate for therapeutic strategies to counter bacterial infection in animals. the aim of this study is to identify the genes encoding for amps in 39 zebrafish. by analysing zebrafish est and genome databases we identified those sequences homologous to known antimicrobial peptides genes from higher vertebrates: 1) mucosal (parasin i, chatepsin d3 and d5); 2) liver/organs expressed (defensin beta-db1, leap-2); 3) circulating phagocytes (bactericidal permeability-increasing protein, bpi); 4) the neuroendocrine/immune system–related peptides, chromogranin a and b (cga, b). we designed specific primers sets that allowed the amplification of the full or partial length of these genes. the lengths of the seven cdnas (obtained by retro-trascription of total rna extracted from adult samples) range from 242 bp (dbi) to 504 bp (cgb). they have been cloned and sequenced and the deduced amino acids sequences have been aligned to the well known antimicrobial peptides to confirm successful amplification of the genes of interest. here we report the comparison analysis the deduced amps amino acid sequences from danio rerio with those of other fishes. further analysis will be performed in order to investigate the expression of these molecules during ontogeny. more than three decades since the use of mussels as pollution biosensors, their genes are still almost unknown. in the frame of the european integrated project food-ct-2005-007103, est sequencing and gene expression profiling are providing new interesting evidences. the former refers to the extension of the dna microarray of m. galloprovincialis defined at cribi. (university of padova) and currently used for investigating mussel gene transcription. among a number of selected stress factors, mussels of different origin have been challenged with bacterial antigens and the total rna subsequently purified from haemolymph samples was used to build primary cdna libraries. the abundant expression and molecular variability of transcripts identified as antimicrobial peptides emphasize the importance of such molecules in the response to bacterial pathogens. in silico processing and robust sequence annotation is in progress on the whole bulk of the new mussel ests. parallel evaluation of gene expression is performed by dna microarray analysis on indigenous mussels from the venice lagoon, a coastal transition system (4 sites, summer samplings 2005-2007). a relatively small number of immune-specific probes is present in the available cdna microarray and, with exception of one defensin, they appear down-regulated in the lagoon compared to offshore mussels. results will be discussed according to the published literature. experimental evidences from est sequencing and gene expression profiling in mytilus galloprovincialis l varotto1, a pallavicini2, f bernante3, s domeneghetti1, s cagnin3, g lanfranchi1,3, p venier1 1department of biology, university of padua, padua, italy this work was also supported in part by corila (consortium for the management and coordination of the activities related to the venice lagoon system). 2department of biology, university of trieste, trieste, italy 3cribi, university of padua, padua, italy 40 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj105.pdf 64 isj 3: 64-76, 2006 issn 1824-307x review regeneration in echinoderms: repair, regrowth, cloning md candia carnevali department of biology, university of milan, milan, italy accepted june 16, 2006 abstract regenerative potential is expressed to a maximum extent in echinoderms. it is a common phenomenon in all the classes, extensively employed to reconstruct external appendages and internal organs often subjected to amputation, self-induced or traumatic, rapidly followed by complete successful re-growth of the lost parts. regeneration has been studied in adult individuals as well as in larvae. in armed echinoderms, regeneration of arms is obviously frequent: in many cases, the detached body fragments can undergo phenomena of partial or total regeneration independently of the donor animal, and, in a few cases (asteroids), the individual autotomised arms can even regenerate to produce new complete adults, offering superb examples of cloning strategies. in the species examined so far most results throw light on aspects related to wound healing, growth, morphogenesis and differentiation, even though in most cases many crucial questions remain unanswered. the present paper provides an overview of the current understanding of the phenomenon and covers the main biological aspects of regeneration giving an idea of the “state of the art” across the phylum in terms of experimental approaches and representative models. key words: regenerative development; repair; regrowth; cloning introduction to regeneration development is a broad-spectrum process intrinsic of life which does not always start from the egg, fertilized or not, but can involve significantly all the stages of the life cycle, including not only the embryonic and post-embryonic periods, but also the adult phase. regeneration is in fact a distinct type of developmental process typically occurring in adults or larvae: it can involve limited processes of cell turnover and tissue repair, replacement of lost parts or organs, cast off following self-induced or traumatic mutilations, and even complete regrowth of whole individuals from body fragments (thus contributing to typical asexual reproductive processes). corresponding author: md candia carnevali dipartimento di biologia, universita’ degli studi di milano via celoria 26, 20133 milano, italy e-mail: daniela.candia@unimi.it in terms of general significance, regeneration is an important regulatory phenomenon with wide implications on reproductive biology, as previously suggested by spallanzani in his historical studies on head regeneration in snails (1768). it does not represent the first expression of new combination of genes resulting from sexual processes, but starts from cells, which have an already tested genetic program. it is fundamentally a conservative asexual process. in fact, thanks to its evident close relation with fission phenomena and cloning processes, regeneration can be regarded as the necessary and specific developmental complement to asexual reproduction, in analogy, and in parallel, to what happens for embryogenesis which is the established developmental strategy complementary to sexual gametic reproduction. therefore, although regeneration unavoidably involves analogous problems in terms of basic mechanisms and often superficially resembles embryogenesis, fundamental differences in its intrinsic asexual start and meaning makes regeneration a significantly diverse biological process. in embryogenesis, the whole organismic structure is totally created de novo; in contrast, in regeneration an anatomically defined part of the 65 organism, small or large, is reformed after its loss or severe injury and the new cells develop in an established context of mature tissues and differentiated cells in individuals (adult or larval) well characterized in terms of morphology and functions. therefore, in all animals, the regenerative processes related to different organs and structures should be regarded as fundamentally distinct developmental processes, which can not be considered an accelerated version of ontogenetic processes (candia carnevali and bonasoro, 2001a; reik and dean, 2002). although a response to injury is evoked in all animals, there is a remarkable variability in terms of degree of morphological and functional recovery, not only between unrelated groups, but also between closely related species, and even between organs and parts of the same individual. in contrast to old traditional views regarding the regenerative potential as a unique prerogative of the simplest and most primitive animals, regeneration is actually a common and widespread phenomenon through phylogeny, and its quite heterogeneous distribution from the lowest to the highest phyla appears to be independent of their organization and complexity level (ferretti and géraudie, 1997; thouveny and tassava, 1997; candia carnevali and bonasoro, 2001a). in fact, the regenerative capabilities appear to depend upon the individual potential for histogenetic and morphogenetic plasticity expressed in terms of recruitment of stem cells and/or dedifferentiated cells, cell proliferation and migration, supply of specific regulatory/trophic factors, and finally expression, or reexpression, of a specific developmental program (weissman, 2000; wadman, 2005). despite the differences between the species, repair and regrowth are common to all organisms, and differentiated tissues (liver, muscles) can be replaced de novo even in adult mammals, at least to a certain degree. obviously the regenerative potential can vary significantly with the stage of the life cycle (embryonic, larval, adult) and the age of the individual, the regenerative capabilities being higher in larval tissues and organs in comparison with those of adults, and in many cases present only in the early embryonic stages (mammals). it is not a case that traditional studies of regeneration in vertebrates have often employed larval stages of amphibians. if a close correlation between the regenerative potential of the individual and its possibility of survival can be inferred easily, self-repair abilities appear not only an obvious advantage for the individual, but gives a fundamental contribution to the adaptive capacities of the species and its fitness, since they increase the individual’s chances of reproducing, sexually or asexually, even when its body integrity is dramatically compromised. the universal character of regeneration is expressed in the famous aphorism by goss (1969): “if there were no regeneration there could be no life, if everything regenerated there would be no death. all organisms exist between these two extremes “. on the other hand, there are still unsolved fundamental questions concerning why regenerative potential is expressed to such different extents in different organisms and whether this phenomenon has been selected for or against in the course of evolution. according to a modern authoritative hypothesis (goss, 1988, 1992; thouveny and tassava, 1997) which takes into account the evolution of regeneration abilities in the organisms, regeneration should be regarded as a primary attribute of life: all organisms in principle possess a latent potential for regeneration, that could have been suppressed in response to specific selection pressures, i.e. inhibited/eliminated wherever its expression does not help survival and reproduction and does not counterbalance the disadvantages of its high metabolic cost. in other words, as far as the animal kingdom is concerned, the suppression of regeneration should not be regarded as a negative adaptation but rather as a pleiotropic epiphenomenon related to specific constraints and linked to more useful but incompatible adaptations (goss, 1988, 1992). in spite of the wide choice of potential models for studying regeneration, this phenomenon has been appropriately explored only in a few animal models traditionally and successfully employed by developmental biologists (hydrozoans and planarians among invertebrates and amphibian urodeles among vertebrates). surprisingly, with regard to many other animal groups well known for their spectacular regenerative capabilities, the actual knowledge is very poor not only in terms of cellular and molecular aspects of regeneration, but also in terms of basic mechanisms (candia carnevali and bonasoro, 2001a). with regard to these latter, according to a traditional view, regeneration involves two alternative basic mechanisms, epimorphosis and morphallaxis (following the terminology introduced by morgan, 1901). in epimorphosis, new tissues arise from undifferentiated cells (stem cells or de-differentiated cells), which are recruited to develop a typical blastema. this pool of new cells represents a discrete centre of proliferation activity from which all the regenerated structures are derived. in morphallaxis extensive phenomena of rearrangement/recycling from differentiated tissues take place and no blastema is formed: only limited and localized proliferation activity involves cells derived from existing tissues by reversal of differentiation and/or migration. in spite of this schematic and apparently well-established difference between epimorphosis and morphallaxis, there is recent experimental evidence (candia carnevali and bonasoro, 2001b) that the mechanisms at the tissue/cellular level can be much more flexible and largely overlapped to each other, and that the dichotomic view of these two alternative processes is too reductive. the regenerative potential in echinoderms regeneration is a physiological phenomenon in echinoderms, common in all classes (hyman, 1955). thanks to their spectacular regenerative capabilities, echinoderms were favourite models for the pioneer regenerationists of the 19th and early 20th centuries. after an unexplainable long scientific oblivion, they were recently re-proposed to the attention by a series of papers (for review see candia carnevali, 2005; candia carnevali and bonasoro, 2001b; thorndyke and candia carneval, 2001) exploring the basic 66 mechanisms of the regenerative phenomenon, its cellular and molecular aspects and also its potential for applied research. regeneration is extensively employed to reconstruct external parts (arms or appendages such as spines or pedicellariae) and internal organs (gonads, gut, whole visceral mass) often subjected to predation or amputation, selfinduced or traumatic, rapidly followed by complete regrowth of the lost parts; it is also part of the life cycle as an indispensable complement of the programme of asexual reproduction and is extensively employed in cloning processes. repair, regrowth and cloning processes can involve both the adult and the larval stages (eaves and palmer, 2003). regenerative potential in echinoderms is developed to a very broad extent: contradicting goss’s paradigm about the “non-regenerability” of the truly vital structures (1965, 1969), it enables vital organs such as the whole visceral mass to be regenerated completely after evisceration. it is largely a predicted phenomenon and in most cases follows autotomic self-induced mutilations, which can be considered the most important proximate cause of structural loss and depends on the presence and unique properties of “mutable collagenous tissues” (mcts, see for a review wilkie, 2001, 2005) at the level of the autotomy plane. it is relevant that in physiological conditions regeneration is always prompted by autotomy and proceeds from the retained side of a fractured autotomy plane. adults. the capabilities to regenerate body parts represent an obvious advantage for echinoderms. firstly, for the replacement of tissues following predation, an ability that echinoderms show to a greater extent than other invertebrates. reconstitutive regeneration is particularly frequent and extensive in crinoids and ophiuroids which both have long, fragile arms often subjected to predation or mutilations, selfinduced or traumatic, rapidly followed by complete regrowth of the lost parts. these regenerative phenomena are so frequent that specimens collected in natural environments always have two or more regenerating arms at different regrowth stage. in many cases, the detached body fragments can survive for a long time and undergo phenomena of partial or total regeneration independently of the donor animal (candia carnevali et al., 1998). these phenomena, quite common also in asteroids, provide clear evidence for the wide exploitation and implications of regenerative potential in echinoderms. in particular in asteroids, besides the extensive application in common repair mechanisms, arm regeneration offers in fact the most complete examples of cloning strategies carried out by adult specimens. as well known, in some starfish (linkia sp., coscinasterias sp.), new complete adults can be regenerated from individual autotomised arms. this extreme case clearly shows that, in echinoderms, regeneration is employed as part of a programme of asexual reproduction leading to the development of new individuals through specific fission mechanisms (emson and wilkie, 1980; mladenov and burke, 1994). besides asteroids, also many ophiuroids and holothuroids undergo asexual propagation involving the splitting of adults into two or three pieces with subsequent regenerative development of new complete individuals from each isolated portion. fission phenomena appear to be correlated with seasonal and metabolic factors as well as to ecological factors, and also depend on the age and size of the individuals: they are particularly frequent in small specimens, whereas the larger ones appear to prefer sexual processes. in terms of epimorphosis versus morphallaxis, echinoderms appear to employ in regeneration one or the other of these two processes according to apparently established criteria which depend on the specific start-conditions and requirements; however, just in echinoderms there is the clearest evidence that epimorphic regeneration often involves a significant contribution of morphallactic processes and that the borderline between these two processes is not so defined (candia carnevali and bonasoro, 2001a, b). if we consider the distribution of typical epimorphic or morphallactic processes in the different echinoderm groups (bonasoro et al., 1998, candia carnevali and bonasoro, 2001a; thorndyke et al., 1999), the emerging pattern can be summarized as follow. typical epimorphic processes with blastema formation appear to be employed in those situations where regeneration is a widely predicted, rapid and effective phenomenon, which takes place following autotomy (for instance in crinoids and ophiuroids, candia carnevali and bonasoro, 2001a, b; thorndyke et al., 2001b). in contrast, morphallactic regeneration seems to be a more complicate and slower process, which tends to follow traumatic mutilations (for instance in arm tip regeneration of asteroids, mladenov et al., 1989; moss et al., 1998): in this case amputation is not a predictable event and the regenerative mechanisms imply phenomena of substantial rearrangement of the old structures. in spite of their apparently different meaning and alternative employment in echinoderm regeneration processes, recent results (see crinoid regeneration in different experimental conditions, candia carnevali and bonasoro, 2001b) show that the same individual can employ both epimorphic and morphallactic mechanisms, modulating their different contributions according to its specific needs. this suggests that previous interpretations of the mechanisms at the tissue/cellular level need to be revised substantially. although occurring in all echinoderm classes, regeneration was investigated by a modern approach only in a few echinoderm models. rather detailed investigations are related to crinoids, ophiuroids, asteroids, and recently also holothuroids, which were explored both in terms of mechanisms at the tissue/cellular level and ecological significance (thorndyke et al., 1999; candia carnevali and bonasoro, 2001b; thorndyke and candia carnevali, 2001; dolmatov and ginanova, 2001; garcia-arraras and greenberg, 2001). regeneration also occurs in echinoids, but is less spectacular in terms of extent and degree of capabilities and only a few examples have been investigated so far (bonasoro et al., 2004; dubois and ameye, 2001). larvae. it is well known that traditional studies of regeneration in vertebrates have often employed larval stages of amphibians. this interest in larval stages is justified by the much higher regenerative potential of larval tissues and organs in comparison with those of 67 adults. as a general rule, any regeneration, which begins during larval life, appears to be inhibited or retarded after metamorphosis (wallace, 1981). in spite of the many obvious reasons for exploring the problem of the regenerative potential of echinoderm larvae, and apart from some fascinating descriptions provided by historical reports (mortensen, 1921; hörstadius, 1925a, b , c), this aspect of echinoderm regeneration has been neglected until recent times, when some exciting results have renewed interest in this phenomenon and in particular have focused attention on its possible implications for evolution, phylogeny and clonal populations. current research has been specifically addressed to regeneration of larvae: the comparative analysis of the different potential exploited by the diverse groups shows that the phenomenon of regeneration has an unexpected plasticity in the larvae and indicates its direct and close relationship to asexual reproduction and cloning (vickery et al., 2001). as is the case for all other regenerating systems, the basic goal in echinoderm regeneration research is to answer a few crucial questions regarding how regeneration processes are initiated, which sets of genes are activated (or reactivated), what is the origin of the cells involved in reconstruction or repair of the damaged or lost structure, and which factors (morphogens and/or mitogens) regulate growth, morphogenesis and differentiation at the right time and at the correct place to ensure a complete reestablishment of anatomical pattern and functional integrity (carlson, 1998; ferretti and géraudie, 1998; thouveny and tassava, 1998). in the species examined so far most results throw light on aspects related to wound healing, growth, morphogenesis and differentiation, but in most cases many fundamental questions remain unanswered, especially those related to specific cellular and molecular aspects and much work is still requested. in spite of the widespread and successful employment of echinoderms for molecular studies based on embryonic or larval development, there is at present a large gap in our knowledge of regeneration and only a few recent data are available. the following synthetic overview, although far to be exhaustive, offers an account of the phenomenological aspects of regeneration through the echinoderm phylum, presenting a summary of results obtained by diverse perspectives and different experimental approaches in representative models, and highlighting the biological relevance and the evolutionary implications of the phenomenon. crinoids adult: regeneration of body parts (crown and stalk) following self-induced or traumatic amputation in sealilies; regeneration of appendices (arms, pinnulae, cirri) or body parts following self-induced or traumatic amputation in feather stars; regeneration of individual internal organs (gonads, gut) or whole visceral mass following self-induced or traumatic mutilation/evisceration in feather stars. the main cellular and molecular aspects of the regenerative processes are summarized in table 1. crinoids are well known for their spectacular regenerative potential. feather stars extensively employ regeneration to reconstruct both external parts, namely arms, pinnules and cirri, and internal organs, such as digestive apparatus, gonads, and even complete visceral mass, which can be frequently lost following traumatic injury, predation or spontaneous autotomy (perrier, 1873; minckert, 1905; reichensperger, 1912). specimens collected in nature always show regenerating arms at different stages of growth. these regenerative phenomena can be easily reproduced in the lab mimicking the autotomy conditions and amputating the arms at the level of the autotomy plane (sutures). the overall regenerative potential of comatulids was accurately tested by przibram (1901) in experiments of severe mutilations. according to these studies, the animal appears to be able to survive and subsequently regenerate the lost parts in a number of traumatic conditions: for instance, when the body is halved and even when reduced to only one fifth. crinoid sea lilies (metacrinus rotundus) are also spectacular in their regeneration powers, and not only a new stalk can be regenerated following partial or complete removal (nakano et al., 2004), but also a completely new and functional crown can be regenerated following total ablation (amemiya and oji 1992). regeneration is also documented in fossil crinoids, particularly in extinct crinoids of the paleozoic, mesozoic and cenozoic, which provide evidence of additional branchings in regenerated arms, underlining the biological, ecological and evolutionary implications of the regenerative phenomenon in echinoderm phylogeny (oji, 2001). arm regeneration in feather stars represents the most thoroughly explored model in echinoderm regeneration studies (candia carnevali and bonasoro, 2001b). in these recent years a comprehensive study of the overall process of arm regeneration was carried out in antedon mediterranea, a flexible experimental model previously successfully employed in old classical studies (minckert 1905; perrier 1873; reichensperger 1912), which was re-explored in all its aspects from the macroscopic to the molecular level. this phenomenon can be described on the whole as a typical blastemal regeneration in which new structures develop from migratory pluripotent, actively proliferating cells in the presence of presumptive regulatory factors, which are responsible for both repair and regenerative phenomena. the overall process can be subdivided into three main phases: a repair phase, an early regenerative phase and an advanced regenerative phase, whose crucial aspects are related to common fundamental mechanisms such as 1) intervention of stem cells and/or dedifferentiated cells; 2) cell migration and proliferation; 3) contribution of putative growth factors, particularly in terms of specific neurally derived factors; 4) mechanisms of pattern formation. the data obtained so far are derived from an integrated approach, which utilizes different methods on experimentally induced arm regenerations (standard or abnormal) obtained in significantly different experimental conditions, including extreme mutilations (explants). the regenerative response was also employed in applied research as a new valuable model for ecotoxicological studies addressed to the e f f e c t s of the e x p o s u r e t o s p e c i f i c c l a s s e s o f 68 table 1. crinoids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -epimorphic process with main contribution of undifferentiated cells -blastema -nerve-dependent regeneration -stem cells (amoebocytes, coelomocytes) -differentiated cells (phagocytes, (granulocytes) -dedifferentiated cells (myocytes) -differentiation -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -extensive proliferation -neurotrasmitters (dopamine, serotonin) -neuropeptides (s1, s2, substance-p) neural growth factors (tgfß, bmp, ngf, fgf-2) anbmp2/4 (tgf-ß superfamily environmental contaminants (candia carnevali, 2005). in particular, the normal mechanisms and pattern of the regenerative processes in standard conditions have been established in a series of experiments on regeneration at different stages following pseudo-autotomic amputations (candia carnevali et al. 1993; 1995; 1997). a parallel analysis has been carried out on the regenerative processes of both the normal regenerating arms and the respective amputated arm segments (explants) (candia carnevali et al., 1998; bonasoro et al., 1999; candia carnevali and bonasoro, 2001b): the explants can be maintained in good living conditions for weeks and represent excellent models for testing the arm regenerative potential in terms of autonomy of resources and control and for comparing regenerative mechanisms in a same individual. different types of isolated explants have been successfully employed: during the culture period they are able to undergo extensive repair and regenerative processes in parallel with their donor arms. comparison between the regenerative processes of arm explants and normal regenerating arms of corresponding stages highlights that beside general similarities in the basic regenerative processes there are some meaningful differences in terms of mechanisms employed and cellular/tissue elements involved. in terms of mechanisms, there is clear evidence that the epimorphic blastemal regeneration can involve a significant contribution of morphallactic processes (candia carnevali and bonasoro, 2001a, b). the regenerative potential, mechanisms and pattern have been also explored and compared with regard to aberrant regenerations resulting from arms deliberately subjected to traumatic mutilations which do not reproduce autotomy (candia carnevali and bonasoro, 2001b). the bulk of the results obtained so far in crinoids not only contribute to throw light on the most relevant aspects related to wound healing, morphogenesis, differentiation and growth in echinoderm regeneration, but also strongly suggest to employ this fascinating and promising experimental model for a successful applied approach. in terms of the specific cellular contribution, most of the cell types involved in regeneration are morphologically undifferentiated migratory elements, which are produced at the level of a) the brachial nerve (amoebocytes) and b) the coelomic epithelium (coelomocytes) respectively. available evidence suggests that the migratory amoebocytes produce the blastemal cells and all the blastema-derived differentiated cells, whereas the coelomocytes give rise to all the differentiated elements related to coelomic tissues. other types of migratory cell involved include phagocytes and granulocytes (“wanderzellen”, reichensperger, 1912). these latter are considered to be a source of putative growth factors. the growth processes are supported by extensive cell cycle activity as evidenced by brdu incorporation studies showing sites of extensive cell proliferation in the blastema and in the coelomic epithelium (candia carnevali et al., 1995, 1997, 1998). the nervous system with its differentiated components (ectoneural, entoneural, hyponeural) plays a crucial role in regeneration: this is related to its striking capacity to regenerate itself, to its pilot-action as a promoter/inductor of the overall regenerative processes, particularly for the development of the musculo-skeletal components, and finally to its contribution in terms of release of regulatory factors (candia carnevali et al., 1989, 1996; thorndyke and candia carnevali, 2001; patruno et al., 2002, 2003). a number of neurohumoral factors with paracrine or autocrine action are involved in regenerative development: neurotransmitters, particularly monoamines such as dopamine and serotonin; neuropeptides, such as substance-p, salmfamide 1 (s1), salmfamide 2 (s2), nerve-derived growth factors, particularly tgf-ß and related peptides (bmp), ngf, fgf-2. recent specific results are related to differential localization and levels of tgf-β1 and tgf-β-type ii receptor expression during regeneration in a. mediterranea (patruno et al., 2002), and to the cloning of native growth factors in crinoids and their implications for regenerative processes (patruno et al., 2003). in particular, the gene identified so far in antedon bifida, is a new member of the tgf ß superfamily, anbmp2/4, which shows a sequence similarity with other echinoderm and human bmps. according to the expression pattern of this gene a plausible role can be suggested in specification of migratory stem cells, blastemal growth and tissue differentiation, particularly skeletogenesis (patruno et al., 2003). it is relevant to remind that, in general, the active gradient established by bmp ligands is considered as one of the main factors responsible for 69 table 2. asteroids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -morphallactic processes: substantial contribution of old tissues -no blastema nerve-dependent regeneration -dedifferentiated cells -stem cells (coelomocytes?) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -limited proliferation -neurotransmitters (dopamine, serotonin) -neuropeptides (s1, s2) -arhox1 (homeoboxcontaining gene) generating the positional information during development, particularly in regeneration. visceral regeneration. if arm regeneration in crinoids can be considered the representative model of complete regeneration of a complex body part, the regenerative potential of the internal organs is well illustrated by the visceral regeneration process. in all crinoids, the loss of the visceral mass does not seem to be a very traumatic event and, in spite of the apparent complexity of the organs and tissues involved, can be easily repaired by prompt regeneration (dendy, 1886; clark, 1921; hyman, 1955). current research (dolmatov et al., 2003; mozzi et al., 2004) is actually focusing on the overall process of visceral regeneration (involving gut and associated tissues and organs) in a. mediterranea, a phenomenon which was explored in the past by the historical study by dendy (1886). the preliminary results collected so far show that visceral regeneration is a very rapid and effective process during which a small gut, functionally and anatomically complete, is reformed de novo in a loose context of new tissues (mainly coelomic cavities and hemal lacunae). in terms of responsible cellular elements, the migratory cells usually employed in regeneration (see arm regeneration) are involved (amoebocytes, coelomocytes, granulocytes, phagocytes). in addition, recent experiments on transplantation of the tegmen and related viscera also show an unexpected plasticity and adaptability of tissues and organs to extremely traumatic conditions. in terms of repair and reconstitutive processes at the tissue level, the mechanisms involved appear to be only partly comparable to those described in visceral regeneration: all the migratory cells seen above contribute to regeneration and are involved in mutual cell exchange between donor and acceptor tissues (mozzi et al., 2004). these results show a striking potential of cell plasticity and tissue histocompatibility in crinoids and confirm their remarkable repair/regenerative capabilities. on the whole, regeneration appears to be a quick and effective process when the mutilation involves a vital organ or the injury is particularly traumatic: in contrast, it seems to be a slower and less effective phenomenon when the part involved is not so indispensable for survival (reichensperger, 1912). interestingly, there is no apparent correlation between availability/assimilation of food and regenerative capabilities which seem to be comparably rapid and effective in both well-fed and starved animals. larvae: partial regeneration of body parts following traumatic amputations. surprisingly, larval regeneration was only rarely recorded in crinoids. the available data refer to phenomena of partial regeneration of body parts following traumatic amputations in both swimming and sessile larval stages (runnström, 1915, 1925), which have been re-explored also in recent preliminary studies (barbaglio et al., unpublished). nothing is known at the moment about possible phenomena of larval cloning, which is actually a field of topical interest and of expanding potential for future research. asteroids adults: regeneration of body parts ( arms) following selfinduced or traumatic amputation; regeneration of internal organs (pyloric caeca, cardiac stomach) following self-induced or traumatic mutilation; fission processes. the main cellular and molecular aspects of the regenerative processes are summarized in table 2. in asteroid arm regeneration (for instance in leptasterias hexactis and asterias rubens), no discrete blastema as a centre of cell proliferation is evident. this appears to apply to both post-traumatic and post-autotomic regenerations (mladenov et al., 1989; moss et al., 1998). typical morphallactic processes seem to be employed and most cell cycle activity is concentrated in the epidermal layer and in the epithelium of the coelomic canals, the two different populations of cells, epidermal and coelomicmesothelial, providing a significant contribution in terms of development/regrowth of the tissues. migratory cells (coelomocytes or others) recruited at distance contribute to a minor extent to tissue 70 regeneration. as noted above, the morphallactic process in asteroids is rather slow when compared to the epimorphic processes of ophiuroids and crinoids; in particular, the initial repair phase may last for a week or more depending on temperature and species, the cell cycle activity being rather low at this stage (moss et al., 1998) and more effective at more advanced stages of arm regrowth. it is also at this period that the regenerating tip begins to display the organization and features of an adult arm complete with distal optic cushion and terminal tentacle. the results obtained so far suggest: a) reversal of differentiation, proliferation and redifferentiation and/or b) direct transdifferentiation of committed cells which can switch from their ‘default' pathway to another and generate all the other cell types (moss et al., 1998). asteroid arm regeneration is a nerve-dependent process. in model species (asterina gibbosa), arm regeneration can not occur if the radial nerve has been removed and the neurotrophic action of the nervous system is needed throughout the whole course of regeneration (huet, 1975; huet and franquinet, 1981; thorndyke and candia carnevali, 2001). in some species, as stated above, following autoamputation isolated arms (comets) can regenerate to produce completely new adults. in coscinasterias muricata the phenomenon is reproducible in the lab: two isolated arms, including a minimum portion of the original central disk, are able to regenerate two complete new starfishes following a process which is perfectly simultaneous in both uniparental comets and similar in terms of regeneration rate and histological aspects (ducati et al., 2004). this phenomenon is regulated by both exogenous (seasonal changes, environmental stimuli) and endogenous factors (body size, humoral and nervous factors). recent results in asterias rubens (thorndyke et al., 2001a) give a further insight in the promising field of the molecular approach to echinoderm regeneration, with particular reference to the characterization and implication of homeoboxcontaining genes (arhox1) and their spatial and temporal expression patterns during starfish arm regeneration. larvae ( all stages): regeneration of body parts following traumatic amputation; processes of larval cloning . in asteroid larvae, post-traumatic regeneration was explored in several past and recent studies (hörstadius, 1925a, b, c, 1973; vickery and mcclintock, 2000; vickery et al., 2001). in some species (luidia foliolata), more substantial regeneration can take place whereby two new, completely independent larvae can be produced following surgical bisection of the original (vickery and mcclintock, 1998). in planktonic asteroid larvae, there is also convincing evidence for regeneration in terms of true asexual reproduction and potential for clonal growth (bosch, 1988; vickery et al., 2001; eaves and palmer, 2003). planktotrophic bipinnaria larvae of some starfish species have been shown to undergo clonal reproduction whereby posterolateral arms transform into new larvae and are released by fission, leaving the arm stump to regenerate in its entirety (bosch et al. 1989). in optimized laboratory conditions, up to 24% bipinnaria larvae of pisaster ochraceus undergo asexual reproduction (vickery and mcclintock, 2000); in samples of field collected larvae of other species up to 90% cloning was recorded (eaves and palmer, 2003). ophiuroids adults: regeneration of body parts (arms) following selfinduced or traumatic amputation; regeneration of internal organs (pyloric caeca, cardiac stomach) following self-induced or traumatic mutilation; fission processes. the main cellular and molecular aspects of the regenerative processes are summarized in table 3. ophiuroids also show a great capacity for regeneration, since they frequently cast off their arms at all levels, with breakage occurring at all the autotomy planes after damage, predation, or handling. regeneration is usually rather rapid and can involve several arms. the cellular aspects of the regeneration process in ophiuroids are poorly understood. most recent work has focused on the ecological benefits and impacts of the extensive regeneration seen in many species (stancyk et al., 1994; thorndyke et al., 2001b). these animals are certainly ideal subjects for studying the implications of regrowth on other physiological processes. they are also valuable models for experiments concerning the cellular basis of regeneration: present evidence suggests that a combination of morphallactic and epimorphorphic processes is responsible for regeneration in ophiuroids. thus morphallactic wound-healing processes involving initial expansion and migration of epidermal cells are followed by the rapid formation of an epimorphic blastemal structure due to a local accumulation of coelomocytes (dobson, 1988, thorndyke et al., 2001b; bannister et al., 2005). several specific studies on cell cycle activity (dobson 1988; thorndyke et al., 2001b) clearly indicate the importance of cell division in this regenerative process. moreover, the primary location of the blastema at the end of the severed nerve cord provides clear support for the idea of an important role for the nervous system in regrowth, as previously suggested by early studies (zeleny, 1903; morgulis, 1909;). recent molecular studies in amphiura filiformis (bannister et al., 2005) has led to identify and clone afuni, a novel tgf-ß gene, expressed in regenerating tissues and showing a sequence similarity with sea urchin univin (85 % identity). on the basis of the differential spatiotemporal expression of this gene in regenerating arm of advanced stages, a presumptive role in arm growth and segmentation, as well as in neurogenesis and skeletogenesis was suggested. larvae (pre-metamorfic stages): regeneration of body parts following traumatic amputation; 71 table 3. ophiuroids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -epimorphic regeneration with main contribution of undifferentiated cells -blastema formation nerve-dependent regeneration -stem cells (coelomocytes) -dedifferentiated cells (? ) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -extensive proliferation -neural factors (neuropeptides s1, s2) afuni (novel tgf-ß gene cloned) table 4. holothuroids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -epimorphic-like regeneration with presumptive contribution of undifferentiated cells pseudo-blastema formation nerve-dependent regeneration -dedifferentiated cells (myocytes) stem cells (coelomocytes) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -limited local proliferation -neural factors (?) -ependimin-related protein (?) epenhg (ependymi n-related gene) processes of larval cloning (secondary larvae) from larval posterolateral arms detached from primary ophiopluteus. in ophiuroids, larval regeneration has been described in pre-metamorphic stages. the most frequent phenomenon is the regeneration of secondary larvae from the posterolateral arms which are lost or released on settlement of the metamorphosed juvenile (mortensen, 1921; balser, 1996, 1998; eaves and palmer, 2003), which can even involve the continuous and progressive development of serial clones of regenerating larvae (tertiary larvae, etc.) in a sort of strobilation process. the overall phenomenon appears to involve also pseudogastrulation morphogenetic events, whereby cell proliferation, de-differentiation, migration and differentiation take place to produce larval clones (balser, 1998). holothuroids adults: regeneration of body parts and appendages (tentacles) following self-induced or traumatic amputation; regeneration of whole visceral mass or individual internal organs (gut, gonads, haemal system, respiratory trees, cuvierian tubules) following self induced or traumatic amputation; asexual fission processes. the main cellular and molecular aspects of the regenerative processes are summarized in table 4. in holothurians the regenerative potential can differ a lot between different groups (dendrochirota, aspidochirota, apoda) and also vary with the age of the individuals. since the first half of the 20th century, much attention has been focused on visceral regeneration (gut and related structures, bertolini, 1930; dolmatov, 1992; garcia-arraras et al., 1998; 1999; garcia-arraras and greenberg, 2001) and on muscle regeneration (dolmatov et al., 1996). visceral regeneration. recent work (garcia-arraras et al., 1998, 2001; garcia-arraras and greenberg, 2001; dolmatov, 1992, 1996) has indicated that in holothuria glaberrima, following evisceration the new digestive tube develops from the mesentherial lamina, which anchored the original gut to the body wall. this phenomenon is apparently a typical epimorphic process, where a blastema-like structure is formed as a thickening of the mesentherial edge of the lamina. in the following stages the regrowth of the intestinal tract can imply two alternative mechanisms of cell recruitment (garcia-arraras and greenberg, 2001): a) from the remnants of the oesophagus and cloaca, through morphallactic mechanisms of tissue 72 rearrangement and migration/proliferation of endodermally derived cells (garcia-arraras et al., 1998; mashanov and dolmatov, 2001); b) from coelomic epithelium, through direct and exclusive proliferation/migration of new mesodermally derived progenitor cells, this phenomenon typically occurring when endodermally derived tissues are completely lost by evisceration (mosher, 1956; mashanov et al., 2004). on the basis of indirect evidence, the overall process is considered to be a nerve-dependent regeneration. the employment of immunocytochemical methods underpin specific activities in the mesentherial septum: at early stages, extensive ecm (extracellular matrix) degradation (collagen, fibronectin, laminin) and metalloproteinase (mmps) activity or effects of their inhibitors; at advanced stages, extensive rearrangement of the ecm (collagen, fibronectin, laminin) and myocyte dedifferentiation (quinones et al., 2002; garciaarraras et al., 2001). although more specific mechanisms at cellular level have still to be explored, particularly by developing appropriate methods for cell culture (odintsova et al., 2005), a plausible interpretation about elements involved and their possible roles has been proposed, with particular reference to the fundamental role of the mesothelium and the organogenetic potential of the coelomic epithelium, which also implies a presumptive mesodermal derivation of the luminal epithelium of the new gut. recent work was carried out on the molecular aspects of regeneration and has allowed identification of at least one gene, epenhg (ependymin-related gene), which is expressed in many tissues and overexpressed during regeneration. this gene shows a sequence similarity with holothurian, sea-urchin and vertebrate (frog and mammalian) ependiminrelated sequences. in terms of specific roles, its presumptive involvement as promotor of proliferation and differentiation, neurotrophic factor and inductor of axonal regrowth was proposed (suarez-castillo et al., 2004) muscle regeneration. a specific aspect of regeneration which is extremely interesting per se in terms of morphogenetic and histogenetic processes and particularly relevant for the functional recovery of the injured individual, is the regenerative development of the muscles. this problem has been explored in holothurians, the echinoderm group in which muscles are most developed, as far as myogenesis of somatic and visceral muscles is concerned (dolmatov et al., 1996; dolmatov and ginanova, 2001). here the regeneration process appears to be due to the coelomic epithelial cells which apparently dedifferentiate, migrate and invade the muscle bands where they differentiate into muscle bundle rudiments, finally giving rise to new muscle cells. the myogenesis appears to start from two different cell precursors: with regard to somatic muscles, myogenesis starts from undifferentiated coelomocytes, in their turn derived from epithelial cells of the coelothelium (perytoneocytes) via migration, proliferation, differentiation; with regard to visceral muscles, from myocytes of the coelothelium, via partial dedifferentiation/transdifferentiation and migration (dolmatov and ginanova, 2001; murray and garciaarraras, 2004). on the basis of the resulting pattern in terms of occurrence and distribution of dedifferentiation, migration, proliferation and redifferentiation at tissue and cellular level, a basic mechanism for holothurian muscle regeneration is proposed which excludes a significant role for cell division but is based fundamentally on the recycling and transdifferentiation of cells from the coelomic epithelium. larvae (different stages): regeneration of body parts following traumatic amputations; processes of larval cloning. larval regeneration after surgical transection was previously described in holothurians in earlier and recent papers (hörstadius, 1925a, b, c, 1973; dolmatov, 1991, 1996). in eupentacta fraudatrix, the pentactula juvenile stage, after bisection, can regenerate the posterior part including the intestinal tract (mashanov and dolmatov, 2001). besides these processes of post-traumatic regeneration, frequent phenomena of larval cloning have been also observed (eaves and palmer, 2003), particularly development of secondary auricularia larvae (parastichopus californicus). in addition, asexual budding is also frequently observed in early pentactulae. echinoids adult: regeneration of external appendages (spines, pedicellariae) post-fracture, removal or self-induced release; regeneration of test after surgical treatment or traumatic mutilations. the main cellular and molecular aspects of the regenerative processes are summarized in table 5. echinoids show an apparent reduced requirement for regeneration as they do not reproduce by fission and do not show large exposed appendages vulnerable to predation. nevertheless, if arm regeneration is a typical feature of long-armed echinoderms, traumatic or self-induced loss and subsequent regeneration of external appendages, such as spines and pedicellariae, are frequently occurring in sea urchins and recent studies have concentrated on regeneration of spines and pedicellariae in the common sea urchin paracentrotus lividus (carpenter, 1847; dubois and ameye, 2001) after fracture or total removal. interestingly, a pattern emerges from the detailed comparative analysis of diverse situations, which suggests that the regenerative processes in these external appendages can vary significantly in terms of basic mechanisms and processes and can be modified in response to different types of injury. these results are strongly consistent with the idea of a fundamentally different significance of post-traumatic and post-autotomic regeneration and confirm the wide plasticity/adaptability of the repair and regenerative processes in echinoderms in relation to the specific conditions of local damage. 73 table 5. echinoids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -morphallactic processes with substantial contribution of migratory cells -annular blastema, centripetal growth -dedifferentiated cells -stem cells (coelomocytes?) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -limited local proliferation only a few reports have been focused on test regeneration after injury (bonasoro et al., 2004; ameye and dubois, 1995; shimizu et al., 1994) and the process has still to be explored in its detail. in terms of timing and modalities, sea-urchin test regeneration appears to vary a lot, depending on many intrinsic and extrinsic factors. nevertheless, the main events tend to follow a constant sequence: in paracentrotus, after surgical removal of a small test portion (one or two skeletal plates), the experimental animals are able to repair their wounds by employing regenerative mechanisms which involve three main stages (repair, early and advanced regeneration) and lead to the complete closure of the wound. the overall regeneration process implies the formation of an annular blastema and follows a concentric centripetal regrowth (bonasoro et al., 2004): the new tissues are progressively reformed due to the substantial contribution of migratory cells presumably derived through a morphallactic rearrangement of old tissues, with only a minor local employment of cell proliferation. larvae: regeneration of body parts after surgical division or amputation; larval cloning. the regenerative potential of sea-urchin larvae after surgical or traumatic amputation was described in historical and recent papers (runnström, 1915; 1925; hörstadius, 1925a, b, c, 1973; vickery et al., 1999, 2001). in lytechinus variegates, after bisection, different larval stages, particularly echinoplutei, can completely regenerate the anterior or posterior body portion (vickery et al., 2001). in addition, in different larval stages frequent phenomena of larval cloning by budding have been also described (for instance in strongylocentrotus purpuratus, eaves and palmer, 2003), leading also in echinoids to the regular formation of secondary larvae. concluding remarks the extensive and strategic employment of regenerative phenomena throughout the phylum indicates that in echinoderms regeneration actually represents an essential component of the life-cycle and has a wide range of relevant biological implications: not only it increases the potential of survival of the individuals, but it can also be considered the indispensable requisite for performing fissiparous reproduction, thus allowing the rapid colonization of new environments through the production of multiple copies of genotypes (clones) well adapted to local conditions. on the basis of its evident contribution to the adaptive capacities of the species and of its biological and ecological implications for echinoderm phylogeny the regeneration phenomenon must be considered one of the most important responsible factor for the evolutionary success of the groups and for the striking success of the whole echinoderm phylum throughout the marine ecosystem. unfortunately what we actually know about the biology of echinoderm regeneration is far to be exhaustive. much work is requested and many questions are still to be answered. results obtained so far strongly encourage a wider employment of molecular approaches in regeneration research (odelberg, 2004) and promote the hope that, with the powerful tools provided by molecular biology, the key of the striking regenerative performances of echinoderms can be soon appropriately studied and completely explained. we should not forget that what we learn from echinoderms can be relevant in applied research: regenerative medicine can in fact receive a significant improvement if echinoderm models are extensively studied in parallel with traditional mammal models, in the reasonable hope that what echinoderms can do so easily may eventually become easy also for other animals, humans included (lagasse et al., 2001). acknowledgements this work was carried out thanks to the support of a number of specific projects financed through the years by cnr, murst (cofin) and university of milano (first) programs. the author is particularly grateful to the following colleagues for their indispensable help and valuable collaboration during the research work: a barbaglio, a biressi, f bonasoro, c di benedetto, d mozzi, l parma, m patruno and m sugni. 74 references ameye l, dubois p. resorption and calcification during regeneration of echinoid test. in: emson rh, smith ab, campbell ac (eds), echinoderm research 1995, rotterdam, balkema, pp 231-235, 1995. amemiya s, oji t. regeneration in sea lilies. nature 357: 546-547, 1992. balser ej.mortensen vs mcbride: evidence for asexual reproduction in ophiuroid larvae. am.zool. 36: 70a, 1996 balser ej. cloning by ophiuroid echinoderm larvae. biol. bull. 194: 187-193, 1998. bannister r, mcgonnell im, graham a, thorndyke mc, beesley pw. afuni, a novel transforming growth factorbeta gene is involved in arm regeneration by the brittle star amphiura filiformis. dev. genes. evol. 215: 393401, 2005. bertolini f. rigenerazione dell’apparato digerente nello stichopus regalis. pubb. staz. zool. napoli 11: 439-448, 1930 bonasoro f, candia carnevali md, moss c, thorndyke mc. epimorphic versus morphallactic mechanisms in arm regeneration of crinoids and asteroids: pattern of cell proliferation/differentiation and cell lineage. in: mooi r, telford m (eds), echinoderms, san francisco, balkema, rotterdam, pp 13-18, 1998. bonasoro f, candia carnevali md, sala f, patruno m, thorndyke mc. regenerative potential of crinoid arm explants. in: candia carnevali md, bonasoro f (eds), echinoderm research 1998, balkema, rotterdam, pp 133-138, 1999. bonasoro f, ferro p, di benedetto c, sugni m, mozzi d, candia carnevali md. regenerative potential of echinoid test. in: heinzeller t & nebelsick jh (eds), echinoderms: munchen, taylor & francis group, london, pp 97-103, 2004 bosch i, replication by budding in natural populations of bipinnaria larvae of the sea star genu luidia. in: burke rd, mladenov pv, lambert p, parsley rl (eds), echinoderm biology, rotterdam, balkema, pp 789, 1988. bosch i, rivkin rb, alexander sp. asexual reproduction by oceanic planktotrophic echinoderm larvae. nature 337: 169-170, 1989 candia carnevali md. regenerative response and endocrine disrupters in crinoid echinoderms: an old experimental model, a new ecotoxicological test. in: matranga v (ed), echinodermata. progress in molecular and subcellular biology (marine molecular biotechnology), springerverlag, heidelberg, vol.39, pp 167-198, 2005. candia carnevali md, bonasoro f. introduction to the biology of regeneration in echinoderms. microsc. res. tech. 55: 365-368, 2001a candia carnevali md, bonasoro f. a microscopic overview of crinoid regeneration. microsc. res. tech. 55: 403426, 2001b candia carnevali md, bonasoro f, biale a. pattern of bromodeoxyuridine incorporation in the advanced stages of arm regeneration in the feather star antedon mediterranea. cell tissue res. 289: 363-374, 1997. candia carnevali md, bonasoro f, invernizzi r, lucca e, welsch u, thorndyke mc. tissue distribution of monoamine neurotransmitters in normal and regenerating arrns of the feather star antedon mediterranea. cell tissue res. 285: 341-352, 1996. candia carnevali md, bonasoro f, lucca e, thorndyke mc. pattern of cell proliferation in the early stages of arm regeneration in the feather star antedon mediterranea. j. exp. zool. 272: 464-474, 1995. candia carnevali md, bonasoro f, patruno m, thorndyke mc. cellular and molecular mechanisms of arm regeneration in crinoid echinoderms: the potential of arm explants. dev. genes evol. 208: 421-430, 1998. candia carnevali md, bruno l, denis donini s, melone g. regeneration and morphogenesis in the feather star arm. in: kiortsis v, koussoulakos s, wallace h (eds), recent trends in regeneration research. nato asi series, ser a, vol 172, plenum press, new york, london, pp 447-460, 1989. candia carnevali md, lucca e, bonasoro f. mechanism of arm regeneration in the feather star antedon mediterranea: healing of wound and early stages of development. j. exp. zool. 267: 299-317, 1993. carlson bm. development and regeneration, with special emphasis on the amphibian limb. in: ferretti p, géraudie j (eds), cellular and molecular basis of regeneration: from invertebrates to humans, john wiley and sons, chichester, new york, pp 45-61, 1998. carpenter wb. report on the microscopic structure of shells. br. assoc. advan. sci. lond. rept.17 (pt.ii): 93-143, 1847. clark ah. a monograph of the existing crinoids. vol 1. the comatulids. part 1. bull. u.s. natn. mus. 82: 1-389, 1921. dendy a. on the regeneration of the visceral mass in antedon rosaceus. stud. biol. lab. owens college, manchester, vol i, 299, 1886. dobson we. early post-autotomy tissue regeneration and nutrient traslocation in the brittlestar microphiopolis gracillima (stimpson) (echinodermata: ophiuroidea). ph.d. thesis, university of south carolina, usa, 1988. dolmatov iy. regeneration of digestive tract in the pentactulae of the far-eastern holothurian eupenctacta fraudatrix (holothuroidea, dendrochirota). inv. repr. dev. 39: 143-151, 1991. dolmatov iy. regeneration of the aquapharingeal complex in the holothurian eupenctacta fraudatrix (holothuroidea, dendrochirota). in: taban ch, boilly b (eds) keys for regeneration. monogr. dev. biol. 23: 40-50, 1992. dolmatov iy. asexual reproduction, evisceration and regeneration in holothurians. russ. j. dev. biol. 27: 211-219, 1996. dolmatov i, bonasoro f, ferreri p, candia carnevali md. visceral regeneration in the crinoid antedon mediterranea. feral jp (ed), echinoderm research 2001, balkema, rotterdam, pp 215-220, 2003. dolmatov iy, eliseikina mg, bulgakov aa, ginanova tt, lamash ne, korcaghin vp. muscle regeneration in the holothurian stichopus japonicus. roux’s arch.dev.biol. 205: 486-493, 1996. dolmatov iy, ginanova tt. muscle regeneration in holothurians. microsc. res. tech. 55: 452-463, 2001. dubois p, ameye l. regeneration of spines and pedicellariae in echinoderms: a review. micr. res. tech. 55: 427-437, 2001. ducati cc, barker mf, candia carnevali md. regenerative potential and fissiparity in the forcipulate starfish coscinasterias muricata.in: echinoderms: münchen (heinzeller t & nebelsick jh (eds), taylor & francis group, london, pp113-118, 2004. eaves aa, palmer ar. widespread cloning in echinoderm larvae. nature 425: 146, 2003. emson rh, wilkie ic. fission and autotomy in echinoderms. oceanogr. mar. biol. ann. rev. 18: 155-250, 1980. ferretti p, géraudieu j. preface. in: ferretti p, géraudie j (eds), cellular and molecular basis of regeneration: from invertebrates to humans, john wiley and sons, chichester, new york, xix-xx, 1997. garcia-arraras je, diaz-miranda l, torres-vasquez i, file s, jimenez l, rivera-bermudez k, arroyo e, cruz w. regeneration of the enteric nervous system in the seacucumber holothuria glaberrima. j. comp. neurol. 406: 461-475, 1999 garcia-arraras je, estrada-rodgers l, santiago r, torres ii, diaz-miranda l, torres-avillan i cellular mechanisms of intestine regeneration in sea-cucumber, holothuria glaberrima selenka (holothuroidea: echinodermata). j. exp. zool. 281: 288-304, 1998. 75 garcia-arraras je, rojas-soto m, jimenez lb, diaz-miranda l. the enteric nervous system of echinoderms : unexpected complexity revealed by neurochemical analysis. j. exp. biol. 204: 865-873, 2001. garcia-arraras je, greenberg mj. visceral regeneration in holothurians. micr. res. tech. 55: 438-451, 2001 goss rj. mammalian regeneration and its phylogenetic relationships. in: kiortsis v, trampush hal (eds), regeneration in animals and related problems, northholland publishing company, amsterdam, pp 33-38, 1965. goss rj. principles of regeneration, academic press, new york and london, 1969. goss rj. regeneralities. in: inoue set al (eds), regeneration and development, okada printing and publishing company, maebashi, japan, pp 3-12, 1988. goss rj. the evolution of regeneration: adaptive or inherent? j. theor. biol. 159: 241-260, 1992. hörstadius s. entwicklungsmechanische studien an asterina gibbosa forbes. ark. zool. b17, 6: 1-6, 1925a. hörstadius s. entwicklungsmechanische studien an astropecten aurantiacus l. ark. zool. b17, 7: 1-6, 1925b. hörstadius s. entwicklungsmechanische studien an holothuria poli dalle chiaje. ark. zool. b17, 8 :1-6, 1925c. hörstadius s. experimental embriology of echinoderms. oxford: clarendon press, 1973. huet m. le róle du système nerveux au cours de la régénératíon du bras chez une étoile de mer, asterina gibbosa penn (echinoderme, astéride). j. embryol. exp. morph. 33: 535-552, 1975. huet m, franquinet r. histofluorescence study and biochemical assay of catecholamines (dopamine and noradrenaline) during the course of arm-tip regeneration in the starfish asterina gibbosa (echinodermata, asteroidea). histochemistry 72: 149-154, 1981. hyman hl. the invertebrates: echinodermata. mcgraw-hill, new york, toronto, london, vol iv, p 763, 1955. lagasse e, shizuru ja, uchida n, tsukamoto a, weissman il. toward regenerative medicine. immunity 14: 425436, 2001. mashanov vs, dolmatov iy. ultrastructural peculiarities of gut regeneration in 5month old pentactulae of a holothurian eupentacta fraudatrix (holothuroidea, dendrochirota) after artificial transverse bisection. russian j. mar. biol. 39: 143-151, 2001. mashanov vs, dolmatov iy. functional morphology of the developing alimentary canal in the holothurian eupentacta fraudatrix (holothuroidea, dendrochirota), acta zool. 85: 29-39, 2004. minckert w. über regeneration bei comatuliden nebst ausführungen über die auffassung und bedeutung der syzygieen. arch. f. naturg. jahrg. 71: 163-244, 1905. mladenov pv, burke rd. echinodermata: asexual propagation. in: adiyodi kg, adyodi rg (eds) reproductive biology of invertebrates, vol vi part b. asexual propagation and reproductive strategies. new delhi: oxford and hill, pp 339-383, 1994. mladenov pv, bisgrove b, asotra s, burke rd. mechanisms of arm-tip regeneration in the sea star, leptasterias hexactis. roux’s arch. dev. biol. 189: 19-28, 1989. morgan th. regeneration. macmillan, new york, 1901. morgulis s. regeneration in the brittlestar ophiocoma pumilia, with reference to the influence of the nervous system. proc. am. acad. arts & sci., 44: 655-659, 1909 mortensen t. studies on the development and larval forms of echinoderms. copenhagen: gec gad,1921. mosher c. observations on evisceration and visceral regeneration in the sea-cucumber, actinopyga agassizi selenka. zoologica 41: 17-26, 1956. moss c, hunter j, thorndyke mc. pattern of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish asterias rubens. phil. trans. r. soc., london b, 353: 421-436, 1998. mozzi d, ferreri p, petrillo p, dolmatov iy, bonasoro f, candia carnevali md. graft and regeneration of viscera in the crinoid antedon mediterranea. in: echinoderms: münchen (heinzeller & nebelsick eds), taylor & francis group, london, pp135-139, 2004 murray g, garcia-arraras je. myogenesis during holothurian intestinal regeneration. cell tissue res. 318: 515-24, 2004. nakano h, hibino t, hara y, oji t, amemiya s. regrowth of the stalk of the sea lily, metacrinus rotundus (echinodermata: crinoidea). exp. zoolog. 301 a: 464471, 2004. odelberg sj. unraveling the molecular basis for regenerative cellular plasticity. plos biol. 2: e232, 2004. odintsova na, dolmatov iyu, mashanov vs. regenerating holothurian tissues as a source of cells for long-term cell cultures. mar. biol. 146: 915-921, 2005. oji t. fossil records of echinoderm regeneration with special regard to crinoids. micr. res. tech. 55: 397-402, 2001 patruno m, mcgonnell im, graham a, beesley p, candia carnevali md, thorndyke m. anbmp2/4 is a new member of the tgf-b superfamily isolated from a crinoid and involved in regeneration. p. roy. soc. lond. b. 270: 1341-1347, 2003 patruno m, smertenko a, candia carnevali md, bonasoro f, beesley pw, thorndyke mc. expression of tgf-b-like molecules in normal and regenerating arms of the crinoid antedon mediterranea: immunocytochemical and biochemical evidence. proc. r. soc. lond. b 269: 17411747, 2002. perrier e. l'anatomie et la régénération des bras de la comatula. arch. zool. exp. gen. 2: 29-86, 1873. przibram h. experimentelle studien über regeneration. arch. f. entwm. mech. bd. xi, pp 321-345, 1901. quinones jl, rosa r, ruiz dl, garcia-arraras je. extracellular matrix remodeling and metalloproteinase involvement during intestine regeneration in the sea cucumber holothuria glaberrima. dev. biol. 250: 181197, 2002. reichensperger a. beiträge zur histologie und zum verlauf der regeneration bei crinoiden. ztschr. wiss. zool. 101: 1-69, 1912. reik w, dean w. back to the beginning. nature 420: 127, 2002. runnström j. analytische studien über die seeigelentwicklung.ii. arch. entwmec. org. 41:1-56, 1915. runnström j. zur experimentaellen analyse der entwiglung von antedon. w. roux arch. entw.mech.org. 105: 63113, 1925. shimizu m, ishii n, watambe y. protein, calcium levels and acid phosphatase activity in regenerative test tissues of the sea urchin, strongylocentrotus intermedius. comp. biochem. physiol. 109a: 721-726, 1994. spallanzani l. prodromo di un opera da imprimersi sopra le riproduzioni animali. montanari ed, modena, 1768. stancyk se, golde hm, pape-lindstrom pa, dobson we. borne to lose. i. measures of tissue loss and regeneration by the brittlestar microphiopholis gracillima (echinodermata: ophiuroidea). mar.biol.118: 451-462, 1994. suárez-castillo ec, medina-ortíz we, roig-lópez jl, garcía-arrarás je. ependymin, a gene involved in regeneration and neuroplasticity in vertebrates, is overexpressed during regeneration in the echinoderm holothuria glaberrima. gene 334: 133-43, 2004. thorndyke mc, candia carnevali md. regeneration neurohormones and growth factors in echinoderms. can. j. zool. 79: 1171-1208, 2001. thorndyke mc, chen wc, moss c, candia carnevali md, bonasoro f. regeneration in echinoderms: cellular and molecular aspects. in: candia carnevali md, bonasoro 76 f (eds), echinoderm research 1998, balkema, rotterdam, pp 159-164, 1999. thorndyke mc, wc chen, beesley pw, patruno m. molecular approach to echinoderm regeneration. micr. res. tech. 55: 474-485, 2001a. thorndyke mc, patruno m, moss c, beesley pw. cellular and molecular bases of arm regeneration in brittlestars. in: barker m (ed), echinoderms 2000: new zealand. balkema, rotterdam, pp 323-326, 2001b. thouveny y, tassava ra. regeneration through phylogenesis. in: ferretti p, géraudie j (eds), cellular and molecular basis of regeneration: from invertebrates to humans. john wiley and sons, chichester, new york, pp 9-43, 1997. vickery mcl, mcclintock jb. regeneration in metazoan larvae. nature 394: 140, 1998. vickery mcl, mcclintock jb. effects of food concentration and availability on the incidence of cloning in planktotrophic larvae of the sea star pisaster ochraceus. biol. bull. 199: 298-304, 2000. vickery mcl, vickery ms, amsler cd, mcclintock jb. regeneration in echinoderm larvae. micr. res. tech. 55: 464-473, 2001. vickery mcl, vickery ms, mcclintock jb. regeneration in echinoid larvae. am.zool. 39: 51a, 1999. wadman m. scar prevention: the healing touch. nature 436: 1079-1080, 2005. wallace h. vertebrate limb regeneration. john wiley and sons, chichester, new york, brisbane, toronto, 1981. weissman il. stem cells: units of development, units of regeneration, ancd units in evolution. cell 100: 157168, 2000. wilkie ic. mutable collagenous tissue: overview and biotechnological perspective. in: matranga v (ed) echinodermata. progress in molecular and subcellular biology (marine molecular biotechnology), springerverlag, heidelberg, vol.39, pp 221-250, 2005. wilkie ic. autotomy as a prelude to regeneration in echinoderms. micr. res. tech. 55: 369-396, 2001. zeleny c. a study of the rates of regeneration of the arms in the brittlestars ophioglypha lacertosa. biol. bull. 6: 1217, 1903. djslk is required for the survival of planarian dugesia japonica 1 isj 14: 1-8, 2017 issn 1824-307x research report rnai of cns-expressed gene djslk induces morphogenetic malformation and death in planarian dugesia japonica x chen1,2, h zhen1, s wu1, q lu1, q pang1, b zhao1 1laboratory of developmental and evolutionary biology, school of life sciences, shandong university of technology, zibo 255049, p.r. china 2translational medicine center, the sixth people′s hospital of zhengzhou, zhengzhou 450000, p. r. china accepted december 13, 2016 abstract ste20-like kinases are critically multifuctional proteins which play important roles in varieties of celluar processes and physiological events. here, we characterized a ste20-like kinase gene (djslk) in planarian dugesia japonica. whole-mount in situ hybridizations revealed that djslk was expressed in the central nervous system (cns) including cephalic ganglia and ventral nerve cords (vncs) in intact and regenerating animals. after rna interference (rnai) of djslk, adult planarians became immobilized and wrinkled, then swelled and lysed eventually. djslk rnai treated regenerating planarians could form the entire animals, and then displayed the similar phenotype transformation. these results suggest that loss of function of djslk leads to morphogenetic malformation of planarian d. japonica probably via regulating cell volume instead of disrupting the balance between cell proliferation and apoptosis. key words: cns; djslk; expression; morphogenetic malformation; cell volume introduction endowed with abundant pluripotent stem cells named neoblasts which proliferate and differentiate to all cell types in response to amputation and/or injury ， planarians can regenerate the complete worms with all organs from almost any tiny fragments (newmark and sánchez alvarado, 2002; reddien and sánchez alvarado, 2004). neoblast is the only source to provide new cells during turnover and regeneration (wagner de et al., 2011). elimination of neoblasts by irradiation, planarians demonstrate the typical phenotype defects such as head regression, dorsal curling and lesions during homeostasis and fail to shape the whole animals for amputated pieces (newmark and sánchez alvarado, 2002; reddien and sánchez alvarado, 2004; saló et al., 2009; scimone et al., 2010). planarians can acquire similar phenotypes by silencing some genes related to neoblast maintenance, proliferation, differentiation, and apoptosis (reddien et al., 2005; guo et al., 2006; pearson and sánchez alvarado, 2010; li et al., 2011). ___________________________________________________________________________ corresponding author: bosheng zhao school of life sciences shandong university of technology 266 xincun w estern road, zibo 255049, p.r. china e-mail: zhaobosheng@sdut.edu.cn sterile 20 (ste20) is originally found as mitogen-activated protein kinase kinase kinase kinase (map4k) involved in the mating response of haploid yeast saccharomyces cerevisiae (wu et al., 1995). its homologs in other organisms form the ste20-like kinase (slk) superfamily and are mainly regarded as upstream regulators of the mapk pathways (dan et al., 2001; ling et al., 2008). ste20-like kinases play crucial roles in various cellular processes including cell growth, cell migration, apoptosis, cell-cycle control, cell shape change and stress responses (dan et al., 2001; strange et al., 2005; ling et al., 2008). in this paper, we identified a slk gene in planarian dugesia japonica and studied its tempospacial expression pattern and loss-of-function phenotype in intact and regenerating animals. materials and methods animals the planarians dugesia japonica collected in boshan, shandong, china, are maintained in autoclaved tap water at 20 ℃ and 6 10 mm long animals were starved for at least one week before use in experiments. cloning and sequence analysis of cdna the djslk cdna was derived from random mailto:zhaobosheng@sdut.edu.cn 2 sequencing of a planarian cdna library. comparison against the genbank protein database was performed using the blast network server at the national center for biotechnology information (ncbi). multiple protein sequences were aligned using the megalign program by the clustal method in dnastar software package (burland, 2000). whole-mount in situ hybridization whole-mount in situ hybridizations were performed as described previously (pearson et al., 2009). the digoxigenin (dig)-labelled antisense rna probes were synthesized in vitro. hybridizations were carried out at 56 ℃ for 17 h, the bcip/nbt mixture solution (roche) was used for color development. for regeneration experiments, animals were amputated preand post-pharyngeally and left to regenerate, the head-, trunk-, and tail-pieces were collected at the times indicated. quantitative real-time pcr quantitative real-time pcr was used to monitor the quantitative expression of the djslk as described previously (yu et al., 2015) in intact planarians, regenerating trunk fragments， and regenerating trunk fragments of rnai-treated planarians at different times after amputation. the cdna was synthesized using a first-strand system kit from invitrogen after total rnas were extracted using rnaiso reagent (takara). qpcr reactions were performed using fast start universal sybr green master (rox) (roche, switzerland) according to the manufacturer’s protocol. three samples for each condition were run in parallel by a 7,500 real time pcr system (applied biosystems). data were normalized to the expression of the internal control djef2. the following sets of specific primers were used: djslk mrna, 5′-cgaaggacaaaggcacat-3′, 5′-gagcgaacaccaggaact-3′. djef2 mrna, 5 ′ t t a a t g a t g g g a a g a t a tg t t g 3 ′; 5 ′ g t a c c a t a g g a t c t g a t t t t g c 3 ′. the data were analyzed using spss 16.0 software. the significance of differences was analyzed by one-way analysis of variance (anova) followed by a tukey's post-hoc analysis to identify differences between the experimental and intact planarians. data presented are means ± sd. values of p < 0.05 were considered to be significant. rna interference djslk was cloned into the l1440 plasmid with two t7 primers, and then dsrna was synthesized according to the manufactrue’s instructions (megascript® rnai kit). animals were injected at day 0, day 2, 4, and 6. for regeneration studies, animals were cut at day 10. control animals were injected with deionized sterile water. immunostaining animals were killed in 2 % hcl for 5 min at rt and fixed in 4 % paraformaldehyde solution for 3 h at 4 °c, then dehydrated in 100 % methanol solution for 1 h at -20 ℃. the following procedures were processed as described elsewhere (robb and sánchez alvarado, 2002; inoue et al., 2007; cebria, 2007, 2008). the primary antibody anti-synorf1, a mouse monoclonal antibody specific for synapsin (developmental studies hybridoma bank) was used at a dilution of 1:25. the secondary antibody dylight 594 affinipure goat anti-mouse igg(h+l) (earthox) was used at a dilution of 1:200. results sequence analysis of djslk the cdna clone obtained from planarian d. japonica cdna library is about 1,100 bp with the longest open reading frame of 879 bp. it encodes for a deduced protein of 292 amino acids with predicted molecular mass of approximately 32.4 kda (fig. 1) fig. 1 the nucleotide and deduced amino acid sequence of djslk. 3 fig. 2 alignment of subdomain xi of ste20-like kinases, including djslk using the megalign program (dnastar) by the clustal w method. shaded (with solid black) residues are the amino acids that match the consensus. genbank accession numbers: aplysia californica (xp_005111485.1), ailuropoda melanoleuca (xp_002914907.1), crassostrea gigas (ekc21462.1), canis lupus (xp_003433112.1), clonorchis sinensis (gaa52112.1), drosophila melanogaster (nm_142339.2), danio rerio (xp_005165982.1), felis catus (xp_003980538.1), latimeria chalumnae (xp_005992350.1), loa loa (ejd76726.1), schistosoma japonicum (cax753595.1), salmo salar (aci33699.1). initial blastp search at ncbi revealed that this gene belongs to ste20-like kinase. however, its 5′ end is missed and only subdomain xi is entire (hanks and hunter, 1995). the deduced amino acids aligned with other subdomain xi of ste20-like kinases showed that djslk shares 39.2 % 72.2 % similarity with its homlogs in other organisms (fig. 2). ste20 kinases consist of the p21-activated kinase (pak) and germinal center kinase (gck) families according to the relative location of kinase domain, these two families can be further subdivided into pak i and pak ii and gck i to viii subfamilies, respectively (dan et al., 2001; strange et al., 2005; ling et al., 2008). due to loss of most subdomains, the closest homlogs can not be ascertained and the gene is termed ste20 like kinase (djslk) in this study. djslk expression pattern in adult and regenerating planarians in order to analyse the expression pattern of the planarian djslk gene we performed whole mount in situ hybridization on intact and regenerating animals. in intact planarians, djslk was expressed in central nervous system which possesses an inverted u-shaped pair of cephalic ganglia and two longitudinal ventral nerve cords that project posteriorly along the worm (cebrià et al., 2002; cebrià, 2007; agata and umesono, 2008) (fig. 3b). and djslk localized in both the central spongy region and the lateral branches in the cephalic ganglia (fig. 3b). in regenerating animals, djslk transcripts could always be detected in cns in the head-, trunk-, and tail-pieces. during the initial regeneration stages after amputation, djslk expression was not detected fig. 3 expression of djslk in intact planarian (a) an intact planarian processed and hybridized using the djslk sense probe. no signal was seen in the control. (b) ventral view of intact planarian, expression of djslk is mainly present in the cns. anterior is to the left. scale bar: 500 μm. 4 fig. 4 expression of djslk during regeneration. expression of djslk in regeneration of day 1 (a c), day 3 (d f), day 5 (g i), day 7 (j l), and day 9 (m o) after amputation. djslk is detected in the pre-exiting and newly regenerated cns. anterior is to the left. scale bar: 300 μm. within the head and tail blastema, but it was detected in the preexisting cns (figs 4a c). at 3 and 5 days of regeneration, new neural cells in front of the commissure differentiated, and cns recovered most of its function (cebria, 2007). djslk expression was detected in the preexisting and newly regenerated cns (figs 4d i). with the development of regeneration, the original expression was gradually reestablished (figs 4j o). the relative quantitative real-time pcr analysis was performed to investigate the change of expression of djslk mrna during planarian regeneration. we examined rna samples from normal intact planarians and trunk fragments regenerated for 1 day, 5 fig. 5 qrt-pcr analysis of djslk expression in intact and regenerating truck fragments at 1, 3, 5, 7, 9 days after amputation. data was expressed as the ratio of djslk to djef2α mrna. error bars represent the  sd for three independent pcr amplifications and quantifications. *p < 0.05 or **p < 0.01 compared to control intact planarians. fig. 6 qrt-pcr analysis of djslk rnai efficiency in adult intact planarians. error bars represent the  sd for three independent pcr amplifications and quantifications. **p < 0.01 is the comparison between control intact animals and rnai intact animals. template 6 fig. 7 abnormal appearance change in intact (b-e) and regenerating planarians (b′-e′) after djslk rnai. (a) control animal, microinjection of water in adult animal after 2 months. (b-e) after rnai, planarians became immobilized and wrinkled (b), swelled (c), lysed (d), and died (e). (a′) contral animal, day 30 of regeneration after amputation. (b′-e′) the appearance transformation in regenerating trunk fragments after rnai-treated planarians. anterior is to the top. scale bar: a e = 800 μm, a′e′ = 200 μm. 3 days, 5 days, 7 days, and 9 days, respectively. the resluts indicated that djslk mrna was gradually increased during regeneration compared to normal intact planarians and achieved to the maximal leval at 7 days (p < 0.01) after amputation. then it declined to almost normal level at regeneration day 9 (fig. 5). djslk rnai induces morphogenetic defects and death of planarians to study the role of djslk gene in the homeostasis and regeneration of the planarian, we knocked down the endogenous expression of djslk using rnai. real-time pcr analysis of djslk mrna showed that the rnai-treatment efficiently down-regulated the expression of djslk in intact planarians ( p< 0.01) (fig. 6). after 5 days of injecting djslk dsrna, the intact animals became immobilized and wrinkled (fig. 7b, n = 6/13). then the planarians swelled and started to lyse at day 9 and day 15, respectively (figs 7c and d, n = 6/13). and the animals completely lysed at about 25 days after the treatment (fig. 7e, n = 6/13). in contrast, control animals lived without any abnormal change even for 2 months (fig. 7a, n = 10/10). djslk rnai treated trunk fragments could regenerate the whole bodies, and then showed the same appearance change starting at 14 days after amputation (figs 7b’ e’). immunostaining with an anti-synorf1 antibody against synapsin revealed that djslk rnai didn′t interfere with cns intactness and regeneration (fig. 8). discussion ste20 kinases function in morphological events in different organisms (strange et al., 2005). in yeast mating pathway, cell shrinkage activates ste20 kinase and suppresses mating defects (strange et al., 2005). one ste20 kinase named proline-alanine-rich ste20-related kinase (pask) is strongly expressed in neurons and transporting epithelia in rats (ushiro et al., 1998). pask can interact with and phosphorylate na-k-2cl (nkcc) and k-cl (kcc) cotransporters to regulate cell volume (piechotta et al., 2002; dowd and forbush, 2003; strange et al., 2005). during cell swelling, loss of kinase activity of pask results in dephosphorylation of both cotransporters which lead to inhibit nkcc and activate kcc (strange et al., 2005). in this study, djslk was expressed in cns and rnai enventully induced swelling and lysing of planarians. and the defects probably resulted from cell swelling and osmotic lysis after loss of kinase 7 fig. 8 immunostaining with anti-synorf1 in djslk-rnai-treated planarians during homeostasis (a b and a' b') and regeneration (c d and c' d'), the defects of cns including brain and vnc were not detected. (a) intact planarian was injected with water as control. (b) djslk-rnai-treated intact planarian at 9 days. (c) immunostaining of normally regeneration of the cutting head sample was detected at 15 days after amputation. (d) djslk-rnai-treated intact planarian was cut head to regenerate at 15 days. a ′d′ the magnification of head in a d, respectively. anterior is to the up. scale bars: a d = 500 μm; a′ d′ = 100 μm. activity of djslk. meanwhile, the irradiation-treated typical phenotype change did not occur in intact planarians, and amputated fragments could regenerate the whole bodies after silencing djslk. all these results suggest that, just like pask, djslk regulates cell volume instead of involving in neoblast maintenance, proliferation, differentiation, and apoptosis like other neoblast-related genes. acknowledgments we thank dr. yuqi huo from translational medicine center, the sixth people′s hospital of zhengzhou, for reading and discussing the manuscript. this work was supported by the national natural science foundation of china (grant numbers 31572263, 31172074) and shandong province natural science foundation of china (grant number zr2013cm011). references agata k, umesono y. brain regeneration from pluripotent stem cells in planarian. phil trans r soc b. 363: 2071-2078, 2008. burland tg. dnastar's lasergene sequence analysis software. methods mol. biol. 132: 71-91, 2000. cebria f. regenerating the central nervous system: how easy for planarians! dev. genes evol. 217: 733-748, 2007. cebria f. organization of the nervous system in the model planarian schmidtea mediterranea: an immunocytochemical study. neurosci. res. 61: 375-384, 2008. cebria f, nakazawa m, mineta k, ikeo k, gojobori t, agata k. dissecting planarian central nervous system regeneration by the expression of neural-specific genes. dev. growth differ. 44: 135-146, 2002. dan i, watanabe nm, kusumi a. the ste20 group kinases as regulators of map kinase cascades. trends cell biol. 11: 220-230, 2001. dowd bfx, forbush b. pask (proline-alanine-rich ste20-related kinase), a regulatory kinase of the na-k-cl cotransporter (nkcc1). j. biol. chem. 278: 27347-27353, 2003. guo t, peters hfm, newmark pa. a bruno-like gene is required for stem cell maintenance in planarians. dev. cell 11: 159-169, 2006. hanks sk, hunter t. protein kinase 6. the eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. faseb j. 9:576-596, 1995. 8 li y, zeng a, han x, wang c, li g, zhang z et al.argonaute-2 regulates the proliferation of adult stem cells in planarian. cell res.151: 1-5, 2011. ling p, lu t, yuan c, lai m. biosignaling of mammanlian ste20-related kinases. cell signal. 20: 1237-1247, 2008. newmark pa, sánchez alvarado a. not your father’s planarian: a classic model enters the era of functional genomics. nat. rev. genet. 3: 210-219, 2002. pearson bj, sánchez alvarado a. a planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages. development 137: 213-221, 2010. pang q, liu x, zhao b, jiang y, su f, zhang x, et al. detection and characterization of phenoloxidase in the freshwater planarian dugesia japonica. comp. biochem. physiol. 157b: 54-58, 2010. pearson bj, eisenhoffer gt, gurley ka, rink jc, miller de, sánchez alvarado a. formaldehyde-based whole-mount in situ hybridization method for planarians. dev. dyn. 238: 443-450, 2009. piechotta k, lu j, delpire e. cation chloride cotransporters interact with the stress-related kinases ste20-related proline-alanine-rich kinase (spak) and oxidative stress response 1 (osr1). j. biol. chem. 277: 50812-50819, 2002. reddien pw, sánchez alvarado a. fundamentals of planarian regeneration. annu. rev. cell. dev. biol. 20: 725-757, 2004. reddien pw, oviedo nj, jennings jr, jenkin jc, sánchez alvarado a. smedwi-2 is a piwi-like protein that regulates planarian stem cells. science 310: 1327-1330, 2005. robb smc, sánchez alvarado a. identification of immunological reagents for use in the study of freshwater planarians by means of whole-mount immunofluorescence and confocal microscopy. genesis 32: 293-298, 2002. strange k, denton j, nehrke k. ste20-type kinases: evolutionarily conserved regulators of ion transport and cell volume. physiology 21: 61-68, 2005. saló e, abril jf, adell t, cebrià f, eckelt k, fernàndez-taboada e, et al. planarian regeneration: achievements and future directions after 20 years of research. int. j. dev. biol. 53: 1317-1327, 2009. scimone ml, meisel j, reddien pw. the mi-2-like smed-chd4 gene is required for stem cell differentiation in planarian schmidtea mediterranea. development 137: 1231-1241, 2010. takano t, pulvers jn, inoue t, tarui h, sakamoto h, agata k, et al. regeneration-dependent conditional gene knockdown (readyknock) in planarian: demonstration of requirement for djsnap-25 expression in the brain for negative phototactic behavior. dev. growth differ. 49: 383-394, 2007. ushiro h, tsutsumi t, suzuki k, kayahara t, nakano k. molecular cloning and characterization of a novel ste20-related protein kinase enriched in neurons and transporting epithelia. arch. biochem. biophys. 355: 233-240, 1998. ultanir sk, yadav s, hertz nt, oses-prieto ja, claxton s, burlingame al, et al. mst3 kinase phosphorylates tao1/2 to enable myosin va function in promoting spine synapse development. neuron 84: 968-982, 2014. wagner de, wang ie, reddien pw. clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration. science 332: 811-816, 2011. wu c, whiteway m,thomas dy, lerberer e. molecular characterization of ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (mek) kinase kinase from saccharomyces cerevisiae. j. biol. chem. 270: 15984-15992, 1995. yu s, chen x, yuan z, zhou l, pang q, mao b, et al. planarian myosin essential light chain is involved in the formation of brain lateral branches during regeneration. mol. genet. genomics 290: 1277-1285, 2015. isj096.pdf 32 isj 2: 32-40, 2005 issn 1824-307x review flow cytometry as a tool for analysing invertebrate cells a cossarizza1, m pinti1, l troiano1, el cooper2 1 department of biomedical sciences, university of modena and reggio emilia, modena, italy 2 department of neurobiology, ucla school of medicine, los angeles, ca, usa accepted april 8, 2005 abstract flow cytometry (fcm) is a powerful tool that allows analysis of thousand of cells in a few seconds, at the single cell level. in the last 15 years, researchers have used fcm to investigate the cellular machinery of invertebrates. analyses have focused on functions linked to innate immunity, such as phagocytosis and natural killer cell activity, as well as on the sensitivity of invertebrate cells to a particular stress or to a toxic agent. further, fcm has been employed to recognize antigens, or at least immunodominant epitopes, shared in common with mammalian cells, including human leukocytes. in this review, main studies that have utilized fcm to investigate either phenotype and functions of invertebrate cells are reported and discussed. keywords: invertebrate; flow cytometry; immunology introduction: diversity of approaches and principles of flow cytometry (fcm) recently, a consistent number of scientists working in different fields have utilized flow cytometry (fcm) for numerous analyses. fcm has indeed impacted both basic cell biology and clinical medicine in a significant manner, and instruments are present almost everywhere. according to the essential principle of fcm, single cells (or particles, or organelles) suspended within a stream of liquid pass through a light source (usually a laser beam tuned at 488 nm) focused on a minute region. during this passage, cells are interrogated individually thus providing relevant numbers of information. basically, signals generated by cells passing through the laser beam are spectral bands of light in the visible spectrum, which represent the detection of various chemical or biological components, mostly fluorescence. since flow cytometers can analyze single particles or single cells, it is possible to separate them into populations based upon differences in any of the variables that can be measured on each particle/cell. corresponding author: andrea cossarizza dept. biomedical sciences, university of modena and reggio emilia,via campi 287, 41100 modena, italy. e-mail: cossarizza.andrea@unimore.it currently, the number of these variables is consistently high, and several instruments allow identification of more than a dozen different parameters in the same particle/cell. it is then possible to separate these populations electronically, and to identify them using multivariate analysis techniques. fcm uses several fluorescent molecules that are attached by one means or another to the particle or cell of interest. the fluorescent probe might be membrane bound, cytoplasmic, or attached to nuclear material. to recognize specific receptors present on the plasma membrane, as well as to identify intracellular antigens or to calculate the amount of a given molecule within a cell, it is a common practice to use monoclonal or polyclonal antibodies directly conjugated to fluorescent dyes. particles of almost any nature can be evaluated by flow cytometry. they can be very small, even below the resolution limits of visible light, because they can be detected by their fluorescent signatures. similarly, depending on the structure of the flow cell and fluidics, particles as large as several thousand microns can be evaluated. fcm allows the evaluation of thousands of events in a very short time. for example, the most common instruments can detect hundreds of cells per second, measuring up to 5 parameters; sophisticated systems exist that can run particles at rates approaching 100,000 events per second while collecting 10 to 20 parameters from each particle. finally, it is possible to separate single particles/cells physically from mixed 33 populations and, by using the sorting option eventually present in the instrument, to place them into a defined location for cloning, or for further molecular analysis. fcm analysis of cells from planorbarius corneus using antisera to vertebrate bioactive peptides the first studies that employed fcm in invertebrates were devoted to the analysis of epitopes/molecules present in cells from the mollusc p. corneus that were able to cross-react with human molecules. the starting point was represented by immunohistochemical data, when the attention was devoted to the detection of immunoreactive molecules in round and spreading hemocytes of p. corneus (ottaviani and cossarizza, 1990). antisera to vertebrate (mainly human) bioactive peptides were used, and it was found that spreading hemocytes had alpha 1-antichymotrypsin-bombesin-, calcitonin-, cck-8 (inc)-, cck-39-, gastrin-, glucagon-, metenkephalin-, neurotensin-, oxytocin-, somatostatin-, substance p-, vip-, and vasopressin-immunoreactive molecules, while round hemocytes were only positive to anti-bombesin, anti-calcitonin, anti-cck-8, anticck-39, anti-neurotensin, anti-oxytocin, antisubstance p and anti-vasopressin antibodies. fcm, whose sensibility is clearly much higher than that of a microscope, was then used to confirm the presence of acth and â-endorphin immunoreactive molecules in the same invertebrate (ottaviani et al., 1990). in the hemolymph of these animals, two main types of cells exist, i.e. round (rh) and spreading hemocytes (sh) (ottaviani, 1991). sh but not rh were positive to antisera recognizing vertebrate acth and âendorphin. interestingly, only by means of analyzing physical characteristics (dimension, granularity) of cells from invertebrate hemolymph, fcm allowed identification of the same cell types that had been described morphologically long before (fig. 1). interestingly, similar data on the presence of acthimmunoreactive molecules were obtained in another mollusc, such as lymnea stagnalis (ottaviani et al., 1991), and in leukocytes from the frog rana esculenta (ottaviani et al., 1992). the functional capacity of p. corneus spreading hemocytes, along with the presence of acth-like molecules (i.e., reactive with antibodies directed against human acth) in other species, including carassius carassius var. auratus and coris julius (pisces), hymenochirys gillii (amphibia), podarcis muralis (reptilia), and gallus domesticus (aves) suggested that cells with phagocytic activity, likely the oldest natural immune response, may represent a suitable model to unravel the tangled web of the common ancestor of the immune and the neuroendocrine systems. evidence of cytotoxicity in molluscs the investigation on natural immunity in p. corneus continued by evaluating the capacity of its cells to kill targets in a non-mhc restricted manner (franceschi et al., 1991). natural killer (nk) cells represent an important form of cell recognition that results in cytotoxicity, and it was predicted that nk-like activity had to be preserved throughout phylogenetic development (cooper et al., 2002). nonadherent round hemocytes were able to lyse the k562 human target cell line in a short-term nk cytotoxicity test. this nk-like activity, severely reduced after 18 hour incubation at 24° c, was maintained by the presence of human recombinant interleukin-2 (il-2). a further analysis performed by mouse anti-human monoclonal antibodies and cytofluorimetric analysis revealed that both sh and rh reacted with several antibodies, including those directed against epitopes typical of mammalian nk cells, and against different cell adhesion molecules (fig. 2). these data suggested the hypothesis that a primitive nk-like activity appeared early in evolution, and is not shared by phagocytic cells. possible role of interleukins a support for this hypothesis came from the cytofluorimetric observation that il-2 could compete with corticotropin releasing factor (crf) by binding to the receptor that is able to bind human il-2, present on the plasma membrane of molluscan hemocytes (fig. 3). at the functional level, pre-incubation of hemocytes with il-2 or anti-il-2 monoclonal antibody (mab) significantly reduced or completely eliminated the crf-induced release of biogenic amines. these findings are compatible with the presence of a unique (ancestral?) receptor on molluscan hemocytes, capable of binding both crf and il-2, two key molecules of the neuroendocrine and immune system (ottaviani et al., 1994). subsequently, using fcm it was observed that spread cells present in the hemolymph of the mussel mytilus galloprovincialis expressed three il-2 receptor (il-2r) subunits: á, â and ã. mussel il-2rá and il2râ subunits displayed a molecular weight similar to those in vertebrates tissues, whereas mussel il-2rã was smaller than that in vertebrates. both lipopolysaccharide (lps) and il-2 induced il-2rá expression, and such induction depended on the concentration of both agonists (barcia et al., 1999). stimulation with lps, il-2 or platelet-derived growth factor also resulted in an increased release of dopamine, adrenaline and noradrenaline in the culture medium (cao et al., 2004). identification of cell shape and surface antigens in other species, and relevance to tumor cells and environmental chemicals fcm proved to be a powerful technique for the analysis of the heterogeneity of cell populations in a variety of invertebrates. studies were performed on hemocytes derived from individual l. stagnalis, and revealed that cell sizes were comparable in all 40 specimens studied, and was not affected by age or by infection with trichobilharzia ocellata (amen et al., 1992). a monoclonal antibody of the clam was generated to normal hemocytes of mya arenaria (white et al., 1993). the antibody, designated 2a4, was evaluated by elisa, immunocytochemistry, western blotting, and finally fcm. m. arenaria is a very interesting model, because it develops a leukemia 34 fig. 1 presence of immunoreactive acth molecules on the cell surface of spreading (sh) and round (rh) hemocytes from planorbarius corneus and from lymnea stagnalis. a= control, b= stained cells. modified from ottaviani et al., 1991. fig. 2 expression of epitopes recognized by mouse anti-human monoclonal antibodies in round (rh) or spreading (sh) hemocytes from planorbarius corneus. a= control, b= stained cells. modified from franceschi et al., 1991. 35 fig. 3 expression of il-2 like molecules on the plasma membrane of planorbarius corneus revealed by anti-il-2 mabs, and inhibition of the staining with exogenous il-2 or crf. modified from ottaviani et al., 1994. detected first in the hemolymph and, as the disease progresses, in solid tissue. the 2a4 antigen was detected on 87 % normal adherent cells, but was lost as nonadherent leukemia cells proliferated. the mature leukemia cell neither expresses 2a4 nor can 2a4 be detected in the leukemia cell lystate. since 2a4 reacts with a 130-kda protein, it was suggested that p130 may be involved in the regulation of cell adhesion. subsequently, the same investigators evaluated the reactivity of both normal and tumor cells from m. arenaria to a polyclonal antibody to polychlorinated biphenyls (pcbs) (harper et al., 1994). fcm was indeed used to ascertain both leukemia prevalence and pcb reactivity. analytical chemistry was used to quantitate the amount of aroclor 1254 (a widely studied pcb), per tumor cell population and compared directly to fcm results. the prevalence of leukemia consistently exceeded 60 % when clams were retrieved from new bedford harbor, a site heavily contaminated with pcbs. both normal circulating cells and tumor cells were extremely reactive with the pcb antibody. when clams from two other sites were compared with clams from new bedford harbour, both disease prevalence and cell reactivity to the pcb antibody were significantly reduced. this study was the first that demonstrated the presence of pcbs in cell populations of marine invertebrates, and showed that the presence of pcbs in vivo was directly correlated with environmentally linked leukemia. exposure to ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons, may affect the earthworm cellular immune-defense system. after exposure to soil contaminated with 7,12dimethylbenzanthracene (dmba), cellular functions of coelomocytes from the earthworm eisenia fetida were examined by flow cytometry, that allowed to classify them as small and large cells (komiyama et al., 2004). sexually mature animals were kept in dark at 18° c with various doses of dmba contaminated artificial soil for 7 days. coelomocytes were harvested from earthworms and processed for in vitro phagocytosis and h2o2 activity. phagocytosis was assessed by ingestion of fluorescence beads and h2o2 activity examined using 2’,7’-dichloro-fluorescin diacetate. cell functions were down regulated in a dose dependent manner after exposure to sublethal doses of dmba. this study demonstrates the utility of flow cytometry to evaluate the biological activity of earthworm cells, and the effect of soil contamination for ecotoxicological studies. age-related changes in the immunocyte population in m. galloprovincialis have been reported (ottaviani et al., 1998). in this mollusc, a young and an old stage belonging to the same cell type have been described. interestingly, either in young or adult animals these cells were positive to the staining with mouse anti-human mabs anti-cd5, -cd11b and cd16. other molecules, such as cd10, a surface antigen known to be identical to neutral endopeptidase-24.11 (nep), have been recently found in immunocytes of m. galloprovincialis (caselgrandi et al., 2000). computer-assisted microscopic image analysis revealed nep functional activity, i.e. the capacity to deactivate the pdgf-aband tgf-â1induced changes in immunocyte shape. cytotoxicity in earthworms: the model of eisenia foetida a cytometric approach has been used to investigate the main features of natural cytotoxicity in coelomocytes from e. foetida, that were used as effector cells against the human tumor target cell line k562 (cooper et al., 1995). to first assess the viability of effectors, dna synthesis was tested and was higher in autogeneic (cells from one animal) than in 36 fig. 4 allogeneic inhibition of cell proliferation. left panel: cell viability in freshly collected coelomocytes from eisenia fetida. right panels: a, proliferation of coelomocytes from single animals or pooled from different animals after stimulation with phytohemagglutinin (pha), concanavalin a (cona) or inhibition with mitomycin-c (mmc). note that pooled cells showed an allogeneic inhibition. b and c: cytofluorimetric analysis shows the comparison between unstimulated cells from a single animal (b) or cells collected and pooled from different animals (c). note the difference in the percentages of cells in the s or g2/m phases of the cell cycle. modified from cossarizza et al., 1996. fig. 5 expression of epitopes recognized by mouse anti-human monoclonal antibodies in small and large cells from eisenia fetida. k= control, unstained cells. modified from cooper et al., 1995. 37 allogeneic (cells from two animals) coelomocytes. cell cycle analysis revealed that autogeneic cultures showed significantly greater numbers of cells in s, g2, or m phases than allogeneic ones (fig. 4). when autogeneic or allogeneic cells were kept together in culture, no significant cell killing occurred in either, while autogeneic but not allogeneic cultures could kill k562 target cells. cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding the lectin phytohemagglutinin. the heat labile supernatant from autogeneic but not allogeneic cultures killed k562 targets. recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms. subsequently, it was found that earthworm coelomocytes effect cytotoxicity at significantly high levels also against the nk-resistant target cells (such as the u937, bsm, and cem cell lines) (cossarizza et al., 1996). then, using fcm and mouse anti-human monoclonal antibodies, the two main types of cells present in e. foetida coelom were better characterized: small (8-11 µm), electron-dense cells (sc) were: cd11a+, cd45ra+, cd45r0+, cdw49b+, cd54+, hla class ii (dr)+, â2-µ+ and thy-1+ (cd90+) (fig. 5); large (12-15 µm), electron-lucent cells (lc) were negative for these markers. both cell types were negative for other cd and mhc class i and class ii markers. subsequently, it was found that coelomocytes were positive to the staining with rat anti-mouse mabs for cd90, cd5, cd8, cd45ra, cd45r0 and anti-perforin, negative for ia, cd4 and cd11c (komiyama et al., 2002). in general, only sc and to a lesser extent, lc reacted with these antibodies. sc were active during recognition, rapidly binding to targets; lc were phagocytic (cossarizza et al., 1996). sc were able to exert rapid, significant, and equal levels of killing at 4, 20, and 37°c, suggesting that, as for phagocytosis, also primitive nk-like activity appeared early in evolution. fcm can be used to analyze cell organelles and different cellular functions the presence and functionality of mitochondria in terms of mass and membrane potential (mmp) have been investigated in both sc and lc from e. foetida at the single cell level by different fcm techniques (cossarizza et al., 1995). in comparison with sc, lc have a higher number of mitochondria, and, accordingly, showed a greater fluorescence intensity when mitochondrial mass was measured by nonyl acridine orange. to measure mmp, both the lipophilic cationic probe jc-1 and rhodamine (rh) 123 were used. using jc-1, mmp was analyzed separately on sc and lc, and significant percentages of coelomocytes (> 95 % of sc and about 90 % of lc) displayed a high mmp. adding 0.1 µm valinomycin (val) caused most sc to depolarize, while this occurred in only a few lc. rh123 gave different results: no effects of val were observed either in sc or in lc. it was concluded that in coelomocytes there may be several energy-independent rh123-binding sites, and that it is possible to analyze mitochondrial parameters by fcm in intact invertebrate cells. earthworms possess also non-specific responses found in other complex metazoans. coelomocytes from the e. foetida were further characterized by electron microscopy and fcm analyses, and the structural changes that occur when effector coelomocytes and target cells interact during cytotoxic activity were deeply investigated (quaglino et al., 1996). it was found that using in vitro cultures: 1) the two aforementioned earthworm cell types retained their morphological features; 2) their dna content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line k562; 3) significant percentages of coelomocytes were found to be in s or g2/m phases of the cell cycle. it is noteworthy that these two parameters were investigated by direct staining of dna with fluorescent probes, as well as by the classical staining with bromodeoxyuridine (dolbeare et al., 1983). when cultivated alone, coelomocytes formed no aggregates. however, when mixed with k562 target cells, as described above, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. these results are the first to describe this type of earthworm non-specific "inflammatory" response in vitro against xenogeneic tumor cells. fcm analysis of dna content and cell cycle fcm has been used to quantify dna content in invertebrate cells (ulrich, 1990). cell material from the diptera species chironomus thummi, drosophila melanogaster, calliphora vicina and musca domestica was analyzed using an impulse cytophotometer with a special quartz objective, that was especially manufactured for cytofluorometric investigations with the dna-specific fluorochrome dapi in combination with the protein fluorochrome sulforhodamine 101. the occurrence of heterogenous cell populations with aneuploid and polyploid dna content within the cell material of different developmental stages of diptera species have been determined, whereby in larvae polyploid cell populations and in imagos aneuploid cell populations predominate. more recently, studies were carried out to determine the alteration in dna cell cycle characteristics of hemocytes from m. galloprovincialis collected at 17 different locations along the adriatic coast, croatia (bihari et al., 2003). fcm was used to connect possible genomic manifestation to urban and/or industrial waste, and the incidence of altered dna profiles was investigated. different alterations in cell cycle, mirroring either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte dna, were found. among these, the most relevant were intraindividual genome size variability, aneuploidy and accidental apoptotic processes; normal cell cycle dna profiles were obtained in 60.9 % individuals from all 17 sites. molecular assays were used along with cytofluorimetric analysis of cell cycle, and confirmed the presence of dna damages, such as alkali-labile sites and single-strand breaks, interstrand cross-links and dna-protein cross-links (bihari et al., 2002). 38 a cell line from the insect spodoptera frugiperda (sf9) was used to investigate the capacity of azadirachtin to alter the mitotic index (salehzadeh et al., 2003). fcm demonstrated that cells accumulated in the g2/m phase of the cell cycle, and that the effect was concentration-dependent. azadirachtin had the same effect on c6/36 mosquito cells, but failed to affect l929 murine fibroblasts even at high concentrations. experiments with colchicine and taxol showed similarities of action between azadirachtin and colchicine, and azadirachtin was apparently able to displace colchicine from specific binding-sites present in living insect cells. in vitro analysis of the effects of 20hydroxyecdysone (20e) on the cell cycle in ial-pid2 cell line established from imaginal wing discs of plodia interpunctella were reported (mottier et al., 2004). it was found that 20e induced an arrest of cells in g2 phase, accompanied by a sharp decrease in the levels of cyclin a and b expression. studies on dna damages and apoptosis several groups have investigated by fcm either dna damages or the induction of apoptosis in invertebrate cells. the terminal dutp nick-end labeling technique (tunel) was used to detect m. galloprovincialis cells displaying dna fragmentation within gill structures after treatment with tri-n-butyltin (tbt) (micic et al., 2001). dna degradation as well as decreased number of cells in g0/g1 were detectable after 1.5 hour of tbt incubation. presence of apoptotic cells in mussels' gills was indicated by the selective loss of g2/m cells concomitant with the appearance of cells with decreased dna content in the s and g0/g1 regions of cell cycle. the effect of the tbt on cell cycle in a mussel gill was depending upon dose and exposure time. metabolites of salsolinol (sal), an intraneuronal, dopamine-derived tetrahydroisoquinoline (tiq), induce apoptosis in human dopaminergic neuroblastoma cells, and likely contribute to the killing of nigrostriatal dopaminergic neurons occurring in parkinson's disease. since insects employ dopamine and related catecholamines in a variety of processes including cuticular sclerotization and cellular immune reactions, the capacity of their cells to metabolize exogenous sal was investigated (ottaviani et al., 2002). iplb-ldfb cells from lymantria dispar exhibited no significant increase in apoptosis when incubated for 48 hours with low concentrations of sal (up to 1 mm). apoptosis was observed only with the highest concentration of sal tested (5 mm), but only 12.4 % of the cells manifested this form of cell death. the resistance of iplb-ldfb cells to sal-induced apoptosis was attributed to the ability of these insect cells to metabolize and/or detoxify the dopaminederived catecholic tiqs. the toxicity of the mycotoxins nivalenol (niv), deoxynivalenol (don), and fumonisin b1 (fb1) was studied in the lepidopteran s. frugiperda sf-9 cells by fcm (fornelli et al., 2004). niv was significantly more toxic than don, and both were significantly more toxic than fb1. cell cycle distribution showed an arrest of cells in the g0/g1 phase in the presence of all of the three compounds. morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic niv induced late apoptosis, whereas don and fb1 produced less-severe morphological changes characteristic of early apoptosis. it was concluded that niv is more toxic than don, which in turn is more toxic than fb1, and that these mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process. assessing the response of invertebrate cells to fungi and bacteria innate immunity is strongly regulated by microbial products, and fcm can be added to the list of available methods to investigate this activity. furthermore, fcm can provide a simple, reproducible, and sensitive method for evaluating invertebrate hemocyte responses to immunological stimuli. the response of hemocytes from the white river crayfish procambarus zonangulus to stimulation by fungal cell walls (zymosan a) were recently measured by fcm (cardenas et al., 2000). changes in physical characteristics were assessed using forwardand side-scatter light parameters, and viability was measured by two-color fluorescent staining with calcein-am and ethidium homodimer 1. the main effects of zymosan a on crayfish hemocytes were reduction in cell size and viability. adding trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade played a key role in generating signal molecules which mediate these cellular responses. also lps from gram-negative bacteria strongly stimulate hemocytes from p. zonangulus in vitro (cardenas et al., 2004). fcm revealed that treating hemocytes with lps caused a conspicuous and reproducible decrease in cell size as compared to control hemocytes. this physical modification was accompanied by a reduction in hemocyte viability, that was assessed as described above. the onset of cell size reduction was gradual and occurred prior to cell death. interestingly, hemocytes treated with lps from salmonella minnesota without the lipid a moiety (detoxified lps) decreased in size without a reduction of viability, while the addition of trypsin inhibitor to the lps treatments caused noticeable delays in cell size and viability changes. it was concluded that crayfish hemocytes react differently to the polysaccharide and lipid a moieties of lps, where lipid a is cytotoxic and the polysaccharide portion is stimulatory. these effects concur with the general pattern of mammalian cell activation by lps, thereby indicating common innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. fcm reveals cellular responses to stress such as environmental pollution the use of invertebrates as bioindicators in environmental monitoring is of relevant interest for its practical implications. tunicates are filter feeding marine invertebrates that are susceptible to 39 environmental contamination by toxic metals and polyaromatic hydrocarbons (radford et al., 2000). their immune reactions are profoundly affected by exposure to tributyltin (tbt) and copper, both of which are components of marine antifouling paints. immunofluorescence labeling with an anti-hemocyte monoclonal antibody whose binding to plasma membrane was revealed by fcm demonstrated that the antigenic structure of the circulating hemocyte population was substantially affected by tbt and copper. antigen-positive hemocytes were also found to accumulate in the pharyngeal papillae of tbtexposed tunicates, confirming that exogenous metals can have profound effects on tunicate hemocytes. the effects of mercuric chloride and methylmercury on the phagocytic capacities of m. arenaria hemocytes were evaluated (fournier et al., 2001). clams were exposed to single metal in water for up to 28 days at different concentrations (10-9 to 10-5 m), and phagocytic activity of hemocytes was determined by uptake of fluorescent microspheres and fcm. the phagocytic capacity is clearly proportional to ingestion of these particles, and thus to the total fluorescence that can be detected from each cell. clams exposed to high concentrations died by day 7 of exposure, while the viability of hemocytes was decreased only in clams exposed for 28 days to a 10 times lower concentration. a significant decrease in phagocytic activity of hemocytes was also present, and a clear correlation was established between body burden of mercury and effects on phagocytic activity of hemocytes. recently, the presence of immunoreactive inducible nitric oxide synthase molecules (ir-inos) has been demonstrated in the l. dispar iplb-ldfb cell line. in these cells, ir-inos were induced after 18 hour-incubation with sodium nitroprusside (snp). the increase in no provoked by snp in turn induced apoptosis, that was further increased by n-acetyl-lcysteine. apoptosis was quantified by fcm through the identification of nuclei showing the typical hypodiploic peak present in cells with less dna content, after staining with the fluorescent probe propidium iodide (ottaviani et al., 2001). the analysis of apoptosis by fcm is a very simple assay, that can be performed in almost all cells from all species, since the basic principle is that of staining dna with a stoichiometric fluorescent molecule. changes in fluorescence, along with modifications of the physical parameters of the cell, typically occur in those that lose dna, as during apoptosis. conclusions and perspectives on analysis of invertebrate cells by fcm a variety of fcm techniques currently exist that allow analysis of almost all cellular functions and parameters, with all of the possible advantages of this approach. from this point of view, until now the world of invertebrates has received a poor attention, and is almost completely virgin territory for more research. since invertebrate cells are obviously eukaryotic, researchers have indeed the possibility to use all of the methodologies that have been developed for studies on mammalian and human cells. similarly, almost all reagents and fluorescent dyes that have been developed to investigate different cellular activities can be successfully used in invertebrates. even if a variety of functional parameters have still to be investigated, the few data available already provide relevant information not only on the phenotype or the physical characteristics of a given invertebrate cell, but also on its capacity to perform a given function or to respond to a given stress. considering the relevant importance that comparative studies are assuming, either for the importance of these biological models and their low cost, and the relevant decrease in the cost of flow cytometers (that fortunately are becoming always more affordable), it is easy to predict that in the next few years we will assist in an unprecedented development of cellular studies in the field invertebrate biology and immunology. references amen ri, aten ja, baggen jm, meuleman ea, de lange-de klerk es, sminia t. trichobilharzia ocellata in lymnaea stagnalis: a flow cytometric approach to study its effects on hemocytes. j. invertebr. pathol. 59: 95-98, 1992. barcia r, cao a, arbeteta j, ramos-martinez ji. the il-2 receptor in hemocytes of the sea mussel mytilus galloprovincialis lmk. iubmb life. 48: 419-423, 1999. bihari n, hamer b, jaksic z, fafandel m, micic m, batel r. application of alkaline elution, fast micromethod and flow cytometry in detection of marine contamination. cell mol. biol. (noisy-le-grand). 48: 373-377, 2002. bihari n, micic m, batel r, zahn rk. flow cytometric detection of dna cell cycle alterations in hemocytes of mussels (mytilus galloprovincialis) off the adriatic coast, croatia. aquat. toxicol. 64: 121-129, 2003. cao a, ramos-martinez ji, barcia r. in vitro effects of lps, il-2, pdgf and crf on haemocytes of mytilus galloprovincialis lmk. fish shellfish immunol. 16: 215225, 2004. cardenas w, dankert jr, jenkins ja. flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides. fish shellfish immunol. 17: 223233, 2004. cardenas w, jenkins ja, dankert jr. a flow cytometric approach to the study of crustacean cellular immunity. j. invertebr. pathol. 76: 112-119, 2000. caselgrandi e, kletsas d, ottaviani e. neutral endopeptidase-24.11 (nep) deactivates pdgfand tgf-beta-induced cell shape changes in invertebrate immunocytes. cell biol. int. 24: 85-90, 2000. cooper el, cossarizza a, suzuki mm, salvioli s, capri m, quaglino d, franceschi c. autogeneic but not allogeneic earthworm effector coelomocytes kill the mammalian tumor cell target k562. cell immunol. 166: 113-122, 1995. cooper el, kauschke e, cossarizza a. digging for innate immunity since darwin and metchnikoff. bioessays. 24: 319-333, 2002. cossarizza a, cooper el, quaglino d, salvioli s, kalachnikova g, franceschi c. mitochondrial mass and membrane potential in coelomocytes from the earthworm eisenia foetida: studies with fluorescent probes in single intact cells. biochem. . res. commun. 214: 503-510, 1995. cossarizza a, cooper el, suzuki mm, salvioli s, capri m, gri g, quaglino d, franceschi c. earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. exp. cell res. 224: 174-182, 1996. dolbeare f, gratzner h, pallavicini mg, gray jw. flow cytometric measurement of total dna content and incorporated bromodeoxyuridine. proc. natl. acad. sci. usa 80: 5573-5577, 1983. fornelli f, minervini f, mule g. cytotoxicity induced by nivalenol, deoxynivalenol, and fumonisin b1 in the sf-9 40 insect cell line. in vitro cell dev. biol. anim. 40: 166171, 2004. fournier m, pellerin j, clermont y, morin y, brousseau p. effects of in vivo exposure of mya arenaria to organic and inorganic mercury on phagocytic activity of hemocytes. toxicology 161: 201-211, 2001. franceschi c, cossarizza a, monti d, ottaviani e. cytotoxicity and immunocyte markers in cells from the freshwater snail planorbarius corneus (l.) (gastropoda pulmonata): implications for the evolution of natural killer cells. eur. j. immunol. 21: 489-493, 1991. harper dm, flessas da, reinisch cl. specific reactivity of leukemia cells to polyclonal anti-pcb antibodies. j. invertebr. pathol. 64: 234-237, 1994. komiyama k, kauschke e, cooper el. evidence for a perforin-like cell mediated cytolysis by earthworm coelomocytes. in: el cooper, a beschin and m bilej (ed), ios press. amsterdam, the netherlands, 73-81, 2002. komiyama k, okaue m, miki y, ohkubo m, moro i, cooper el. non-specific cellular function of eisenia fetida regulated by polycyclic aromatic hydrocarbons. pedobiologia. 47: 717-723, 2004. micic m, bihari n, labura z, muller we, batel r. induction of apoptosis in the blue mussel mytilus galloprovincialis by tri-n-butyltin chloride. aquat. toxicol. 55: 61-73, 2001. mottier v, siaussat d, bozzolan f, auzoux-bordenave s, porcheron p, debernard s. the 20-hydroxyecdysoneinduced cellular arrest in g2 phase is preceded by an inhibition of cyclin expression. insect biochem. mol. biol. 34: 51-60, 2004. ottaviani e. immunorecognition in the gastropod molluscs with particular reference to the freshwater snail planobarius corneus (l.) (gastropoda, pulmonata). boll zool. 59: 129-139, 1991. ottaviani e, barbieri d, malagoli d, franchini a. nitric oxide induces apoptosis in the fat body cell line iplb-ldfb from the insect lymantria dispar. comp. biochem. physiol. 128b: 247-254, 2001. ottaviani e, cossarizza a. immunocytochemical evidence of vertebrate bioactive peptide-like molecules in the immuno cell types of the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). febs lett. 267: 250-252, 1990. ottaviani e, cossarizza a, ortolani c, monti d, franceschi c. acth-like molecules in gastropod molluscs: a possible role in ancestral immune response and stress. proc. r. soc. lond. b biol. sci. 245: 215-218, 1991. ottaviani e, franchini a, barbieri d, kletsas d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two agingrelated hemocytes stages. it. j. zool. 65: 349-354, 1998. ottaviani e, franchini a, caselgrandi e, cossarizza a, franceschi c. relationship between corticotropinreleasing factor and interleukin-2: evolutionary evidence. febs lett. 351: 19-21, 1994. ottaviani e, franchini a, cossarizza a, frenceschi c. acthlike molecules in lymphocytes. a study in different vertebrate classes. neuropeptides 23: 215-219, 1992. ottaviani e, nappi aj, vass e. resistance of the insect cell line iplb-ldfb to salsolinol-induced apoptosis. arch. insect biochem. physiol. 49: 1-9, 2002. ottaviani e, petraglia f, montagnani g, cossarizza a, monti d, franceschi c. presence of acth and beta-endorphin immunoreactive molecules in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata) and their possible role in phagocytosis. regul. pept. 27: 1-9, 1990. quaglino d, cooper el, salvioli s, capri m, suzuki mm, ronchetti ip, franceschi c, cossarizza a. earthworm coelomocytes in vitro: cellular features and "granuloma" formation during cytotoxic activity against the mammalian tumor cell target k562. eur. j. cell. biol. 70: 278-278, 1996. radford jl, hutchinson ae, burandt m, raftos da. effects of metal-based environmental pollutants on tunicate hemocytes. j. invertebr. pathol. 76: 242-248, 2000. salehzadeh a, akhkha a, cushley w, adams rl, kusel jr, strang rh. the antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells. insect biochem. mol. biol. 33: 681-689, 2003. ulrich w. aneuploid and polyploid cellular dna heterogeneity in insect cell material of diptera species analyzed by flow cytometry. z. naturforsch. 45: 1027-1030, 1990. white mk, miosky d, flessas da, reinisch cl. the expression of an adhesion-related protein by clam hemocytes. j. invertebr. pathol. 61: 253-259, 1993. isj 6: sxx-syy, 2009 isj 6: 22-31, 2009 issn 1824-307x report of meeting xth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 18 20 february 2009, department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy organizers: m betti, m balsamo, s papa department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy session 1 1999-2009: a 10-year apprenticeship in comparative immunology. the immunocytes as leading actors in the responses of the bivalve mytilus to environmental challenge l canesi1, m betti2, c ciacci2, c pruzzo1, g gallo1 1department of biology, university of genoa, genoa, italy 2department of human, environmental and natural sciences, università di urbino “carlo bo”, urbino, italy in bivalve molluscs, circulating hemocytes are responsible for the innate immune defence, and are also involved in the transport of nutrients from the digestive gland to the gonad during gametogenesis. although it has long been known that the activity of these cells can be modulated by multiple signals, only in recent years the mechanisms involved in the responses of hemocytes to different stimuli, from both the internal and the external environment, have been investigated. in the marine bivalve, the edible mussel mytilus, studies carried out both in vitro and in vivo on the effects of challenge with different bacterial species and strains, heterologous cytokines, as well as with a number of environmental contaminants, lead to the identification of the main signaling pathways involved in immune activation, as well as of the main mechanisms of cellular damage and consequent immunodepression. moreover, endogenous estrogens were identified as physiological modulators of the hemocyte function. the results obtained added force to the hypothesis that the mechanisms of innate immunity are extremely conserved between invertebrate and mammalian systems. overall, the data collected so far allowed the functional characterization of mussel hemocytes as a model for investigating both the basic processes of the immune response, and the mechanisms toxicity of emerging environmental contaminants. moreover, these studies demonstrated that mytilus hemocytes represent a sensitive target for environmental stimuli, and that changes in their function are related to significant changes in the physiological status of bivalves, an ecologically and economically relevant group of invertebrates. effects of bacterial challenge on mytilus digestive gland biomarkers c ciacci1, b citterio2, r fabbri1, c barmo3, m betti1, p roch4, l canesi3 1department of human, environmental and natural sciences, università di urbino “carlo bo”, urbino, italy 2dipartimento di scienze biomolecolari, università degli studi di urbino “carlo bo”,urbino, italy 3department of biology, university of genoa, genoa, italy 4jru ecosystèmes lagunaires, cnrs-universitè de montpellier 2-france in bivalve molluscs, responses to bacterial challenge have been largely investigated in the immune cells, the hemocytes, whereas little is known about the possible effects at the tissue level. in this work, the effects of in vivo challenge of mussels (mytilus spp.) with different bacteria (micrococcus lysodeikticus, vibrio anguillarum, vibrio splendidus) on different biomarkers of stress were evaluated in the digestive gland at different times post injection (p.i.). all bacteria induced a significant decrease in lysosomal membrane stability lms (about -50 %) with respect to controls (pbs-nacl-injected mussels) from 3 to 24 h p.i. when expression of antioxidant genes was evaluated by quantitative rtpcr with both m. lysodeikticus and v. anguillarum a general downregulation of both metallothionein isoforms, mt10 and mt20, was observed, with the exception of a large increase in mt20 mrna induced by v. anguillarum and a smaller increase in mt10 by m. lysodeikticus at 6 h p.i. both bacteria downregulated glutathione s-transferase gst-π at all time p.i. v. anguillarum decreased the expression of catalase, wherease no effects were observed with m. lysodeikticus. at 48 h p.i. the activity of 22 catalase was increased (2-fold with both m. lysodeikticus and v. anguillarum; +60 % with v. splendidus) and total gst activity was decreased (30, -40 %) with all bacteria. both m. lysodeikticus and v. splendidus also decreased gsh reductase gsr activity (about -24 %) and total glutathione content (-47 %) whereas no effects were observed with v. anguillarum. flow cytometry as a tool for analyzing invertebrate immunocytes: our experience with mytilus hemocytes b canonico, m betti, c ciacci, m arcangeletti, s papa, l canesi1 department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy 1department of biology, university of genoa, genoa, italy flow cytometry (fc) represents a powerful tool generally utilized in biomedical research. fc has been also increasingly applied in invertebrate immunology; in particular, in bivalve molluscs, fc has been largely utilized to characterize hemocyte populations and to evaluate total cell count and oxyradical production. in addition, fc can be employed to recognize antigens, or at least immunodominant epitopes, shared in common with mammalian cells, including human leukocytes. in addition to traditional methods such as microscopy and protein chemistry, fc can provide a simple, reproducible, and sensitive method for evaluating invertebrate hemocyte responses to different immunological stimuli. data are here summarized obtained in different in vitro and in vivo studies carried out on the hemocytes of the marine bivalve mytilus galloprovincialis. fc was successfully utilised to evidentiate, utilizing annexin v binding and mitochondrial markers, pre-apoptotic processes in mussel hemocytes in response to cytokines (tnfα) and emerging contaminants (nanoparticles), as well as proliferation/differentiation processes in response to growth factors (stem cell factor-scf). furthermore, we have recently investigated the presence of different antigens (cd34, cd117 and cd11b) in mussel immunocytes and changes in their expression following in vivo challenge with both gram(+) bacteria and autoctonous vibrio species. these data support the importance of developing the utilization of fc in comparative and environmental studies on invertebrate immunocytes. new knowledge of antimicrobial peptides in mediterranean mussel u rosani1, l varotto1, s domeneghetti1, a pallavicini2, g lanfranchi1,3, p venier1 1department of biology, university of padoa, padoa, italy 2department of biology, university of trieste, trieste, italy 3c.r.i.b.i., university of padoa, padoa, italy marine invertebrates are constantly surrounded by potentially invading microorganisms. their defence mechanisms involved circulating haemocytes that infiltrate injured tissues, encapsulate or phagocyte microbial cells and release cytotoxic factors such as lectins, complement factors and antimicrobial peptides (amps). mussels especially seem more resistant to infections and diseases than other edible bivalves. in the frame of the european integrated project food-ct-2005-007103, est production and gene expression profiling in m. galloprovincialis (lmk., 1919) are providing new interesting evidences. massive est sequencing of primary cdna and normalized libraries of immuno-stimulated mussels revealed a high sequence variability of amps, especially in haemolymph. these results confirm the importance of such molecules in innate immune response of marine invertebrates. selected immune-specific and immune-related trancripts were arrayed in the first mussel immunooligochip. preliminaries microarray and real time experiments showed various gene expression changes in mussels injected with bacterial antigens. moreover, the new generation sequencing technologies provide us plentiful and precise knowledge of the variability of these natural antibiotics, that can explicate better the ancient host-pathogen interactions and the adaptation strategies. proliferation of mussel haemocytes: effects of stem cell factor (scf) m betti, p gobbi, b canonico, c ciacci, s papa, l canesi1 department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy 1department of biology, university of genoa, genoa, italy stem cell factor (scf) is a member of mammalian haematopoietic cytokines, a group of glycoproteins that regulate the growth and differentiation of haematopoietic cells and functionally activate mature neutrophils or macrophages. in bivalve molluscs, circulating haemocytes resemble the monocyte/macrophage lineage. however, in these organisms, no information is available on the physiological control of haematopoiesis. in this work, the in vitro effects of heterologous scf on the haemocytes of mytilus sp. were investigated. flow cytometry (fc) analysis of control haemocytes identified 3 main cell subpopulations: granulocytes, agranulocytes, blast-like cells. a significant proportion of total haemocytes showed immunoreactivity towards anti-cd34 and anticd90 antibodies, utilised as markers of haematopoietic stem cells. scf induced significant changes in haemocytes functional parameters, indicating increase in phagocytic activity and reduction in lysosomal membrane fusion processes. moreover, fc analysis showed that scf significantly affected 23 both haemocyte number and cell cycle, mitochondrial activity, and immunoreactivity towards different anti-cd-antibodies. in particular, increases in the number of granular (phagocytic) haemocytes were observed; the effects of scf involved the activation of c-kit tyrosine kinase-like receptors. ultrastructural morphological studies in colcemide-treated haemocytes confirmed that the large majority of cells are able to enter the mitotic phase of the cell cycle. the results support the hypothesis that common pathways involved in modulating activity, proliferation and differentiation of immune cells are conserved from molluscs to mammals. effects of the protein pheromone er-1 isolated from the ciliate euplotes raikovi, on the phagocytic activity of the bivalve mytilus galloprovincialis l casarini, d malagoli, a vallesi1, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy 1dipartimento di biologia molecolare cellulare e animale, university of camerino, camerino, italy phagocytosis plays a central role in the cellmediated immune response and the study of cytokine-like molecules capable of modulating the phagocytic activity of immunocytes may contribute insightful information on the evolution of the immune response. previous experiments on the immunocytes of the mussel mytilus galloprovincialis suggested that regulation of the phagocytic activity involves campand pka-pathways. we have now analyzed the response of these immunocytes to the effects of a protein pheromone, denoted er-1, produced by the protozoan ciliate euplotes raikovi and characterized by an exclusively helical structure like il-2 and its cytokine family members. our results indicate that er-1 increases the immunocyte phagocytic activity, and this increase follows camp and pka-dependent signal transduction pathways. this indication is consistent with the activity of ciliate pheromones in mechanisms of self-nonself recognition and cell-cell adhesion. mytilus hemocytes as a model for nanoparticle toxicology in marine invertebrates c ciacci1, b canonico1, m betti1, g pojana2, a marcomini2, l canesi3 1department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy 2department of environmental sciences, university ‘ca foscari’ of venice, venice, italy 3department of biology, university of genoa, genoa, italy the potential for human and ecological toxicity associated with nanomaterials is a growing area of investigation. in mammalian cells, nanoparticles (nps) have been shown to induce inflammation and oxidative stress, and changes in cell signalling and gene expression. as the nanotechnology industries increase production, nanoscale products and by products will enter the aquatic environment, posing a possible threat to aquatic organisms. in particular, filter-feeding invertebrates may represent a unique target group for nanoparticle toxicology, since they have highly developed processes for the cellular internalisation of nanoand microscale particles, endocytosis and phagocytosis, that are integral to key physiological functions such as intracellular digestion and cellular immunity. in this work we show that in the hemocytes of the marine bivalve mytilus different types of engineered nanoparticles (carbon black, c60 fullerene, tio2, sio2) induce activation of different immune parameters (lysozyme release, activation of oxidative burst and no production) to a different extent depending on the np type and concentration, without significant cytotoxicity. only at higher concentrations mitochondrial damage was observed, this indicating pre-apoptotic processes. the inflammatory effects of nps were mediated by rapid activation of the stress activated p38 mapk. the results indicate that bivalve immunocytes represent a suitable model for investigating the potential effects of nps in aquatic organisms. session2 are really males the sterner sex? the immune responses of the clam tapes philippinarum as a case study v matozzo, m marin department of biology, university of padoa, padoa, italy for the first time, gender-related differences in immune responses of the clam tapes philippinarum were investigated. haemocytes from male, female and sexually undifferentiated clams were collected, and total haemocyte count (thc), haemocyte volume, capability of haemocytes to assume the vital dye neutral red (indicative of endocytotic activity), acid phosphatase and lysozyme-like activities in both haemocyte lysate (hl) and cell-free haemolymph (cfh), were evaluated. no statistically significant differences in thc values were observed. however, differing haemocyte size frequency distribution was found: the fraction of larger haemocytes (6-8 µm diameter, 200 fl volume) markedly increased in females, whereas the fraction of smaller haemocytes (< 5 µm diameter, < 200 fl volume) increased in both male and undifferentiated clams. significantly increased neutral red uptake was recorded in haemocytes from females. this was most likely related to the higher fraction of larger haemocytes in females, these cells being more actively involved in phagocytosis. no significant variations in lysozymelike activity was observed in hl, whereas in cfh enzyme activity resulted significantly higher in females with respect to male and undifferentiated animals. hl acid phosphatase activity was significantly higher in males with respect to females and undifferentiated clams, whereas no significant variations in enzyme activity was observed in cfh. overall, results obtained demonstrated that gender 24 related differences in immune responses occurred in t. philippinarum, and indicated that females had more active haemocytes than both male and undifferentiated clams, at least on the basis of the cellular parameters investigated. rhamnose-binding lectins in the compound ascidian botryllus schlosseri as multifaceted immune molecules n franchi, f schiavon, l ballarin department of biology, university of padoa, padoa, italy rhamnose-binding lectins are characterised by the presence of common aminoacid motifs (ygr, dpc, kyl) and the presence of eight conserved cysteine residues involved in four disulphide bridges responsible of the compact structure of the proteins. they have been described in many fish and few invertebrates. recently, in the compound ascidian botryllus schlosseri, we identified and characterised a rhamnose-binding lectin (bs-rbl) with opsonic activity, able to enhance phagocytosis of foreign target cells. in the present work, we continued our investigation on bs-rbl and studied the expression of the molecule through immunohistochemical and immunocytochemical analysis, using specific polyclonal antibodies, and in situ hybridisation on both colony sections and haemocyte monolayers. results obtained indicate the phagocytes as the site of synthesis of this kind of molecules as both the protein and the corresponding mrna were located in this cell type. with immunoblot analysis of colony lysates, we studied the expression of bs-rbl during the colonial blastogenetic cycle. the electrophoretic bands recognised by the anti-bsrbl antibody were of higher intensity during the cyclical colony generation change (take-over), characterised by massive apoptosis of zooid tissues, in addition, immunocytochemical analysis showed that that the frequency of haemocyte recognised by the anti-bsrbl antibody significantly increased during the take-over, and that, in addition to phagocytes with a labelled cytoplasm, the plasma membrane of other cell types (e.g., morula cells) resulted immunopositive, indicating that this molecule can recognise sugars on the surface of senescent cells, unexposed in healthy cells, and suggesting its involvement in efferocytosis. the incubation of haemocyte monolayers with purified bs-rbl results in the release in the culture medium of molecules recognised by antibodies raised against mammalian il-1α and tnfα, we have already demonstrated to have an immunomodulatory role. as these molecules are synthesised and released by cytotoxic morula cells (mc), the above observation indicate an immunomodulatory role of bs-rbl on mc activity. in addition, it has been recently demonstrated that fish rbl can recognise lps and lipoteichoic acid, basic components of gram(–) and gram(+) bacterial cell wall, respectively, suggesting an antibacterial role of these proteins. we are carrying out new experiments to verify this aspect in bsrbl. evolutionary analyses of sequence and intronexon structure of two lectin genes in ascidians c gissi, f griggio, f iannelli department of biomolecular sciences and biotechnology, university of milan, milan, italy lectins are a group of sugar-binding proteins that recognize specific carbohydrate structures and agglutinate cells by binding to cell-surface glycoconjugates. these proteins are involved in a variety of distinct role in different cells and species, and in invertebrates they are considered as recognition molecules that trigger defensive activities. lectins show a wide variety of protein architecture, thus they are classified into several families depending on the sugar-binding specificities and the sequence similarities of the carbohydraterecognition domain (crd). we have analysed the evolutionary history of the crd of two lectin genes, the galectin and the rhamnose-binding lectin (rbl), within chordates. in particular, these genes have been identified in the publicly available genomic sequences of ciona, oikopleura (tunicata) and branchistoma (cephalochordata), all protochordate species, and have been annotated using several bioinformatics methods and also with the help of est data. the evolutionary analyses have been carried out in a wide taxonomic sample including the homologous functionally characterized sequences of other deuterostome species, and the human annotated homologs. in addition to the traditional phylogenetic reconstructions based on amino acid sequences, we have also investigated the conservation of the intron-exon structure and evaluated the congruency between the results provided by these two different data types. our results show that the in most cases the single crds are delimited by introns, with linkers and additional protein domains encoded by distinct exons, and that tunicate genes evolve always very fast both at level of sequence and gene structure. individuation of a new metallothionein from the urochordate ciona intestinalis n franchi, l ballarin, e piccinni department of biology, university of padoa, padoa, italy metallothioneins (mts) are metal-chelating proteins occurring in animals, plants and prokaryotes, involved in detosification and immunity. they do not constitute a monophyletic protein family, but rather a superfamily of heterogeneous, low molecular weight, cysteine-rich peptides. current knowledge on mts comes mainly from vertebrate molecules which are composed of approximately 60 amino acids, including 20 cysteine residues, and folded into two independent domains when coordinating divalent metal ions. up to now, there are no descriptions of mts in invertebrate chordates althought it seems that the vertebrate structure is maintained also in other deuterostomes such as the echinoderms. 25 in this study, we present some data on mts of the solitary urochordate ciona intestinalis. we have cloned the transcript and characterized the gene of a new mt, codifying for 39 amino acids, including 12 cys residues (30 % of total amino acids, in accordance with other mts); moreover, the typical organization of cysteine residues in c-x-c motifs is conserved. the gene is composed of two introns (one inside the coding region and the other inside the 3’ utr region) and three exons. the 5’ untrascribed region contains several cis elements similar to those found in vertebrate mt genes such as: metal responding elements (mre), antioxidant responding elements and stat3, which are involved in constitutive and metal-related, ros and cytochines induction, respectively. the amino acid sequence of c. intestinalis mt shows only limited similarity with other mts, e.g., mytilus edulis mt (28.8 % identity), strongylocentrotus purpuratus mt (23.4 % identity) and sparus aurata mt (36.7 % identity). phylogenetic analyses with various methods are in progress. a preliminary study by atomic force microscopy of cytogenetic stability in a mdvintegrated chicken lymphoblastoid cell line s di bucchianico, mf giardi, d botti department of basic and applied biology, university of l’aquila, l’aquila, italy in previous works we have demonstrated that the chicken lymphoblastoid t cell line mdcc-msb1 is able to produce ovotransferrin and nitric oxide to a higher extent than cef (chicken embryo fibroblasts). this ability can be tentatively related to the integration of marek’s disease virus (mdv) genome. in addition, it is well known from literature that lymphocytes infected by mdv produce vil-8, with a chemotactic function towards other lymphocytes, to amplify viral spreading. on the other hand, it is also known that the virus undergoes a fragmentation prior to a random integration, preferentially in subtelomeric regions. the viral integration may induce a novel organization of chromatin architecture, with a modified gene expression. in our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. recently, atomic force microscopy (afm) technique was applied to study the structure of chromosomes at nanoscale level. structural analysis at high resolution of high molecular complexes provide detailed information such as 3dimensional topological data, mechanical behaviours, dynamic processes and molecular interactions. when scanning soft biological surfaces, the afm can achieve a resolution of about 1 nm. the vertical resolution is mostly determined by the afm scanner sensitivity, and typically is 0.01 nm. these features allow to investigate the different structure of chromatin in the regions of the viral insertion. on the basis of these data, the correlation between the localization of viral insertion and the different gene expression due to marek’s disease virus localization is investigated. session 3 self/nonself recognition in the ciliated protozoa: characterization of the pheromone gene family of euplotes nobilii a vallesi1, c alimenti1, a la terza1, g di giuseppe2, f dini2, p luporini1 1dipartimento di biologia molecolare cellulare animale, university of camerino, camerino, italy 2dipartimento di biologia, university of pisa, pisa, italy a family of seven allelic genes encoding self/nonself signal proteins (pheromones) in the polar (cold-adapted) marine hypotrichous ciliate, euplotes nobilii, were cloned from the somatic subchromosomic genome of the cell macronucleus. the determination of their full-length nucleotide sequences shows that their open reading frames specify proteins of 83 to 94 amino acid residues which represent the cytoplasmic pheromone precursors (pre-pro-pheromones). two proteolytic steps would thus remove the pre and pro segments formed by tightly conserved sequences, before the secretion of the structurally more variable mature proteins. at odds with respect to the general structural organization of the macronuclear genes of the hypotrichous ciliates, the 5’ region of all the cloned pheromone genes is significantly longer than the respective coding region (approximately, 350 versus 250 nucleotides). considered jointly with the tight sequence conservation, this feature implies that the 5’ region is site of specific activities in the mechanism of pheromone gene expression. antimicrobial peptide transcription is modulated after repeated exposure to heat-inactivated escherichia coli in drosophila melanogaster sl2 cell line d malagoli, s sacchi, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy recent findings in drosophila melanogaster indicate that the activation of the immune system immediately before an infection increases the resistance to pathogens, suggesting the possibility that a first exposure to an immunogen may strengthen the immune response to a second challenge. by rt-pcr experiments, we have observed in the d. melanogaster sl-2 hemocyte cell line that a second 6 h exposure to heat inactivated escherichia coli significantly increases the expression of the antimicrobial peptides (amps) drosomycin and diptericin, with respect to the first 24 h exposure. conversely, expression levels reached after the first 24 h exposure are not exceeded after a second 6 h immune challenge by the amps defensin and cecropin a1 and by the putative helical cytokine, dhf. surprisingly, the expression of the imd-related kinase dtak1, is increased by the first incubation with bacteria, while its expression is lower than in controls after the second exposure. the augmented expression 26 observed for drosomycin and diptericin is the consequence of the additive effect of the second exposure. in accordance with data present in literature, our observations suggest the existence in d. melanogaster of mechanisms devoted to control the intensity of the immune response. this regulation involves the possibility to modulate the expression of amps, in our case drosomycin and diptericin, and signal transduction regulators, e.g., dtak1. effects of bacterial injection and salinity stress on the humoral immune response of paracentrotus lividus (echinodermata) f drago department of animal biology “marcello la greca”, university of catania, catania, italy the altered expression of inos and lysozyme in the coelomocytes present in the wall of the gut of p. lividus, was observed after bacterial injection and osmotic stress by histochemical and immunocytochemical procedures. after 3 h from injection of bacteria a significant increase expression of inos was observed. this increase remained at the same level until 24 h post inoculum. conversely, the expression of lysozyme in the coelomocytes showed a relevant increase only after 24 h p.i. we also observed that the expression of inos and lysozyme after osmotic stress increased following the pattern registered after bacterial injection. in accordance with data present in literature, the present study identifies the coelomocytes of the sea urchin as the main effectors of defense response and indicate that the expression of inos increases faster than that of the lysozyme. does trichoplax (placozoa) produce resistance stages? l guidi, e cesarini, m balsamo department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy trichoplax adhaerens (placozoa) is currently regarded as the most primitive living metazoan. its mitochondrial and nuclear genomes have been wholly sequenced but its life cycle is still little known. placozoa reproduce asexually, but molecular evidences suggest the occurring of sexual events. under stress conditions, animals enter a degenerative phase (d-phase), and produce few, large oocytes. the existence of spermatozoa has not yet proved with certainty. the d-phase was induced in laboratory cultures of trichoplax, and after four weeks the production of oocytes was observed in vivo. after at least another week eggs were released through animal body disgregation. animals containing oocytes, and isolated eggs at 5, 6 and 10 weeks of age were studied under optical microscopy, sem and tem. the oocyte in the animal body at first shows only the oolemma, then produces an external envelope made up of 2-3 layers of different thickness and nature: at that stage it is released. early stages of cleavage were occasionally seen, in rare cases even in the animal body. very few isolated eggs in cleavage phase and many undivided ones were observed in the cultures: all showed an envelope with an identical morphology. that suggests that sexual reproduction is performed only in adverse environmental conditions, and that both quick-hatching and latehatching eggs might be produced. the formation process of the thick egg envelope and its fine structure appear very close to those reported for the asexual cysts of some ciliata. that supports a possible resistance function of the trichoplax eggs to stress conditions like those induced in the cultures, or those occurring in natural unstable habitats. trichoplax lives as an epibenthic form in the littoral marine environment, where the ecological conditions are quite variable and resistance stages are produced by a number of colonizing organisms (e.g., cysts of protists, gemmules of sponges). identification of a functional motif mobilized by retroelements in mrna from activated human lymphocytes e capelli1, s panelli2, g damiani3 1dipartimento di biologia animale, università di pavia, pavia, italy 2parco tecnologico padano, lodi 3igm-cnr, pavia, italy 3’ untranslated regions (3’ utrs) of eukaryotic mrnas commonly contain conserved repeated motifs involved in the control of mrna stability, i.e., through micro-rna (mirna) mediated regulation. following alignment of functionally related genes (i.e., of genes co-regulated in response to the same stimuli), we identified a number of conserved motives shared by genes involved in the same process (in particular: lymphocyte activation). primers were designed on these putative regulatory motives and used for rt-pcr fingerprintings on cdna from human t lymphocytes before and after pha pulse and il-2 activation. we noticed complex patterns of bands only when using cdna from activated cells. bands were cloned and sequenced. quantitative rt-pcr confirmed that the identified genes, containing the putative regulatory motives, were expressed only in activated cells. searches in mirna databases (mirbase) showed that one of our motives, cactn (3,4,3), corresponds to a sequence of a known mirna family, and thus confirmed its regulatory role. this motif, highly conserved across evolution, in ruminants is part of a short interspersed nuclear element (sine) called bov-a2. this sine is integrated in regulatory regions (promoter, 5’ and 3’ utr, introns) of many immune-related genes. these results are suggestive of connections among retroelements and the sharing of common regulative motives among functionally related genes. in particular, the role of retroelements could be to carry these important motives and help in their spreading among genes (seed) as already suggested by several authors. 27 our results, in turn, suggest a deep involvement of these processes in immunity and in the regulation of genes involved in the response of immune subsets like lymphocytes to external stimuli. session 4 an attempt to re-examine the immune role of ciona intestinalis hemocytes n parrinello, v arizza, m cammarata, a vizzini, d parrinello, m vazzana, tf giaramita, g salerno, m pergolizzi, ma sanfratello department of animal biology, university of palermo, palermo, italy in the last few years, interest in the mutual relationships between ascidians hemocytes and products of innate immunity gene repertoire has led to a more clear-cut knowledge of multifunctional role of hemocytes in ciona intestinalis immunity. this is a suitable approach that may include morphofunctional screening methods allowing us to disclose differentiation of the hemocytes, their activities in immune responses leading to a more precise and reasonable classification taking in account a multi-parameter approach. the genome sequence provided new insight in studying the innate immunity. bioinformatic approach and extensive in silico search have concerned immunorelevant molecules, gene expression patterns and some specific immune properties that contribute to clarify morpho-functional aspects. the involvement in immunity of hemoblasts, lymphocyte-like cells, hyaline amebocytes, and various types of granulocytes have been described. the ciona intestinalis prophenoloxidase activating system during lps inflammatory reaction m cammarata, v mangano, v arizza, c cianciolo, d parrinello, m vazzana, a vizzini, g salerno, n parrinello department of animal biology, university of palermo, palermo , italy phenoloxidases initiate melanin synthesis in almost all organisms, and are involved in different biological activities. in many invertebrates, defense reactions are linked to phenoloxidase activity and/or melanization, an innate response generally observed in wounded tissues. contact with a foreign molecule is able to trigger the prophenoloxidase (propo) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (po). it is generally accepted that the propo system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect propo activation steps has been made. after cells are stimulated by components of the pathogen associated molecular pattern (pamp), propo activation takes place via zimogenic serine proteinase in turn activated by pamp. in the present paper, we report on the ciona intestinalis propo system and related molecules, with particular focus on the biochemical, cellular and molecular components of the propo system in the tunic tissue following lps intratunic injection. tunic homogenate supernatant (ths), assayed with the dopa-mbth reaction, displayed ca2+-independent po activity that was increased by lps and further enhanced by proteases. in vivo experiments were performed by injecting isosmotic medium or lps, and ths was assayed for its po activity. to determine the po response at the injured site, an assay with dopa-mbth was performed in vitro. quinones were mainly contained in the tunic matrix area enriched with inflammatory cells around the injection site. microscopy observations and immunohistochemistry with anti-cinpo2 antibodies showed granulocytes and unilocular refractile granulocytes containing po. immunoblotting with anti-cinpo2 and sds-page zymograms demonstrated po activity linked to different bands as an effect of lps injection. these pos distinguishable by their size are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars. proliferating and propo activity of the hemocytes of ciona intestinalis m pergolizzi¹, i soderhall², h liu², k soderhall², n parrinello¹ ¹department of animal biology, university of palermo, palermo, italy ²department of comparative physiology, evolutionary biology centre, uppsala university, norbivagen 18a, uppsala 75236, sweden hemocytes are essential in tunicate immunity, performing functions such as phagocytosis, encapsulation and lysis of foreign cells. because of the ciona intestinalis phylogenetic taxonomic position between invertebrate and vertebrate, in the last decades, a molecular biology approach has been used to identify the gene expressed in the hemocytes of this tunicate. here, we tried to throw light on hematopoiesis in c. intestinalis. therefore, we used circulating hemocytes, as source of hpt stem cells, and separated them by discontinuous percoll gradient. we characterized the hemocytes with a blood cell staining method, such as maygrunwald-giemsa staining. then we cloned 2 predicted hemocyte genes: pcna, as marker to identify hpt stem cells and their progenitors; propo2, as marker for differentiation and for studying a well known innate defense mechanism of the hemocytes of c. intestinalis. we valued the expression for both the genes by rtpcr, and for pcna gene, by ish. we made primary culture attempts of the circulating hemocytes, and valued their vitality and proliferating activity by mtt assay. the results of the rt-pcr, showed that, the hemocytes of the percoll second band expressed more than those of the other bands both pcna and propo2 genes, whereas, by ish, the transcript for pcna gene was present in 4 of all the hemocyte types: hyaline and granular amoebocytes, urgs and lymphocyte-like cells. the primary culture attempts showed that the hemocytes of the second band seem to proliferate, arrange and form clumps 28 already 30 min after plating, and that these formations become brown within 24 h. session 5 characterisation of a progranulin in the medicinal leech, hirudo medicinalis j vizioli1, a grimaldi2, f croq1, c lefebvre1, c van camp1, g tettamanti2, m salzet1, j pestel1, m de eguileor2, p-e sautière1 1laboratory of annelids neuroimmunology fre 2933 cnrs. university of de lille 1, villeneuve d’ascq, france 2department of biotechnology and molecular science, university of insubria, varese, italy progranulin (pgrn) is a pluripotent growth factor expressed in many animal groups and involved in development, tumorigenesis, inflammation and wound repair. its mechanisms of action remain largely unknown. in animals, the progranulin gene encodes a cystein rich glycoprotein containing several repeated motifs called granulins (grn). granulins are 6 kda peptides produced by proteolytic cleavage of the precursor pgrn. in vertebrates, pgrn is expressed in leucocytes and is involved in defence as well as in wound healing events. its processing is linked to regulation mechanisms of inflammation. to better understand the function(s) of (pro)granulin, we investigated the role of this molecule in an invertebrate model, the medicinal leech hirudo medicinalis. the hirudo progranulin (hmpgrn) gene codes a 150 kda protein containing 16.5 putative grn motifs and showing a high similarity with human and other animal pgrn. hmpgrn is constitutively expressed in leech body and 24 hours after injury the protein accumulates as granules in granulocytes, belonging to fibrovascular tissue (a peculiar hirudinean tissue). these cells, after surgical lesion, increase in number and migrate towards the wound site. in central nervous system (cns) of mammals this molecule is expressed by neurons and microglial cells and probably acts as a neurotrophic factor. in human, mutations of pgrn gene are linked to frontotemporal lobar degeneration and other neurodegenerative pathologies. in leech cns, hmpgrn gene is constitutively expressed in neurons and is upregulated during nerve cord repair. immunohistochemical analysis revealed the accumulation of the protein in neurons cell bodies after injury. taken together, these data suggest that hmpgrn might play a neurotrophic role and contribute to the repair process. since progranulin structure is highly conserved in animals, its study in leech can increase our knowledge on its functions in normal or injured tissues and lead to new therapeutic investigations. session 6 cellular and molecular responses of the teleost fish dicentrarchus labrax against in vivo challenge with nodavirus and photobacterium damselae g scapigliati, f buonocore, e randelli, d casani, s meloni, d pietretti, imaquanim1 consortium dipartimento di scienze ambientali, università della tuscia, viterbo, italy 1www.imaquanim.dfvf.dk the need of high quality farmed fish production in conditions to be safe for the consumer and for the environment requires much efforts to maintain fish health and to avoid spreading of infectious diseases. this, in turn, requires much knowledge of immune responses against most dangerous pathogens that, in the case of mediterranean sea bass (dicentrarchus labrax) are a nodavirus (retrovirus) (nnv) and the gram-negative photobacterium damselae subsp. piscicida (phda). in a concerted scientific action aimed to study cellular and molecular responses of sea bass against these pathogens, fish (20-30 grams) have been challenged intraperitoneally with a modest dose of nnv that caused 12 % mortality (at 30 days), then treated again after 48 days with same virus. alternatively, other fish groups have been treated with virulent phda administered in water, followed by a similar treatment after 30 days. fish from all experimental groups were sampled for molecular analysis by preparing rna from homogenised organs and tissues, copying in cdna and analysing by quantitative pcr for immunomodulatory expressed genes. this analysis was performed through pcr array using a protocol developed by our group. fish were also sampled to obtain leucocytes for cellular analysis to investigate proliferation, lymphocytes profile, and sera analysed for the presence of pathogenspecific antibody by elisa. proliferation of leucocytes in response to inactivated nnv was detected in vitro at day 43 in pbl and gills, and at day 73 in pbl, gills, and head kidney. no detectable in vitro proliferation was observed when adding phda. serum analysis by indirect elisa of pathogenspecific antibody showed that the treatment with nnv induced a specific response observed from day 47 onwards. elisa analysis of phda-treated fish showed the presence of specific antibody at days 10 and 44. however, the observed presence of detectable amounts of antibody against nnv and phda in control fish raises interesting questions. several genes cloned in sea bass have been used for expression array analysis using pcr methodologies, and results obtained with fish challenged with nnv showed modulation of antiviral proteins interferon (type i) and mx, of inflammation-related proteins il-1, cox-2, il-10, and tgf-beta, whereas t cell genes tcr, cd4, and cd8 were not significantly affected. when analysing transcripts from fish challenged with phda, proinflammatory peptides il-1 and cox-2 showed a potent stimulation at 6 h after challenge, whereas t cell genes were only slightly upregulated after boosting. interestingly, none of the immunomodulatory genes analysed was downregulated by nnv and phda challenges. 29 developmental expression of mhc class ii in sea bass dicentrarchus labrax (l) l guerra, l abelli1, e randelli, f buonocore, am fausto, s picchietti department of environmental sciences, tuscia university, viterbo; italy 1department of biology & evolution, section comparative anatomy, university of ferrara, ferrara, italy the major histocompatibility complex (mhc) class i and class ii molecules play a pivotal role in vertebrate immune response to antigenic peptides. mhc class ii genes have been identified in various teleost fish species, but no reports on their expression in sea bass development are still available. sea bass eggs, larvae from 2 to 92 days posthatching (dph) and juveniles, were analysed for the occurrence of mhcii-β transcripts. these were first detected in 4 dph larvae by rt-pcrs, while q-pcr revealed their significant increase until 92 dph (p<0.001). an early role of the mhcii-β molecules in the larval development is therefore suggested. in fact, cd4+ t cells appear later in sea bass (51 dph), when the thymus is well developed. at this stage the in situ hybridization of mhcii-β mrna labelled cells in the inner zones of the thymic paired glands. from 75 dph on, the signal was detected in the thymic cortex and mainly in the outer and inner medulla. in one year old thymus numerous stromal cells were mhcii-β+. this report enlightens possible roles of stromal components in the mechanisms of thymocyte selection in fish. mx protein and interferon in sea bass (dicentrarchus labrax l.): an evolutionary perspective f buonocore, d casani, e randelli, s costantini1, am facchiano1, j zou2, cj secombes2, g scapigliati department of environmental sciences, university of tuscia, viterbo, italy 1laboratory of bioinformatics and computational biology cnr, istituto di scienze dell'alimentazione, avellino, italy 2scottish fish immunology research centre, aberdeen university, aberdeen, uk the interferons (ifns) are a large family of soluble cytokines involved in the immune response against viral pathogens. three families of ifns have been identified in mammals (type i, type ii and type iii) and, recently, homologues of type i and type ii genes have been found in various teleost fish species. our work has been focused on the identification of ifn and mx homologues from sea bass (dicentrarchus labrax), a fish of high economic impact for south mediterranean countries. moreover, we analysed the ifn gene structure and the expression of both ifn and mx by real time pcr after stimulation with poly i:c. finally, we predicted by template-based modelling the 3d structure of ifn and hypothesized its possible residues of interaction with the putative sea bass ifn receptor on the basis of the correspondent human complex. the sea bass ifn cdna consists of 1047 bp that translates in one reading frame to give the entire molecule containing 185 amino acids. the analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential n-glycosylation sites. four mx protein cdnas were obtained and they translated in a putative protein of 652 amino acids. the sea bass ifn gene contains four introns as with other type i ifn teleost genes, except medaka that contains three introns. real time pcr was performed after poly i:c stimulation of dlec cell line and head kidney leukocytes to investigate the expression of sea bass ifn and mx and an induction was observed for both genes. the predicted 3d structure of sea bass ifn is characterized by an “all-alpha” domain that shows an “up-down bundle” architecture made of six helices (abb’cde). the two cysteine residues present in the sequence (i.e. cys23 and cys126) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. these data add new insights on the evolution of the ifn system in teleosts and vertebrates more generally. identification of vaccine candidates against photobacterium damselae subsp. piscicida f andreoni, r boiani, i bianconi, g serafini, f gorini, m magnani department of biomolecular science, university of urbino “carlo bo”, urbino, italy photobacterium damselae subsp. piscicida is the etiological agent of pasteurellosis, which is one of the most devastating bacterial diseases affecting the culture of gilt-head seabream and seabass in mediterranean area. to date, efficient vaccines against the pathogen are not available. the aim of this study is the identification of vaccine candidates against p. damselae subsp. piscicida, using the reverse vaccinology, a new approach which starts from the in silico analysis of the genome sequence. a genomic cosmid library of p. damselae subsp. piscicida strain ncimb 2058 was constructed. sequences, obtained by the shotgun method, were analyzed in silico by glimmer 2.13 and blastp for the identification of the proteins potentially encoded by the bacterium. protein localization was predicted by psortb, tmpred and signalp softwares, to select surface-exposed or secreted proteins. the analysis of 12 cosmid clones (421575 bp) identified 297 orfs, 138 of which have a cellular localization spanning from the inner membrane to outside the bacterium. among these proteins, 35 potential vaccine candidates were selected for expression as recombinant proteins. orfs were cloned into pet-21b vector, expressed as c-terminal his6-tag proteins in e. coli and purified by metal-affinity chromatography. the potentiality of each antigen to become a vaccine ought to be tested by in vitro assay and by challenge experiments in fish. the complete genome sequencing of p. damselae subsp. piscicida will provide us with the inclusive set of 30 proteins potentially encoded by the bacterium and new antigenic molecules to use for vaccination. analysis of the immunoglobulin heavy chain gene locus of the antarctic teleost chionodraco hamatus u oreste, s varriale, v avagliano, mr coscia istituto di biochimica delle proteine, cnr, napoli, italy in teleosts, as in mammals, immunoglobulin heavy chain (igh) primary transcripts are alternatively spliced into mature transcripts encoding either the membrane receptor or the secreted antibody. however, the cryptic donor splice site in the last heavy chain constant domain (ch4), used in mammalian igh gene, is absent in teleosts igh. thus, the exon for the transmembrane region splices to the 3’ end of the ch3 exon. as a result, the synthesized protein lacks the entire ch4 domain. we have previously reported that in chionodraco hamatus, as in the majority of antarctic teleosts, an atypical splicing mechanism generates the membrane form, excluding two entire domains and including two additional 39-nt exons. to investigate what makes this specific splicing type possible, a c. hamatus genomic dna fragment was sequenced. this analysis revealed that the two 39-nt exons are part of reverse complement sequences (rcs) of an upstream gene region (ch3 exon). three rcs regions are present in the gene locus (rcs1, rcs2 and rcs3). each rcs shares, on average, 89.7 % of nucleotide identity with the respective counterpart that is present in the ch3 exon. although sharing all the splicing signatures, the 39-nt sequence, present in rcs1, was missing in the mature transcript. to explain the reason why the rcs1 39–nt element is spliced out from the transcript, the folding of the partial primary transcript encompassing the sequenced genomic region was predicted by using the mfold computational tool. the results showed a very compact structure stabilized by a long duplex comprising the entire ch2 and ch4 exons bound to the rcs1 sequence. the presence of both ch3 exon and the rc1 39-nt element in the duplex may account for the splicing mechanism observed. c. hamatus belongs to the radiation occurred during the most relevant cooling geological period. this may suggest that the environmental changes directed the adaptive evolution of immunoglobulin gene locus. an immunoglobulin transmembrane dimerization motif conserved throughout vertebrate evolution s varriale1.2, a merlino2, mr coscia1, v avagliano1, l mazzarella2, u oreste1 1istituto di biochimica delle proteine, cnr, napoli, italy 2dipartimento di chimica, università di napoli ‘federico ii’, napoli, italy immunoglobulins (ig), the key molecules of the adaptive immune system, are present in all vertebrate classes, but sharing very low sequence identity and differing in many features such as heavy and light chain isotype number, polymeric assembly, heavy chain domains number, carbohydrate content. however ig molecules from different species do share a few important molecular characteristics. these include diversity of the variable domains achieved by somatic recombinatorial events, dimerization of the heavy chain, alternative splicing of premrna to synthesize the secretory or the membrane-bound ig form. the functionality of the membrane-bound ig depends on its ability to link the extracellular antigen recognition event to the cytoplasmic signal transducing machinery. in the present work we are aimed at identifying the structural motif that is universally conserved in vertebrate igs, and is responsible for this activity. structural models of the igtm homodimer were obtained using ig from species of different classes: heterodontus francisci, chionodraco hamatus, pleurodeles waltl, anas platyrhinchos, and homo sapiens. several molecular dynamic simulations were performed in a lipid bilayer using two copies of models of ig transmembrane (igtm) sequences from each species, obtained by homology modeling. all predicted structures of the igtm homodimers displayed similar packing interfaces, characterized by a high degree of surface complementarity. from the analysis of the models, we identified fxxxf as the motif presumptively responsible for the interaction and, in consequence, for the receptor stability and functionality. 31 isj 4: 51-xx, 2007 isj 4: 51-54, 2007 issn 1824-307x visions and perspectives evolution game: which came first, the receptor or the ligand? m mandrioli, d malagoli, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted april 24, 2007 abstract on the basis of a bioinformatic approach, we suggest that in invertebrates many ligands interact with a single, ancestral and generalized receptor driving ligand evolution. in vertebrates, on the other hand, the occurrence of gene/genome duplications induced the shift to a ligand-directed evolution of receptors. key words: cytokines; cytokine receptors; ligand-receptor evolution immunocytochemical approaches revealed that a number of invertebrate species contains mammalian cytokine-like molecules (ottaviani et al., 2004; malagoli et al., 2007). moreover, mammalian cytokines influence some invertebrate immune functions, such as cell motility, chemotaxis, phagocytosis and cytotoxicity (ottaviani et al., 2004) suggesting the presence of cytokine receptors conserved during evolution from invertebrates to vertebrates. il-7 receptor complex (kondo et al., 1993; noguchi et al., 1993; russell et al., 1993). the possibility that receptors able to bind different cytokines may be present in invertebrates prompted us to look at the evolutionary interrelationships between cytokines and their receptors from invertebrates to vertebrates. we took a comparative bioinformatic approach based on the screening of the wholly sequenced drosophila melanogaster, anopheles gambiae and caenorhabditis elegans genomes for the presence of cytokineand related receptor-coding genes. standard tools such us blast and clustalw working at both dna and protein sequence level were used. the presence of cytokine receptors has been reported for platelet-derived growth factor (pdgf)ab and transforming growth factor (tgf)-β1 (kletsas et al., 1998) in molluscan immunocytes, for interleukin (il)-1-, il-2-, il-6and interferon (ifn)-γ in sea star cells (legac et al., 1996) and for ifn-γ in tobacco hornworm larvae (parker and ourth, 1999). interestingly, studies performed on il-2 and corticotrophin-releasing hormone (crh) revealed that the mammalian cytokines il-1α, il-1β, il-2, tumor necrosis factor (tnf)-α, tnf-β and crh may bind the same receptor in molluscan immunocytes, suggesting the existence of an ancestral common receptor on the invertebrate cell membrane (ottaviani et al., 1994, 1995). this result indicates that different cytokines could interact with a single receptor in invertebrates. the question about the lead role of either receptor or the ligand during evolution could have two different replies. the first consists in a liganddirected evolution of receptors, where the former acts as selective agent in the evolution of the latter. in this case, a single ligand may have interacted with different receptors, whose structure has been optimized during evolution in order to increase the specificity of the interaction between ligand and receptor (fig. 1a). if this hypothesis is true, we should find ligands that have been strongly conserved from invertebrates to vertebrates. the second reply envisages the evolution of ligands depending on receptor structure and the receptor acting as a selective agent in the evolution of ligands. in this view, different ligands can interact with a single receptor that serves as a selective agent driving ligand evolution. this would mean that receptors which have been strongly conserved from invertebrates to vertebrates could be identified (fig. 1b). the hypothesis that a single ancestral receptor might bind different ligands is strengthened by data reporting that the mammalian γ chain of the il-2 receptor is also functionally involved in the il-4 and ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology via campi 213/d in order to prove these hypotheses, we studied the evolutionary relationship between crh, tgf-β 41100 modena, italy e-mail: ottaviani.enzo@unimore.it 51 mailto:ottaviani.enzo@unimore.it b. receptor-directed ligand a. ligand-directed receptor a single ligand binds different receptors. a single receptor recognizes and binds different ligands. strong functional constriction of ligand sequence strong ligand conservation during evolution strong functional constriction of receptor sequence strong receptor conservation during l r r r r l l l fig. 1 comparison of the putative mechanisms involved in the evolution of ligands (l) and receptors (r) in invertebrates. family ligands and epidermal growth factor (egf) and their specific receptors. the analysis of genes coding for crh and the two related receptors showed that crh receptors are found in invertebrates with a similarity to mammalian homologues ranging from 43 % to 51 %, whereas the only conserved crh-coding gene was identified in the lepidopteran mamestra brassicae (malagoli et al., 2002). a similar evolutionary pattern was obtained with the tgfβ receptors, which showed a 49 % to 69 % similarity to mammalian homologues. the present data are in agreement with those reported in raftery et al. (1999), who confirmed that more anopheles and drosophila genes code for putative tgf-β family ligands (raftery et al., 1999). overall, these findings indicate that there are far fewer receptor-genes than ligand-coding ones (raftery et al., 1999). egf analysis gives a similar picture: egf receptorcoding genes are conserved in invertebrates with a sequence similarity ranging from 45 to 67 %. our bioinformatic analysis suggests that receptor-coding rather than ligand-coding genes are conserved between vertebrates and invertebrates and that present receptors are more similar to their ancestor than ligands. moreover, data from invertebrates suggest that more ligands may interact with a single receptor (ottaviani et al., 1994), supporting the hypothesis that ligand evolution is receptor-dependent. our assumption founded on cytokineand cytokine receptor-coding sequences is strengthened by data on the evolution of estrogen receptors (schwabe and teichmann, 2004). in particular, schwabe and teichmann (2004) suggested that the steroid receptors are much more ancient than previously thought and that the evolution of nuclear receptors is not liganddirected. using a new structure prediction algorithm, developed to find helical cytokines in human databases (conklin, 2004), we recently have found in d. melanogaster a molecule with a structure similar to that of mammalian helical cytokines (malagoli et al., 2007). this molecule may be involved in the fly immunity, however no information on its receptor is available at present. we, therefore, suggest that receptor structure undergoes a more tights constraint than ligand origin and that receptors drive ligand evolution in invertebrates. this tendency could also be valid in vertebrates, even if, given the occurrence of gene/genome duplications in vertebrate lineage (ohno, 1970; panopoulou et al., 2003), a new element has to be added to the previous receptorligand scenario. the drastic increase in genome size and gene number occurred in vertebrates as a result of two rounds of whole-genome duplication (2r hypothesis) or one complete genome duplication plus many segmental duplications (ohno, 1970; panopoulou et al., 2003). this led to the creation of additional gene copies and the evolution of new protein functions (ohno, 1970; hughes, 1999). the gene/genome duplications at the boundary between invertebrates and vertebrates are responsible for the presence of more genes coding 52 genome duplication receptor specialization for a unique ligand duplication of receptor gene brings to the presence of different receptors allowing the specialization of a receptor towards a single ligand. co-evolution of ligand and receptors r1 l1 l2 l3 r1 r1r1 r1a r1b r1c genome duplication receptor specialization for a unique ligand duplication of receptor gene brings to the presence of different receptors allowing the specialization of a receptor towards a single ligand. co-evolution of ligand and receptors r1 l1 l2 l3 r1 r1r1 r1a r1b r1c fig. 2 transition of the evolution mechanism of ligands (l) and receptors from a receptor(r)-directed mechanism to a ligand-directed one in vertebrates. for receptors in the vertebrate lineage, so allowing the differentiation of receptors towards different ligands. in vertebrates, therefore, we observe a transition from common receptors for different ligands to a specific receptor for each ligand. this phenomenon modifies the invertebrate relationship between receptors and ligands, and permits ligands to drive the evolution of newly duplicated receptors (fig. 2). the transition from a protein with a generalized recognition to more specialized proteins is a common model in evolutionary biology, as observed in the evolution of enzymes (jensen, 1976; o'brien and herschlag, 1999). hughes al. phylogenies of developmentally important proteins do not support the hypothesis of two rounds of genome duplication early in vertebrate history. j. mol. evol. 48: 565–576, 1999. jensen ra. enzyme recruitment in evolution of new function. annu. rev. microbiol. 30: 409-425, 1976. kletsas d, sassi d, franchini a, ottaviani e. pdgf and tgf-β induce cell shape changes in invertebrate immunocytes via specific cell surface receptors. eur. j. cell biol. 75: 362366, 1998. kondo m, takeshita t, ishii n, nakamura m, watanabe s, arai k, et al. sharing of the interleukin-2 (il-2) receptor gamma chain between receptors for il-2 and il-4. science 262: 1874-1877, 1993. in conclusion on the basis of the present bioinformatic findings and reports in the literature, we suggest that in invertebrates many ligands interact with a single, ancestral and generalized receptor driving ligand evolution. in vertebrates, on the other hand, the occurrence of gene/genome duplications induced the transition to a liganddirected evolution of receptors. this transition represented an evolutive advantage because it couples a more refine signalling to a minor sensitivity to receptor gene mutation. legac eg, vaugier l, bousquet f, bajelan m, leclerc d. primitive cytokines and cytokine receptors in invertebrates: the sea star asterias rubens as a model of study. scand. j. immunol. 44: 375-380, 1996. malagoli d, mandrioli m, ottaviani e. cloning and characterisation of a procorticotrophin-releasing hormone in the izd-mb-0503 immunocyte line from the insect mamestra brassicae. peptides 23: 1829-1836, 2002. acknowledgements this work was supported by miur (italy) grants to eo. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. references conklin d. recognition of the helical cytokine fold, j. comput. biol. 11: 1189-1200, 2004. 53 noguchi m, nakamura y, russell sm, ziegler sf, tsang m, cao x, et al. interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor. science 262: 1877-1880, 1993. panopoulou g, hennig s, groth d, krause a, poustka aj, herwig r, et al. new evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes. genome res. 13: 1056-1066, 2003. o'brien pj, herschlag d. catalytic promiscuity and the evolution of new enzymatic activities. chem. biol. 6: r91-r105, 1999. parker ms, ourth dd. specific binding of human interferon-gamma to particulates from hemolymph and protocerebrum of tobacco hornworm (manduca sexta) larvae. comp. biochem. physiol. 122b: 155-63, 1999. ohno s. evolution by gene duplication. springerverlag, new york, 1970. ottaviani e, caselgrandi e, franceschi c. cytokines and evolution: in vitro effects of il-1α, il-1β, tnf-α and tnf-β on an ancestral type of stress response. biochem. biophys. res. commun. 207: 288-292, 1995. raftery la, sutherland dj. tgf-beta family signal transduction in drosophila development: from mad to smads. dev. biol. 210: 251-268, 1999. russell sm, keegan ad, harada n, nakamura y, noguchi m, leland p, et al. interleukin-2 receptor gamma chain: a functional component of the interleukin-4 receptor. science 262: 1880-1883, 1993. ottaviani e, franchini a, caselgrandi e, cossarizza a, franceschi c. relationship between corticotropin-releasing factor and interleukin-2: evolutionary evidence. febs lett. 351: 19 -21, 1994. schwabe jw, teichmann sa. nuclear receptors: the evolution of diversity. sci. stke 217: pe4, 2004. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. 54 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj 5: xxx-yyy, 2008 isj 5: 162-179, 2008 issn 1824-307x review the antimicrobial peptides of the immune response of shrimp xf zhao, jx wang school of life sciences, shandong university, jinan, shandong, china accepted october 23, 2008 abstract the cultivation of penaeid shrimp is a worldwide economic activity which has the potential to contribute to increasing shrimp production. however, penaeid shrimps are susceptible to bacterial and viral diseases, and may thus cause significant losses to the aquaculture industry. in view of this, it is imperative to understand the immune response of shrimp against pathogens as this could help in devising efficient strategies to control, and eventually eradicate, shrimp diseases. at present, a considerable number of research studies on the identification and characterization of antimicrobial peptides/proteins (amps) in penaeid shrimps. such research activities will contribute to finding solutions to shrimp diseases. amps are widespread in animals and plants, involved in their innate immunity, and considered as the front liners of host defense against pathogens. in penaeid shrimps, eight kinds of amps have been found. these are the penaeidins, whey acidic protein (wap) domain containing proteins [crustins and single wap domain containing peptides (swd)], antilipopolysaccharide factors (alfs), lysozymes, a c-type lectin, histones, anionic hemocyanins, and peritrophins. in this study, the structures, distributions, expression profiles, phylogenetic evolution, and functions of some amps are discussed, focusing on the wap-domain containing peptides and alf in penaeid shrimp. key words: antimicrobial peptides; innate defense effectors; innate immunity; penaeid shrimp introduction the cultivation of penaeid shrimp is an important economic activity in the world. this industry, however, has been suffering serious problems brought by viral and bacterial diseases. one specific disease is the white spot syndrome virus (wssv) infection which has caused a drastic decline in production and multi-national economic losses. based on the report of the fisheries and aquaculture department of food and agriculture organization (2007), there is an exceeding 2.4 million tons per annum of shrimp global aquaculture production. however, up to 25 % of this production was estimated to have been lost due to diseases. given that pathogenic diseases are one significant cause of production and economic loss in the shrimp industry, this phenomenon calls for an urgent understanding of the immune defenses of shrimp. at present, research studies are being conducted to ___________________________________________________________________________ corresponding author: jin-xing wang school of life sciences shandong university jinan, shandong 250100, china e-mail: jxwang@sdu.edu.cn examine the innate immunity of shrimp, and such activities have been continuously contributing to the development of shrimp aquaculture. in fact, it has been found that comparable to insects, the innate defense of shrimp is triggered by the pattern recognition receptors, such as the gram-negative binding proteins (vargas-albores et al., 1997; yepizplascencia et al., 1998; jimenez-vega et al., 2002; roux et al., 2002; sritunyalucksana et al., 2002; romo-figueroa et al., 2004; cheng et al., 2005; du et al., 2007; lin et al., 2008) and the c-type lectins (luo et al., 2006; liu et al., 2007; sun et al., 2008). generally, the recognition of non-self activates a proteolytic cascade of serine proteases that amplify the signal and trigger downstream effector responses. this becomes possible through the signal transduction pathways which lead to the elimination of the invader. moreover, the serine proteinase and its inhibitors was found in shrimp (okumura, 2007), and the toll-like receptors and other signal pathway molecules also reported in several shrimp (arts et al., 2007; yang et al., 2007; yang et al., 2008). anti-microbial peptides (amps) are a diverse group of innate immune effector molecules in multi162 cellular organisms. they are considered as effector molecules for immune cells that prevent or withstand microbial infection. similar to those found in other animals, amps are also key factors in the innate immunity of shrimp (bachère et al., 2004). since amps play a significant role in the inherent immunity of shrimp, research studies have been actively focusing on the identification and characterization of amps in penaeid shrimp. in fact, eight kinds of amps have been found in penaeid shrimps. these are the penaeidins (destoumieux et al., 1997, 1999, 2000; cuthbertson et al., 2004; muñoz et al., 2004; kang et al., 2004, 2007), whey acidic protein (wap) domain containing proteins [crustins and single wap containing peptides (swd)] (gross et al., 2001; amparyup et al., 2008a; jia et al., in press), antilipopolysaccharide factors (alfs) (gross et al., 2001; liu et al., 2005; liu 2006; somboonwiwat et al., 2005, 2008; tharntada et al., 2008), histones (patat et al., 2004), hemocyanin (destoumieux-garzon et al., 2001; zhang et al., 2004), lysozymes (hikima et al., 2003 sotelo-mundo et al., 2003; bu et al., 2008; de la re vega et al., 2006; burge et al., 2007; xing et al., in press), a c-type lectin (sun et al., 2008), and peritrophins (loongyai et al., 2007). this study presents a discussion of the structures, distributions, phylogenetic evolution, expression profiles, and functions of some amps, particularly on the wap-domain containing peptides and alf from penaeid shrimp. whey acidic protein (wap)-domain containing peptides (wdps) a major milk protein in most mammals, wap, has eight cysteine residues arranged to form a tightly packed structure called a four-disulphide core (4-dsc) at the carboxyl terminus (hennighausen and sippel, 1982). these wap domain-containing proteins are found to prevail among metazoans (beg, 1995; devinoy et al., 1988; ali et al., 2002; carro et al., 2004; furutani et al., 2004). they are further found to have highly diverse biological functions, including proteinase inhibition (ranganathan et al., 1999; schalkwijk et al., 1999; ota et al., 2002), antimicrobial activity (relf et al., 1999; hagiwara et al., 2003), and association to ovulation (garczynski et al., 1997). in addition, wap-domain proteins have antiviral functions, specifically against the human immunodeficiency virus (alvarez et al., 2008). crustins, which are anti-microbial peptides containing a wap-domain, were first identified in the shore crab carcinus maenas, characterized as cysteine-rich 11.5 kda antimicrobial peptides which function against gram-positive bacteria (relf et al., 1999). there have been more than 50 crustins or crustin-like peptides reported to have been found from a variety of decapods, including crabs, lobsters, shrimp, and crayfish (refer to the review of smith et al., 2008). in this study, they were termed as wap-domain containing peptides (or wdps) and were classified into two sub-families, namely, crustins and swd (the justification for such is discussed below). these wdps are apparently a large family of antimicrobial peptides ubiquitous among penaeid shrimp. in fact, the cdnas of crustins and wdps have been reported to be present in a variety of penaeid shrimp, including litopenaeus vannamei, litopenaeus setiferus (gross et al., 2001; bartlett et al., 2002; vargasalbores et al., 2004), penaeus monodon (chen et al., 2004; supungul et al., 2004, 2008; amparyup et al., 2008a, b), marsupenaeus japonicus (rattanachai et al., 2004), fenneropenaeus chinensis (zhang et al., 2007; jia et al., in press), farfantepenaeus paulensis, farfantepenaeus subtilis, farfantepenaeus brasiliensis, and litopenaeus schmitti (rosa et al., 2007). accordingly, the crustins in shrimp are diverse in amino acid sequences. however, they are conserved with the c-terminus of 12 cysteine residues, thereby leading it to be termed as crustindomain, in which a single wap domain is contained. the swds have no crustin domain, and only contain a single wap-domain (8 cysteine residues). classification of shrimp wdps recently, the crustins (wpds) in crustaceans were comprehensively reviewed by smith et al. (2008). in the review they discussed three main types of crustins (crustin type i, ii and iii) in crustaceans. here, we focused on the crustins and swds in penaeid shrimp, including the new functions of the peptides. there have been several studies that have revealed the presence of crustins and swds in different penaeid shrimp species (refer to the above citations), in this study, most of the sequences wdps in penaeid shrimp and in other crustaceans were collected, including some expressed sequence tags (est) from the genbank database. a multiple alignment analysis for the amino acid sequences (fig. 1) and the phylogenetic analysis of the proteins were performed. the neighbor-joining tree revealed that the wdps in crustaceans could be divided into four different clusters (fig. 2), namely, crustins i and ii, carcinin and carcinin-like peptides, and the swd. it is noteworthy to mention that in this study, our classification is considerably different from that of smith et al. (2008). the crustin type i that they discussed in their study is similar to carcinin and carcinin-like peptide discussed in this study. similarly, the crustin type ii is equivalent to our crustins i and ii, and the crustin type iii is equivalent to our swds. the wdps in the penaeid shrimp are divided into three classes, namely, crustin i, crustin ii, and swds. the first class is characterized by the following: (1) a relatively conserved signal peptide, (2) an n-terminal glycine-rich domain, and (3) a cterminal cysteine-rich domain (12-cysteine crustin domain) with the following signature: c1(x3)c2(xx)c3c4(x16)c5(x6)c6(xn)c7(x5)c8(x5) c9(x5)c9c10(x3)c11(x5)c12. the second class of crustins have similar sequence domains as class i, but in terms of signal peptide, there are sequence differences between them. another difference between them lies in the sequence of their crustin domain, as crustin ii have the following signature: c1(x2)c2(x7)c3c4(x4)c5(x6)c6(xn)c7(x5)c8(x5)c9 c10(x3)c11(x5)c12, specifically in the residue numbers between c4 and c5 (fig.1a). finally, the 163 164 fig. 1 alignment of amino acid sequences of crustins (a), and swds (b), and the domain signature of penaeid shrimp and other crustacea (c). fch, fenneropenaeus chinensis; lse, litopenaeus setiferus; lva, litopenaeus vannamei; mja, marsupenaeus japonicus; pmo, penaeus monodon; the sequences of signal peptides are presented in yellow, the identical cysteines that characterize crustin or wap domain are in purple, and the wap domains are shown in blue. class iii wdps are single wap domain-containing peptides (swd) which are characterized by the following: (1) a highly conserved signal peptide, (2) a prolineand arginine-rich motif between the signal peptide and the wap domain, and (3) a wap-domain (8 cysteine residues) in the c-terminus with the signature: c1(x9)c2(x6)c3(x5)c4(x5)c5c6(x3)c(x3)c (fig.1b). in crustaceans, carcinin and carcinin-like peptides have a signal peptide and a crustin domain, without the n-terminal glycine-rich domain. moreover, swds are significantly different from crustins i and ii, and carcinins because they have no crustin-domain in their sequences. we therefore consider the idea that the four groups of wdps in crustaceans should be divided into two sub-families, namely, the crustins (which present crustin-domain in their sequences) and the swds (which only have wap-domain). as such, these two sub-families also have different functions in vitro (as discussed below). structure comparison of shrimp wdps with other amps to date, a wide variety of amps in metazoans have been identified. on the basis of sequence and structural features, these cationic amps can be grouped into three classes: (i) the linear peptides which form α-helices and do not contain cysteine residues; (ii) the cyclic peptides which contain cysteine residues; and (iii) the peptides with an over-representation in one or two residues, such as proline, glycine, arginine, and tryptophan (bulet et al., 2004). several antibacterial glycine-rich polypeptides have been isolated from various insect species. they are actually considered effective against gram-negative bacteria and are inactive against gram-positive bacteria, yeasts and mammalian cell lines (mackintosh et al., 1998). another example is that of short-chain proline-rich peptides, which are mostly active against gram-negative bacteria, while the gram-positive cells remain generally unaffected (bulet et al., 1999). furthermore, the cyclic peptides containing cysteine residues, like insect defensins, are active against a wide range of gram-positive bacteria and only for a few gramnegative bacteria, fungi and yeasts (bulet et al., 1999). crustins i and ii are composed of an n-terminal glycine-rich domain, and a c-terminal region which contains 12 cysteine residues (crustin domain) organized in two doublets. these crustins are similar with the two classes of insect amps, that is glycine-rich peptides and cyclic peptides containing cysteine residues (bulet et al., 1999). on the other hand, the swds are composed of a short prolinearginine-rich region and a c-terminal region containing 8 cysteine residues (the wap domain). they are also similar in terms of the two classes of insect amps, particularly the cyclic peptides containing cysteine residues and the prolineand arginine-rich peptides. similar to the penaeidins found in shrimp, the crustins and the swd are chimera molecules of glycineor proline-rich amps and cysteine-rich amps. chimera-like features often reflect the multifunctional properties of a molecule, such that each domain performs different functions. for example, crustins i and ii have glycine-rich and crustin domains, and therefore should have anti-gram positive and negative bacterial activities. supungul et al. (2008) reported that crustinpm1 (which belongs to crustin i) exhibited anti-microbial activity against only a gram-positive bacteria, whereas the rcrus-likepm (crustin ii) showed remarkable antimicrobial activity against both gram-positive and negative bacteria (amparyup et al., 2008b). likewise, the crufc (crustin ii) from f. chinensis exhibited high activity against gram-positive bacteria but low activity was exhibited against gram-negative bacteria and fungi (zhang et al., 2007). the swds have proand arg-rich and wap domains. they exhibit the following activities: relatively high against gram-positive and/or negative 165 lsecrus2 af430078 lvacrus2 af430072 lvacrus ay488497 lvacrus ay488494 lvacrus3 af430073 lvacrus ay488495 lvacrus ay488496 lvacrus ay488493 lvacrus ay488492 lvacrus1 af430071 fpacrus ef182747 fsu crusef450744 lsecrus ef182748 fbrcrus ef601055 mjacrus1 ab121740 mjacrus3 ab121742 mjacrus5 ab121744 mjacrus2 ab121741 mjacrus4 ab121743 lsecrus1 af430077 lsecrus3 af430079 fchcrus ay871268 pmocrus3 bi018073 pmocrus1 cf415873 pmocrus4 cf415873 pmocrus2 bi018072 pmocrusee661627 plecrus2 ef523613 cmacar aj237947 cmacari aj821886 cmacarii aj821887 cmacariii aj821888 cmacar aj427538 cmacariv aj821889 fchcrus dq097703 pmocrus ef654658 pmoswd eu623981 mjaswd au176270 lvaswd ay465833 pmoswd eu623980 fchswd ef216349 pmoswd ay464465 pmoswd eu62397973 89 56 77 96 99 68 52 100 9099 100 89 98 84 60 71 76 99 95 56 64 5444 12 10 39 0.2 c ru st in i c ar ci ni nlik e c ru st in ii sw d fig. 2 phylogenetic analysis of wap containing proteins/ peptides in penaeid shrimps by mega 4. five thousand bootstraps were performed for the neighbour-joining trees to verify the reliability of the results. cma, carcinus maenas; ham, homarus americanus; hga, homarus gammarus; par, panulirus argus; ple, pacifastacus leniusculus. others are the same with fig. 1. 166 bacteria, moderate against fungi, and strong antiproteinase activity, especially against the bacterial proteinases (amparyup et al., 2008a; jia et al., in press). therefore, they are bi-function peptides. from above results, we can see that the primary structures of amp are not corresponding to their functions. it need further study for their tertiary structures. expression profiles and functions of shrimp wdps the spatio-temporal expression of wdps in shrimp was not well-understood. most of them seem to be constitutively expressed in the hemocytes. apparently, the expression patterns was only reported during the development of the larvae of shrimp, p. monodon. high level expression of a crustin are recorded at all stages of development from the nauplii stage iv to juvenile period(jiravanichpaisal et al., 2007). furthermore, the expression patterns of wdps to bacterial challenge were reported in several shrimps, but the results showed no consistent patterns of change in expression subsequent to bacterial injection. vargas-albores et al. (2004) found two isoforms of crustin i in l. vannamei, which showed different expression patterns after bacterial inoculation. first, crustin-p seems to be constitutively expressed, and second, the crustin-i mrna concentration drops after 6 h. results also revealed that there was a decrease in the transcribed expression of crustin in p. monodon subjected to bacterial challenge (supungul et al., 2004). in hemocytes, the m. japonicus crustin-like peptide mrna was identified, and the expression level of this peptide mrna increased significantly 1, 3, and 7 days after peptidoglycan feeding (rattanachai et al., 2004). the crus pm1 (crustin i) in p. monodon was also expressed in hemocytes, but the expression profile was not analyzed (supungul et al., 2008). the mrna transcript of a crus-like pm2 (crustin ii) in p. monodon was found to be abundantly expressed in hemocytes and was significantly up-regulated after vibrio harveyi injection (amparyup et al., 2008b). jiménez-vega et al. (2004) reported that the expression of the swd gene in l. vannamei hemocytes increased after 3 to 6 h it was inoculated with v. alginolyticus, but slowly returned to nonstimulated levels within 12 to 24 h. the fc-swd from chinese shrimp is constitutively expressed and increased in hemocytes 24 h after bacterial challenge (staphylococcus aureus and vibrio anguillarum). the results of the reverse transcriptase-polymerase chain reaction (rtpcr) analysis revealed a weak expression in heart and gill and challenged stomachs in addition to hemocytes. moreover, results showed that the signal from challenged tissues was stronger than from those unchallenged. consequently, these results suggest that fc-swd is an inducible gene and is essential in responding to bacterial infection (jia et al., in press). the tissue distribution of swds in p. monodon was as well analyzed through rtpcr. results indicated the presence of all three swd transcripts in hemocytes. the transcript expression of swdpm1 was down-regulated upon injection with s. aureus while no change was recorded in temrs of swdpm2 and swdpm3 expressions. contrastingly, the results obtained from the wssv injection showed that in a biphasic response, there was an up-regulation of the swdpm1 and swdpm2 transcripts at 6 h followed by a significant down-regulation by 24 h after infection (amparyup et al., 2008a). the wdps are a large family of antimicrobial effectors in shrimp immunity. in fact, more than 30 wdps, including isoforms and ests, have been found in shrimp. despite this, many of them are poorly characterized for their functions. one of the p. monodon crustins (crustin i), recombinant expressed in e. coli, exhibited an antimicrobial activity against only gram-positive bacteria, specifically with strong inhibition against s. aureus and streptococcus iniae (supungul et al., 2008). zhang et al. (2007) similarly reported that the recombinant crusfc (crustin ii) had relatively high activities against gram-positive bacteria and low activities against gram-negative bacteria and fungi. moreover, another recombinant crustin ii (ef654658) from p. monodon has been recently reported to have a strong activity not only against gram-positive bacteria, but also against gramnegative bacteria, such as escherichia coli 363 and vibrio harveyi (amparyup et al., 2008b). in penaeid shrimp, there were less than 10 swds found, and two of them were studied for their functions in vitro. the functions of swd molecule from chinese shrimp were analyzed (jia et al., in press). the recombinant fc-swd has manifested antimicrobial activities against gram-positive and gram-negative bacteria and fungi, as well as a strong inhibitory activity against subtilisin a and protein k with an inhibition constant (ki) of 2.14 nm and 2.27 nm, respectively; but a much lesser activity against trypsin was recorded. amparyup et al. (2008a) also analyzed the biological functions of recombinant swdpm. based on the results they obtained, recombinant swdpm exhibits activity against several gram-positive, but not gramnegative bacteria and is a competitive inhibitor of subtilisin a with an inhibition constant (ki) of 1.98 nm. this phenomenon indicates the dual functions of swds, that is antimicrobial activity and antiproteinase activity against pathogenic proteinase. therefore, these swds might have an important role in the immunity of shrimp in vivo. so far, it is generally accepted that both activities could not co-exist in the same (unique) domain. why does swd show both anti-microbial and anti-proteinase activities? in fact, similar situations were found in some peptides with a single domain, which is the avian wap (awap iv) originally found in chicks (townes et al., 2006). this has a broad-spectrum of antibacterial activities against both gram-positive and gram-negative bacteria. in addition to that, the avian wap lysate significantly inhibited the activities of the microbial serine proteinases subtilisin and proteinase k. furthermore, li et al. (2007) reported the occurrence of a small serine proteinase inhibitor with antimicrobial capability in a diskless-fingered odorous frog, odorrana grahami. based on a disulfide-bridged hendecapeptide loop of this serine 167 crustin ii swd double wap domain protein wap domain crustin i fig. 3 the possible divergent evolution of wpds in crustaceans. proteinase inhibitor, a series of peptides have been synthesized. they found that seven synthetic peptides exhibited trypsin inhibitory activity, while the other five have both the trypsin inhibitory and antimicrobial activities. in terms of swds, the findings showed that they have high anti-proteinase activities to bacterial serine proteinases (subtilisin a and proteinase k) and antimicrobial activities. the recombinant wap domain (fc-wapd) shows relatively low activities against the bacterial serine proteinases (jia et al., in press), and manifests quite a low activity against bacteria. comparing their sequence, it was found that fc-swd contain higher positively charged amino acids than the fc-wapd. the net charge of fc-swd is +4, while that of fc-wapd is − 2. in addition, it is generally known that most antimicrobial cationic peptides have the same unique features, that is, they are both polycationic (having a net positive charge of more than +2) and fold into amphipathic structures (having both a hydrophobic and a hydrophilic domain). as such, these characteristics enable them to interact with the negatively charged surface molecule of bacteria and to interact with and penetrate into the negatively charged cytoplasmic membranes of most bacteria (hancock, 1997). this is therefore the reason for the high anti-microbial activity of fc-swd compared to that of fc-wapd’s low activity against bacteria (jia et al., in press). the possible divergent evolution of the wap domain in crustaceans the wap domain was initially identified in the primary milk protein of rats and mice (hennighausen and sippel, 1982). wap proteins have one or more wap domains containing about 50 amino acids with eight highly conserved cysteine residues that form a four-disulphide core (4-dsc). amino acids in the wap domain, except for the conserved cysteine residues, are significantly diverse, and proteins with a wap motif perform variety of functions. in fact, a large biological diversity exists between the proteins that contain one or two wap domains, with many being identified as proteinase inhibitors or amps. the most studied wap proteins, for example, are the elafin and antileukoproteinase, which are two serine-proteinase inhibitors with anti-microbial and inflammatory activity (bouchard et al., 2006). in crustaceans, several wap domain-containing proteins were found. in addition to the aforementioned four wap-containing proteins, double wap domain containing proteins were also reported in shrimp [the l. vannamei secretory leukocyte proteinase inhibitor, ef467169; the m. japonicus double wap domain-containing protein, eu095018; the f. chinensis double wap domaincontaining protein (our unpublished data)]. many identified genes that code for wap proteins in human are clustered on chromosome 20q12–13.1 (bouchard et al., 2006). the results of southern analysis show that a large family of sequences related to the crustins is present in l. vannamei genome (bartlett et al., 2002). this may indicate some similarities in gene locations. based on the domain structure and functions of the wapcontaining proteins in crustaceans, we therefore propose the possible divergent evolution of wap domain in crustaceans (fig. 3). in this proposal, the different groups may have different functions, including antimicrobial and anti-proteinase activities among others. alf factors alf, a basic peptide, was initially found as a potent anticoagulant from horseshoe crabs, limulus polyphemus and tachypleus tridentatus, which inhibited the endotoxin mediated activation of the coagulation cascade (tanaka et al., 1982). thereafter, several studies demonstrated that the alf from hemocytes of the horseshoe crab l. polyphemus have similar characteristics with that of the binding and neutralizing lipopolysaccharide (lps). additionally, alf was indicated to have a strong antibacterial activity, particularly on the growth of gram-negative bacteria (morita et al., 1985; aketagawa et al., 1986; muta et al., 1987). in shrimp, the cdna clones homologous to the horseshoe crab alfs were initially identified in hemocytes of p. monodon and l. setiferus by means of est analysis (gross et al., 2001; supungul et al., 2004). in recent years, there have been a growing number of studies on shrimp alf available to provide pertinent information. to note, several alfs have been isolated and characterized from hemocytes in penaeid shrimp, f. chinensis (liu 168 http://www.ncbi.nlm.nih.gov/pubmed/9033483?ordinalpos=14&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum fig. 4 alignment of alfs-based amino acid sequence. all sequences of alfs are from genbank. fch, fenneropenaeus chinensis; fpa, farfantepenaeus paulensis; ham, homarus americanus; lse, litopenaeus setiferus; lsc, lst; litopenaeus stylirostris:litopenaeus schmitti; lva, litopenaeus vannamei; mja, marsupenaeus japonicus; mol, macrobrachium olfersii; pmo, penaeus monodon; and ple, pacifastacus leniusculus, lpo, limulus polyphemus. 169 et al., 2005; zhou et al., 2008) m. japonicus (nagoshi et al., 2006), p. monodon (supungul et al., 2004; somboonwiwat et al., 2005; tharntada et al., 2008), l. vannamei (de la vega et al., 2008), l. setiferus (gross et al., 2001), f. paulensis (ef601051, ef601054), l. schmitti (dq991357), and l. stylirostris (dq010421). classification of shrimp alf twenty-nine crustacean alf sequences collected from genbank were aligned using the alignment editor of the mega 4 software (tamura et al., 2007). the results obtained by the multiple alignment (fig. 4) revealed that all the molecules of alfs contain two preserved cysteine residues which form a disulfide bridge. it was also found out that alf contained a relatively conserved sequence of a positively-charged amino acid residue cluster within the disulfide loop. this structure of the βhairpin loop in shrimp alf suggests a conservation of the lps binding activity. with the use of neighbor joining method of mega 4, a phylogenetic tree was constructed (tamura et al., 2007). to attain and assess the reliability of the tree, bootstrapping using 5,000 replications was as well performed. the phylogenetic tree analysis categorized the various alf proteins into three main groups. accordingly, most alfs from shrimp belong to cluster i, including the alfs from l. setiferus (white gulf shrimp); then cluster ii contains the l. stylirostris (blue shrimp) and eriocheir sinensis (chinese mitten crab). finally, cluster iii contains the alfs from l. vannamei (pacific white shrimp), p. monodon (black tiger shrimp) and other crustacean pacifastacus leniusculus (signal crayfish), l. polyphemus (atlantic horseshoe crab), and tachypleus tridentatus (horseshoe crab) (fig. 5). it can be observed that in l. vanamei and p. monodon, two kinds of alfs. expression profiles and functions in shrimp innate immunity the transcription of alfs in some species was specified in tissues. in chinese shrimp (f. chinensis), for example, the alf (cluster i) has high expression in hemocytes, gills, and intestine but exhibited low expression in ovary, hepatopancreas, and muscle (liu et al., 2005). similar observations had been reported in p. monodon, in which alf (cluster i) was constitutively expressed in hemocytes, hearts, gills, intestines, and lymphoid organs, while there was no observed transcription in the hepatopancreas (supungul et al., 2004). another example is that in the kuruma shrimp (m. japonicus), in which the alf (cluster i) was reported to be expressed at higher levels in hemocytes, lymphoid organs, hearts, intestines, and gills. the expression of alfs, on the other hand, was found to be at lower levels in stomachs, hepatopancreas, and muscles (nagoshi et al., 2006). in l. vannamei, alf1 (which belongs to cluster iii ) was found to have high mrna levels in the lymphoid organ and heart, intermediate levels in the gills, eyestalk, and hemocytes, and very low levels in the muscle and hepatopancreas (de la vega et al., 2008). the aforementioned reports are considerably in contrast with the patterns of many other amps in shrimp which are carried out primarily in hemocytes (munoz et al., 2002; bachère et al., 2004; kang et al., 2004). it is important to note that in shrimp, however, it is clear that alf transcription, although it is tissue-specific, occurs in multiple organs and could thereby provide systemic protection against pathogens. the transcription of the alf is induced upon bacterial challenge in several shrimp (supungul et al., 2004; liu et al., 2005; nagoshi et al., 2006). in a study, alf was induced upon wssv infection or exposed to uv-inactivated wssv (liu et al., 2006). on the other hand, in l. vanname, the infection with pathogenic bacterium, vibrio penaeicida or fungus, fusarium oxysporum, did not cause any significant change in the lvalf1 mrna levels compared to saline-injected controls. an considerable increase was recorded in the lvalf1 mrna expression in wssv-infected shrimp at 54-h time point (de la vega et al., 2008). similarly, in l. vannamei, alf was significantly up-regulated in the infected viral hepatopancreas (robalino et al., 2007). this upregulation of antimicrobial proteins as a response to viral infection has also been reported in drosophila (zambon et al., 2005). despite these multitude studies, the mechanism in anti-viral responses of alf still needs to be clarified. the results reported by different authors indicate the inconsistent expression patterns of the alfs after the shrimps have been injected with bacteria or viruses. as such, this further indicates the different functions in vivo of the alfs. to date, only a few alfs and their characteristics have been analyzed. in p. monodon, alfpm3, a predominant antimicrobial peptide, was identified in both the unchallenged and v. harveyichallenged shrimp (supungul et al., 2004). in their study, a strong activity against multiple grampositive and gram-negative bacteria and filamentous fungi (somboonwiwat et al., 2005) has been recorded. another study found out that a synthetic peptide, corresponding to the lps-binding domain of mj-alf from m. japonicus, exhibited a lps-neutralizing and hemolytic activities on lpssensitized human red blood cells (nagoshi et al., 2006). moreover, alf was found to have a potential in interfering with the replication of wssv in crayfish p. leniusculus (liu et al., 2006). the in vivo function of alf in protecting shrimp from bacterial, fungal, and viral infections was also studied in l vanamei through the rna interference (rnai) method (de la vega et al., 2008). the injection of double-stranded rna (dsrna) corresponding to the lvalf1 message resulted in a significant reduction of the abundant lvalf1 mrna levels. following knockdown of the lvalf1, the shrimps were challenged with low pathogenic doses of pathogenic v. penaeicida, or f. oxysporum. the results showed a significant increase in the mortality among the lvalf1 low-level shrimps, specifically those with v. penaeicida and f. oxysporum infections, compared to the control shrimps. the result showed that this gene functions in protecting shrimp from both bacterial and fungal infections. in the viral challenge using wssv, the alf dsrna injection caused no significant increase in mortality 170 fig. 5 phylogenetic analysis of alfs from shrimp using the sequence information from fig. 4. compared to the non-specific dsrna controls in the l. vannamei (de la vega et al., 2008). other experiments that focused on alf rnai in freshwater p. leniusculus indicated that alf can protect against wssv infection, as observed in the alf low-level through rnai, which specifically resulted in higher rates of viral propagation (liu et al., 2006). the differences in the two above-mentioned reports emphasize the diverse taxonomic groups of crustacea and their varying responses to infection and immunity mechanisms (de la vega et al., 2008). possible mechanisms of lps binding and antimicrobial activity alfs contain two conserved cysteine residues which form a disulfide bridge. they also contain a relatively conserved sequence of a positively charged amino acid residue cluster within the disulfide loop, which is regarded as alf’s functional domain. hoess et al. (1993) reported the high resolution structure of a recombinant limulus-alf (l-alf). in their report, they stressed that it contains an n-terminal α-helix (that opens into a α-helix in its final turn), a simple four-stranded anti-parallel βsheet, and two c-terminal α-helices. the three helices form a bundle that packs against the β-sheet and encloses a hydrophobic and highly-aromatic core. their study performed a structural analysis of l-alf through the x-ray crystallography, and the results demonstrated the β-hairpin loop with an alternating series of hydrophilic (mainly basic amino acids) in the central disulfide-bonded loop region thus obtaining an amphipathic protein molecule (hoess et al., 1993). this amphipathic loop structure is believed to be a lps-binding motif which can bind a single fatty acid with the phosphoglucosamine portion of lipid a (the membrane anchor of lps). this amphipathic loop of l-alf were compared to the alf sequences (cys1st to cys2nd ) of shrimp, and the results revealed similarity between the alternating residues pattern (fig. 4). as such, the structure of the β-hairpin loop in shrimp alf suggests that there is a conservation of lps binding activity. somboonwiwat et al. (2008) also studied the binding activity of ralfpm3 and found that it could strongly bind to both gram negative and gram positive bacterial cells. further analysis demonstrated that alfpm3 could bind to both the immobilized lps and lipoteichoic acid (lta), with a high dissociation constant (kd) of 1.26×10 −8 and 1.34×10−8 m, respectively. this suggested that they lva dq208701 lva dq208706 lva dq208702 lva dq208705 lva dq208703 lva dq208704 lsc dq991357 fpa ef601054 fpa ef601051 fpa ef601052 fpa ef601053 fch ay859500 fch-alf-2 pmo eu617325 pmo ef523562 pmo ef523559 pmo ef523563 ham eu625516 mol eu289220 ham eu625517 lva alf2 mja ab210110 esi dq793214 lse be846661 lst dq010421 ple ef523760 lpo 1307201a ttr p07087 lva alf1 pmo ef523561 100 100 100 99 96 55 76 58 78 39 76 91 44 25 80 93 93 96 58 51 29 60 71 39 80 89 0.1 171 i ii iii lva af387661 lva ax006138 lva af387662 lva af390145 lva af390139 lva y14926 lva af387663 lva dq206403 lva y14928 lva y14927 fpe eu333491 lva af387660 fpe eu333492 lva af390143 lva dq211700 lva af390142 lva af390140 lva af390144 lva af390141 lst dq010422 lva ay351656 lse ay039204 lse ay039206 lse ay039202 lse ay039203 fch ay260151 fch dq308408 pmo af475082 pmo ay326471 lsc ay956420 lse ay039207 lva dq211701 lva af390149 lva af390147 lva dq206402 fbr ef450745 fsu ef450742 fpa ay956416 fpa ay956417 lsc ay956418 lse ay039205 lsc ay956419 lst ay351655 lva dq211699 lva af390146 lva dq206401 lva ax006136 lva y14925 fch dq153253 fch dq308407 fch ay669323 fch dq154152 64 75 58 99 43 88 99 12 67 29 10 99 97 97 46 99 60 11 2 99 39 19 99 53 31 47 99 99 80 76 92 85 71 67 59 86 50 25 0.000.050.100.150.200.25 iv ii v iii fig. 6 phylogenetic analysis of penaeidins from shrimp using the sequence information from genbank. the upgma tree was obtained using mega with complete deletions of gaps. bootstraps (5000) were performed for the upgma trees to verify repeatability and reliability of results. fch, fenneropenaeus chinensis; fpa, farfantepenaeus paulensis; fpe, fenneropenaeus penicillatus; fsu, farfantepenaeus subtilis; fbr, farfantepenaeus brasiliensis; lva, litopenaeus vannamei; lst, litopenaeus stylirostris; lsc, litopenaeus schmitti; lse, litopenaeus setiferus, and pmo, penaeus monodon 172 are at least one of the target molecules for the alfpm3 on gram-negative and gram-positive bacteria, respectively. assuming that alf binds to bacterial cells before they exterminate the cells, it is still unknown as to how such extermination is performed. alf has both the ability to inhibit the endotoxin or lps mediated coagulation system and to exhibit strong anti-microbial activity against the gramnegative and gram-positive bacteria and fungi. thus, alf is also one of the pivotal effectors in shrimp immunity. nes (bu t al., 2008). -type lectin molecule in the nate immunity of chinese shrimp. ther antimicrobial peptides/proteins penaeidins penaeidins, initially isolated from the pacific white shrimp l. vannamei (destoumieux et al., 1997), are also a large family of amps that have been detected in several penaeid shrimp, including l. setiferus, m. japonicus, p. monodon, f. chinensis (bachère et al., 2004; kang et al., 2004, 2007; cuthbertson et al., 2008), l. stylirostris (aay33770), l. schmitti (aax58698; aax58697), fenneropenaeus penicillatus (aby56821), f. paulensis (aax58696), f. subtilis (abo93321), and f. brasiliensis (abo93324). in fact, among the amp family, the penaeidins are considered the most well-characterized in terms of the level of gene expression and biological activities. there are four classes of penaeidins (penaeidins 2, 3, 4, and 5) that have been characterized so far (bachère et al., 2004; kang et al., 2007) (fig. 6). this classification and characterization of penaeidin isoforms have been summarized in the database, penbase (gueguen et al., 2006) and were reviewed by several authors (bachère et al., 2004; cuthbertson et al., 2008). the evolution pattern of these peptides was likewise analyzed (padhi et al., 2007). table 1 presents the primary characteristics and functions of penaeidins. lysozymes lysozyme (muramidase, ec.3.2.1.17), an important antibacterial protein, catalyzes the hydrolysis of bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens (jollés and jollés, 1984). initially found among eukaryotes and prokaryotes, the lysozymes are classified into six types. these are the chicken-type lysozyme (ctype), goose-type lysozyme (g-type), plant lysozyme, bacteria lysozyme, t4 phage lysozyme (phage-type), and invertebrate lysozyme (i-type) (hikima et al., 2003). similarly, these lysozymes had been reported in several shrimps, such as l. vannamei (sotelo-mundo et al., 2003; de-la-revega et al., 2006; burge et al., 2007; xing et al., in press), m. japonicus (hikima et al., 2003), p. monodon, (xing et al., in press), p. semisulcatus (xing et al., in press) and f. chinensis (bu et al., 2008). furthermore, most lysozymes identified in shrimps belong to a c-type lysozyme, like those from l. vannamei (af425673), m. japonicus (bac57467), p. monodon (b1784440), and f. chinensis (aav83994). in addition, there were i-type lysozymes identified in shrimp, such as lysozymes from l. vannamei (bf023863, bf024192) and l. setiferus (bf024309) (hikima et al., 2003). in some penaeid shrimp, the lysozymes are well-characterized, and they possess lytic activity against a range of gram-positive and gramnegative bacterial species, including pathogenic vibrio spp. (hikima et al., 2003; de-la-re-vega et al., 2006). hikima et al. (2003) used the rt-pcr analysis and reported that the lysozyme from m. japonicus was strongly expressed in samples from hemocytes, moderately expressed in the epidermis, and weakly expressed in the gills, midgut, and muscle. furthermore, the post-infection expression profile of a lysozyme est was analyzed using the macro-array, northern blot, and real-time pcr. the results revealed that the hemocyte lysozyme expression during the v. penaeicida challenge was significantly lower at 12 h after the infection, but had returned to control levels within 24-96 h after the challenge as observed in the surviving shrimps (de lorgeril et al., 2005). similarly, the lysozyme mrna from chinese shrimp (fclyz) was analyzed by semiquantitative rt-pcr. the lysozyme was actually expressed in various tissues of the unchallenged shrimp and the expression of fclyz was increased in the bacterial-challenged tissues of the hemocytes, heart, hepatopancreas, and gills as compared to the mock (saline)-challenged o e c c-type lectins have diverse functions. aside from their agglutinating activity and opsonic effects, some c-type lectins perform antimicrobial activities. a hepatopancreas specific c-type lectin, designated fc-hsl, has been found from the hepatopancreas of the chinese shrimp, f. chinensis (sun et al., 2008). this type of lectin was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated after the bacterial and viral challenge. in addition to fc-hsl’s calciumdependent agglutinating and binding activity to some gram-positive and gram-negative bacteria, it also has high anti-microbial activity against some gram-positive and gram-negative bacteria and fungi. therefore, fc-hsl may act as a pattern recognition receptor and an effector in o hemocyanin, as the main protein component of hemolymph, is prevalent in several invertebrate animals. it typically represents up to 95 % of the total amount of protein (sellos et al., 1997). meanwhile, the hexamer is the predominant form in the most primitive crustacean decapoda, such as penaeus setiferus or p. monodon (sellos et al., 1997). the hemocyanin, in relation to crustaceans, primarily functions as anthropods’ oxygen carrier. it is also the multi-functional proteins involved in physiological processes, such as osmoregulation, protein storage or enzymatic activities. in some reports, the fragments generated from the cterminus of hemocyanin of l. vanamei and l. stylirostris have exhibited a high anti-fungal activity (destoumieux-garzon et al., 2001). contrary to most 173 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=63109228 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002137 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002135 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=164451887 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002133 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=143598606 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=143598723 table 1 antimicrobial peptides/proteins in penaeid shrimp amps expression tissues functions references 1. penaidins penaeidin ii penaeidin iii penaeidin iv penaeidin v primarily in hemocytes and in highly vascular tissues broad spectrum of anti-grampositive and anti-fungal activities and weak activity against gram-negative strains destoumieux et al., 1997, 1999, 2000; bachère et al., 2004; cuthbertson et al., 2004, 2008; kang et al., 2004, 2007 2. wdps crustin i hemocytes anti-gram-positive activity supungul et al., 2008 crustin ii hemocytes anti gram-positive and antigram-negative activities zhang et al., 2007; amparyuo et al., 2008 swd hemocytes anti-gram-positive, antigram-negative, anti-fungal and anti-proteinase activities amparyup et al., 2008; jia et al., in press 3. alfs alf i high expression in hemocytes, gills and intestine and low expression in ovary, hepatopancreas and muscle anti-gram-positive, antigram-negative and antifungal activities; lpsneutralizing and hemolytic activities; interference with the replication of wssv gross et al., 2001; supungul et al., 2004; liu et al., 2005; nagoshi et al., 2006 alf ii ? ? (dq010421, be846661) alf iii high level in lymphoid organ and heart, intermediate levels in the gills, eyestalk and hemocytes and very low levels in the muscle and hepatopancreas anti-gram-positive, antigram-negative and antifungal activities de la vega et al., 2008; somboonwiwat et al., 2005; zhou et al., 2008 4. lysozymes c-type lysozyme hemocytes, lymphoid organ, hepatopancreas, gill, heart, midgut, muscle, epidermis, and eyestalk anti gram-positive and antigram-negative activities sotelo-mundo et al., 2003; hikima et al.,2003; de-lare-vega et al., 2006; burge et al., 2007; xing et al., in press; bu et al., 2008 i-type lysozyme ? ? (bf023863, bf024192) (bf024309) 5. c-type lectin hsl hepatopancreas anti-gram-positive, antigram-negative and antifungal activities sun et al., 2008 6. hemocyanin-derived peptides c-terminal fragment 7.9 kda, 8.3 kda hepatopancreas anti-fungal activity destoumieux-garzón et al., 2001 hemocyanin subunits hepatopancreas anti-viral activity zhang et al., 2004 7. histones h2a, h2b, and h4 hemocytes anti-gram-positive activity patat et al., 2004 8. peritrophin ovary anti gram-positive and antigram-negative activities loongyai et al., 2007 174 hemocytes: penaeidins crustins swd, alf lysozyme histones hepatopancreas: hemocyanin c-type lectin alf lysozyme lymphoid organ: alf lysozyme intestine: lysozyme penaeidins alf gill: lysozyme alf penaeidin epidermis: lysozyme penaeidin fig. 7 distribution of anti-microbial peptides/proteins in shrimp. amps with highly-cationic charges, the antifungal substances present a negative net charge at physiological ph with a pi ranging from 5.65 to 6.54. zhang et al. (2004) also reported that shrimp hemocyanin had antiviral property as it inhibited the virus replication. histone proteins are primarily involved in dna packaging and regulation of dna replication and transcription. a number of reports have shown that histone proteins or histone-derived peptides from various vertebrates possess antimicrobial activity (hirsch, 1958; robinette et al., 1998; fernandes et al., 2002). patat et al. (2004) as well reported that the hemocyte histone h2a, a mixture of histones h2b and h4 and an h1-derived fragment in l. vannamei, have anti-microbial activity against the tested bacterium. they believed that histone proteins or histone-derived peptides can be secreted to the cytoplasm from the nucleus and be localized in it along with other antimicrobial peptides. shrimp peritrophin, a major protein in jelly layer (jl) and cortical rods (crs) (du et al., 2006; loongyai et al., 2007), was inducing expression in hemocytes, heart, stomach, intestine and gill, and was constitutively expressed in ovary (du et al., 2006). the recombinant peritropnin exhibited a chitinase activity and efficiently inhibited the growth of vibrio harveyi and s. aureus, with minimum inhibitory concentrations of 2.4 and 15.7 μm, respectively (loongyai et al., 2007). conclusions to summarize, shrimps have efficiently developed and used their innate immune system in defense against pathogenic microorganisms. to date, eight kinds of amps have been identified in penaeid shrimp, namely, penaeidins, wdps (crustins and swds), alfs, lysozymes, anionic hemocyanin, histones, and a c-type lectin. these kinds of amps have different distribution and expression profiles and functions in shrimp (table 1 and fig. 7). furthermore, there are several subclasses or isoformes for each kind of amps in one species. an example is the three subgroups of wdps (crustin i, ii, and swd) in chinese shrimp. this only suggests that they had different functions in vivo in the shrimp. even though large groups of amps were found in penaeid shrimp, there is still a limited number of studies conducted about the amps’ antibacterial properties and their other functions. in view of this, there is a need to have more studies that will delve on the functions and the anti-bacterial properties of amps, especially with respect to the diverse bioactivities of the natural proteins in vivo. as such, some amps in shrimp can be better candidates for clinical uses in the aquaculture. acknowledgements this work was supported by grants from the national high technology research and development program of china (863 program) (no. 2007aa09z425), the ph. d. program foundation of the ministry of education of china (no. 20060422034), and the major state basic research project (no. 2006cb101806) references aketagawa j, miyata t, ohtsubo s, nakamura t, morita t, hayashida h, et al. primary structure of limulus anticoagulant anti-lipopolysaccharide factor. j. biol. chem. 261: 7357-7365,1986. ali mf, lips rk, knoop cf, fritzsch b, miller c, conlon jm. antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, rana areolata. biochim. biophys. acta 1601: 55-63, 2002. alvarez r, reading j, king df, hayes m, easterbrook p, farzaneh f, et al. wfdc1/ps20 is a novel innate immuno-modulatory signature protein of hiv permissive cd4+ cd45ro+ memory t-cells that promote infection by up-regulating cd54 integrin expression and is elevated in hiv-1 infection. j. virol. 82: 471–486, 2008. 175 amparyup p, donpudsa s, tassanakajon a. shrimp single wap domain (swd)-containing protein exhibits proteinase inhibitory and antimicrobial activities. dev. comp. immunol. 32: 1497-1509, 2008a. amparyup p, kondo h, hirono i, aoki t, tassanakajon a. molecular cloning, genomic organization, and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp penaeus monodon. mol. immunol. 45: 1085-1093, 2008b. arts ja, cornelissen fh, cijsouw t, hermsen t, savelkoul hf, stet rj. molecular cloning and expression of a toll receptor in the giant tiger shrimp, penaeus monodon. fish shellfish immunol. 23: 504-513, 2007. bachère e, gueguen y, gonzalez m, de lorgeril j, garnier j, romestand b. insights into the antimicrobial defense of marine invertebrates: the penaeid shrimps and the oyster crassostrea gigas. immunol. rev. 198: 149-168, 2004. bartlett tc, cuthbertson bj, shepard ef, chapman rw, gross ps, warr gw. crustins, homologues of an 11.5-kda antibacterial peptide, from two species of penaeid shrimp, litopenaeus vannamei and litopenaeus setiferus. mar. biotechnol. 4: 278-293, 2002. beg ou. partial characterization of platypus (ornithorhynchus anatinus) milk proteins. protein pept. lett. 2: 431-434, 1995. bouchard d, morisset d, bourbonnais y, tremblay g.m. proteins with whey-acidic-protein motifs and cancer. lancet oncol. 7: 167-174, 2006. bu xj, du xj, zhou wj, zhao xf, wang jx. molecular cloning, recombinant expression, and characterization of lysozyme from chinese shrimp fenneropenaeus chinensis. chinese j. biotechnol. 24: 723-732, 2008. bulet p, dimarcq jl, hetru c, lagueux m, charlet m, hegy g, et al. a novel inducible antibacterial peptide from drosophila carries an oglycosylated substitution. j. biol. chem. 268: 14893-14897, 1993. bulet p, hetru c, dimarcq jl, hoffmann d. antimicrobial peptides in insects: structure and function. dev. com. immunol. 23: 329-344, 1999. bulet p, stocklin r, menin l. anti-microbial peptides: from invertebrates to vertebrates. immunol. rev. 198: 169-184, 2004. burge ej, madigan dj, burnett le, burnett kg. lysozyme gene expression by hemocytes of pacific white shrimp, litopenaeus vannamei, after injection with vibrio. fish shellfish immunol. 22: 327-339, 2007. carro s, bergo a, mengoni m, bachi a, badaracco g, kilstrup-nielsen c, et al. a novel protein, xenopus p20, influences the stability of mecp2 through direct interaction. j. biol. chem. 279: 25623–25631, 2004. chen jy, pan cy, kuo cm, cdna sequence encoding an 11.5-kda antibacterial peptide of the shrimp penaeus monodon. fish shellfish immunol. 16: 659-664, 2004. cheng w, liu ch, tsai ch, chen jc. molecular cloning and characterization of a pattern recognition molecule, lipopolysaccharideand beta-1, 3-glucan binding protein (lgbp) from the white shrimp, litopenaeus vannamei. fish shellfish. immunol. 18: 297-310, 2005. cuthbertson bj, büllesbach ee, fievet j, bachère e. gross ps. a new class (penaeidin class 4) of antimicrobial peptides from the atlantic white shrimp (litopenaeus setiferus) exhibits target specificity and an independent proline-richdomain function. biochem. j. 381: 79-86, 2004. cuthbertson bj, deterding lj, williams jg, tomer kb, etienne k, blackshear pj, et al. diversity in penaeidin anti-microbial peptide form and function. dev. comp. immunol. 32: 167-181, 2008. de la vega e, o'leary na, shockey je, robalino j, payne c, browdy cl, et al. antilipopolysaccharide factor in litopenaeus vannamei (lvalf): a broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection. mol. immunol. 45:1916-1925, 2008. de la re-vega e, garcía-galaz a, díaz-cinco me, sotelo-mundo rr. white shrimp (litopenaeus vannamei) recombinant lysozyme has antibacterial activity against gram negative bacteria: vibrio alginolyticus, vibrio parahemolyticus, and vibrio cholerae. fish shellfish immunol. 20: 405-408, 2006. de lorgeril j, saulnier d, janech mg, gueguen y, bachère e. identification of genes that are differentially expressed in hemocytes of the pacific blue shrimp (litopenaeus stylirostris) surviving an infection with vibrio penaeicida. physiol. genomics 21: 174-183, 2005. destoumieux d, bulet p, loew d, van dorsselaer a, rodriguez j, bachère e. penaeidins: a new family of anti-microbial peptides isolated from the shrimp penaeus vannamei (decapoda). j. biol. chem. 272: 28398-28406, 1997. destoumieux d, bulet p, strub jm, van dorsselaer a, bachère e. recombinant expression and range of activity of penaeidins, anti-microbial peptides from penaeid shrimp. eur. j. biochem. 266: 335-346, 1999. destoumieux d, muñoz m, cosseau c, rodriguez j, bulet p, comps m, et al. penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge. j. cell sci. 113: 461-469, 2000. destoumieux-garzon d, saulniner d, garnier j, jouffrey c, bulet p, bachère e. crustacean immunity: antifungal peptides are generated from the terminus of shrimp hemocyanin in response to microbial challenge. j. biol. chem. 276: 47070-47077, 2001. devinoy e, hubert c, jolivet g, thepot d, clergue n, desaleux m,et al.. recent data on the structure of rabbit milk protein genes and on the mechanism of the hormonal control of their expression. reprod. nutr. dev. 28: 1145-1164, 1988. du xj, wang jx, liu n, zhao xf, li fh, xiang jh. identification and molecular characterization of a peritrophin-like protein from fleshy prawn (fenneropenaeus chinensis). mol. immunol. 43: 1633-1644, 2006. 176 du xj, zhao xf, wang jx. molecular cloning and characterization of a lipopolysaccharide and beta-1, 3-glucan binding protein from fleshy prawn (fenneropenaeus chinensis). mol. immunol. 44: 1085-1094, 2007. food and agriculture organization fisheries and aquaculture department. the state of world fisheries and aquaculture 2006. food and agriculture organization of the united nations, rome, 2007. fernandes jm, kemp gd, molle mg, smith vj. anti-microbial properties of histone h2a from skin secretions of rainbow trout, oncorhynchus mykiss. biochem. j. 368: 611-620, 2002. furutani y, kato a, kawai r, fibriani a, kojima s, hirose s. androgen-dependent expression, gene structure, and molecular evolution of guinea pig caltrin ii, a wap-motif protein. biol. reprod. 71: 1583-1590, 2004. garczynski ma, goetz fw. molecular characterization of a ribonucleic acid transcript that is highly up-regulated at the time of ovulation in the brook trout (salvelinus fontinalis) ovary. biol. reprod. 57: 856-864, 1997. gross ps, bartlett tc, browdy cl, chapman rw, warr g.w. immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the pacific white shrimp, litopenaeus vannamei, and the atlantic white shrimp, l. setiferus. dev. comp. immunol. 25: 565-577, 2001. gueguen y, garnier j, robert l, lefranc mp, mougenot i, de lorgeril j, et al. penbase, the shrimp antimicrobial peptide penaeidin database: sequence-based classification and recommended nomenclature. dev. comp. immunol. 30: 283-288, 2006. hagiwara k, kikuchi t, endo y, huqun usui k, takahashi m, shibata n, et al. mouse swam1 and swam2 are antibacterial proteins composed of a single whey acidic protein motif. j. immunol. 170: 1973-1979, 2003. hennighausen lg, sippel ae. mouse whey acidic protein is a novel member of the family of ‘fourdisulphide core’ proteins. nucleic acids res. 10: 2677-2684, 1982. hikima s, hikima j, rojtinnakorn j, hirono i, aoki t. characterization and function of kuruma shrimp lysozyme possessing lytic activity against vibrio species. gene 316: 187-195, 2003. hirsch jg. bactericidal action of histones. j. exp. med.108: 925-944,1958. hoess a, watson s, siber gr, liddington r, crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, limulus anti-lps factor at 1.5 å resolution. embo j. 12: 33513356, 1993. hancock re. peptide antibiotics. lancet 349: 418422, 1997. jia yp, wang zh, wang q, zhao xf, wang jx. a single whey acidic protein domain (swd)containing peptide from the fleshy prawn with antimicrobial and proteinase inhibition activities. aquaculture doi:10.1016/j.aquaculture.2008.07.046, (in press). jimenez-vega f, sotelo-mundo rr, ascencio f, vargas-albores f. 1, 3-beta-d glucan binding protein (bgbp) from the white shrimp, penaeus vannamei, is also a heparin binding protein fish. shellfish. immunol. 13: 171-181, 2002. jiménez-vega f, yepiz-plascencia g, soderhall k, vargas-albores f. a single wap domaincontaining protein from litopenaeus vannamei hemocytes. biochem. biophys. res. commun. 314: 681-687, 2004. jiravanichpaisal p, puanglarp n, petkon s , donnuea s, söderhäll i, söderhäll k. expression of immune-related genes in larval stages of the giant tiger shrimp, penaeus monodon. fish shellfish immunol. 23: 815-824, 2007. jollés p, jollés j. what’s new in lysozyme research? always a model system, today as yesterday. mol. cell. biochem. 63: 165-189, 1984. kang cj, wang jx, zhao xf, yang xm, shao hl, xiang jh. molecular cloning and expression analysis of ch-penaeidin, an antimicrobial peptide from chinese shrimp, fenneropenaeus chinensis. fish shellfish immunol. 16: 513-525, 2004. kang cj, xue jf, liu n, zhao xf, wang jx. characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from fenneropenaeus chinensis. mol. immunol. 44: 1535-1543, 2007. li j, zhang c, xu x, wang j, yu h, lai r, et al. trypsin inhibitory loop is an excellent lead structure to design serine protease inhibitors and antimicrobial peptides. faseb j. 21: 2466-2473, 2007. lin yc, vaseeharan b, chen jc. identification and phylogenetic analysis on lipopolysaccharide and beta-1, 3-glucan binding protein (lgbp) of kuruma shrimp marsupenaeus japonicus. dev comp immunol. 32: 1260-1269, 2008. liu h, jiravanichpaisal p, söderhäll i, cerenius l, söderhäll k. antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish pacifastacus leniusculus. j virol. 80: 1036510371, 2006. liu y, liu c, li fh, dong b, xiang jh. molecular cloning and expression profile of putative antilipopolysaccharide factor in chinese shrimp (fenneropenaeus chinensis). mar. biotechnol. 7: 600-608, 2005. liu yc, li fh, dong b, wang b, luan w, zhang xj, et al. molecular cloning, characterization, and expression analysis of a putative c-type lectin (fclectin) gene in chinese shrimp fenneropenaeus chinensis. mol. immunol. 44: 598-607, 2007. loongyai w, avarre j-c, cerutti m, lubzens e, chotigeat w. isolation and functional characterization of a new shrimp ovarian peritrophin with antimicrobial activity from fenneropenaeus merguiensis. mar. biotechnol. 9: 624-637, 2007. luo t, yang h, li f, zhang x, xu x. purification, characterization, and cdna-cloning of a novel lipopolysaccharide-binding lectin from the shrimp, penaeus monodon. dev. comp. immunol. 30: 607-617, 2006. mackintosh ja, gooley aa, karuso ph, beattie aj, jardine dr, veal da. a gloverin-like 177 antibacterial protein is synthesized in helicoverpa armigera following bacterial challenge. dev. comp. immunol. 22: 387-399, 1998. morita t, ohtsubo s, nakamura t, tanaka s, iwanaga s, ohashi k, et al. isolation and biological activities of limulus anticoagulant (antilps factor) which interacts with lipopolysaccharide (lps). j. biochem. 97: 16111620,1985. muñoz m, vandenbulcke f, saulnier d, bachère e. expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp. eur. j. biochem. 269: 2678-89, 2002. muñoz m, vandenbulcke f, garnier j, gueguen y, bulet p, saulnier d, et al. involvement of penaeidins in defense reactions of the shrimp litopenaeus stylirostris to a pathogenic vibrio. cell mol. life sci. 61: 961-972, 2004. muta t, miyata t, tokunaga f, nakamura t, iwanaga s. primary structure of antilipopolysaccharide factor from american horseshoe crab, limulus polyphemus. j. biochem. 101: 1321-1330, 1987. nagoshi h, inagawa h, morii k, harada h, kohchi c, nishizawa t, et al. cloning and characterization of a lps-regulatory gene having an lps-binding domain in kuruma prawn marsupenaeus japonicus. mol immunol. 43: 2061-2069, 2006. okumura t. effects of lipopolysaccharide on gene expression of anti-microbial peptides (penaeidins and crustin), serine proteinase, and prophenoloxidase in haemocytes of the pacific white shrimp, litopenaeus vannamei. fish shellfish immunol. 22: 68-76, 2007. ota y, shimoya k, zhang q, moriyama a, chin a, tenma k, et al. the expression of secretory leukocyte protease inhibitor (slpi) in the fallopian tube: slpi protects the acrosome sperm from inhibitory effects of elastase. hum. reprod. 17: 2517-2522, 2002. padhi a, verghese b, otta sk, varghese b, ramu k. adaptive evolution after duplication of penaeidin antimicrobial peptides. fish shellfish immunol. 23: 553-566, 2007. patat sa, carnegie rb, kingsbury c, gross ps, chapman r, schey k l. antimicrobial activity of histones from hemocytes of the pacific white shrimp. eur. j. biochem. 271: 4825-4833, 2004. ranganathan s, simpson jk, shaw cd, nicholas kr. the whey acidic protein family: a new signature motif and three dimensional structure by comparative modeling. j. mol. graph. mod. 17: 106-113, 1999. rattanachai a, hirono i, ohira t, takahashi y, aoki t. cloning of kuruma prawn marsupenaeus japonicus crustin-like peptide cdna and analysis of its expression. fish. sci. 70: 765-771, 2004. relf jm, chisholm jr, kemp gd, smith vj. purification and characterization of a cysteine-rich 11.5 kda antibacterial protein from the granular haemocytes of the shore crab, carcinus maenas. eur. j. biochem. 264: 350–357, 1999. robinette d, wada s, arroll t, levy mg, miller wl noga ej. antimicrobial activity in the skin of the channel catfish ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins. cell. mol. life sci. 54: 467-475,1998. robalino j, almeida js, mckillen d, colglazier j, trent hf 3rd, et al. insights into the immune transcriptome of the shrimp litopenaeus vannamei: tissue-specific expression profiles and transcriptomic responses to immune challenge. physiol. genomics 29: 44-56, 2007. romo-figueroa mg, vargas-requena c, sotelomundo rr, vargas-albores f, higuera-ciapara i, soderhall k, et al. molecular cloning of a betaglucan pattern-recognition lipoprotein from the white shrimp, penaeus (litopenaeus) vannamei: correlations between the deduced amino acid sequence and the native protein structure dev. comp. immunol. 28: 713-726, 2004. rosa rd, bandeira pt, barracco ma. molecular cloning of crustins from the hemocytes of brazilian penaeid shrimps. fems microbiol. lett. 274: 287-290, 2007. roux mm, pain a, klimpel kr, dhar ak. the lipopolysaccharide and beta-1, 3-glucan binding protein gene is upregulated in white spot virusinfected shrimp (penaeus stylirostris). j. virol. 76: 7140-7149, 2002. schalkwijk j, wiedow o, hirose s. the trappin gene family: proteins defined by an n-terminal transglutaminase substrate domain and a cterminal four-disulphide core. biochem. j. 340: 569-577, 1999. sellos d, lemoine s, wormhoudt av. molecular cloning of hemocyanin cdna from penaeus vannamei (crustacea, decapoda): structure, evolution, and physiological aspects. febs lett. 407: 153-158, 1997. smith vj, fernandes jm, kemp gd, hauton c. crustins: enigmatic wap domain-containing antibacterial proteins from crustaceans. dev. comp. immunol. 32: 758-772, 2008. somboonwiwat k, marcos m, tassanakajon a, klinbunga s, aumelas a, romestand b, et al. recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (alf) from the black tiger shrimp penaeus monodon. dev. comp. immunol. 29: 841-851, 2005. somboonwiwat k, bachère e, rimphanitchayakit v, tassanakajon a. localization of antilipopolysaccharide factor (alfpm3) in tissues of the black tiger shrimp, penaeus monodon, and characterization of its binding properties. dev. comp. immunol. 32: 1170-1176, 2008. sotelo-mundo rr, islas-osuna ma, de-la-re-vega e, hernández-lópez j, vargas-albores f, yepizplascencia g. cdna cloning of the lysozyme of the white shrimp penaeus vannamei. fish shellfish immunol. 15: 325-331, 2003. sritunyalucksana k, lee sy, söderhall k. a beta-1, 3-glucan binding protein from the black tiger shrimp, penaeus monodon. dev. comp. immunol. 26: 237-245, 2002. sun yd, fu ld, jia yp, du xj, wang q, wang yh, et al. a hepatopancreas specific c-type lectin from the chinese shrimp fenneropenaeus chinensis exhibits antimicrobial activity. mol. immunol. 45: 348-361, 2008. 178 supungul p, klinbunga s, pichyangkura r, hirono i, aoki t, tassanakajon a. antimicrobial peptides discovered in the black tiger shrimp penaeus monodon using the est approach. dis. aquat. organ. 61: 123-135, 2004. vargas-albores f, yepiz-plascencia g, jiménezvega f, avila-villa a. structural and functional differences of litopenaeus vannamei crustins. comp. biochem. physiol. 138b: 415-422, 2004. xing y, feng-ying g, qing-mei z, jun-jie b, huan w, hai-hua l, et al. cloning and characterization of the tiger shrimp lysozyme. mol. biol. rep. doi: 10.1007/s11033-008-9303-7, (in press). supungul p, tang s, maneeruttanarungroj c, rimphanitchayakit v, hirono i, aoki t, et al. cloning, expression, and anti-microbial activity of crustinpm1, a major isoform of crustin, from the black tiger shrimp penaeus monodon. dev. comp. immunol. 32: 61-70, 2008. yang c, zhang j, li f, ma h, zhang q, jose priya ta, et al. a toll receptor from chinese shrimp fenneropenaeus chinensis is responsive to vibrio anguillarum infection. fish shellfish immunol. 24: 564-74, 2008. tamura k, dudley j, nei m, kumar s. mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol. biol. evol. 24: 1596-1599, 2007. yang ls, yin zx, liao jx, huang xd, guo cj, weng sp. a toll receptor in shrimp. mol. immunol. 44: 1999-2008, 2007. tanaka s, nakamura t, morita t, iwanaga s. limulus anti-lps factor: an anti-coagulant which inhibits the endotoxin mediated activation of limulus coagulation system. biochem. biophys. res. commun. 105: 717-723, 1982. yepiz-plascencia g, vargas-albores f, jimenezvega f, ruiz-verdugo l m, romo-figueroa g. shrimp plasma hdl and beta-glucan binding protein (bgbp): comparison of biochemical characteristics. comp. biochem. physiol. b. biochem. mol. biol. 121: 309-314, 1998. townes cl, milona p, hall j. characterization of awap iv, the c-terminal domain of the avian protein awak. biochem. soc. trans. 34: 267269, 2006. zhang j, li f, wang z, xiang j. cloning and recombinant expression of a crustin-like gene from chinese shrimp, fenneropenaeus chinensis. j. biotechnol. 127: 605-614. 2007 tharntada s, somboonwiwat k, rimphanitchayakit v, tassanakajon a. anti-lipopolysaccharide factors from the black tiger shrimp, penaeus monodon, are encoded by two genomic loci. fish shellfish immunol. 24: 46-54, 2008. zhang x, huang c, qin q. anti-viral properties of hemocyanin isolated from shrimp penaeus monodon. antiviral res. 61: 93-99, 2004. zhou wj wang s, wang jx. molecular cloning and expression analysis of anti-lipopolysaccharide factor gene from fenneropeneus chinensis. sichuan j of zool. 27: 771-773 2008 (in chinese). vargas-albores f, jiménez-vega f, yepiz-plascencia gm. purification and comparison of beta-1, 3glucan binding protein from white shrimp (penaeus vannamei). comp. biochem. physiol. 116b: 453-458, 1997. 179 analysis of the immune-related transcriptome from an apterygote insect, the firebrat thermobia domestica isj 6: 106-113, 2009 issn 1824-307x review the innate immunity in the cnidarian hydra vulgaris b altincicek interdisciplinary research center, institute of phytopathology and applied zoology, justus-liebig-university of giessen, d-35392 giessen, germany accepted july 29, 2009 abstract hydra vulgaris is currently receiving increased attention as a genetically tractable invertebrate model system for studying important processes of life such as the innate immune defense. similar to complex animals, h. vulgaris polyps respond to injury by abrupt muscle contraction, by limited escape behavior, and by healing the damaged tissue. simultaneously, cellular processes such as phagocytosis and programmed cell death as well as the massive production of antimicrobial peptides are induced. recent studies identified several molecular pathways controlling these responses; however, the interdependence of innate immunity and, for example, regeneration and tissue remodeling is not well elucidated yet. h. vulgaris belongs to the cnidaria representing the phylogenic sister group of bilaterian animals; hence, a better understanding of evolutionarily conserved as well as hydra/cnidaria-specific immune responses will provide deep insight into both origin and evolution of the animal innate immune system. key words: cnidaria; danger signaling; hydra vulgaris, innate immunity; regeneration; model organism introduction a major evolutionary split occurred in the animal kingdom between so-called basal animals, which include cnidaria, and the deuterostomia/protostomia, which include most other animals (fig. 1) (finnerty et al., 2004). the freshwater cnidarian hydra vulgaris has a simple body plan with two cell layers, the ectoderm and the endoderm, separated by an extracellular matrix (mesoglea). hydra is prominent for its ability to regenerate a complete organism from small body fragments. therefore, h. vulgaris has widely been used as feasible model system to study pattern formation, cell differentiation, morphogenesis, and regeneration (trembley, 1744; campbell, 1967; gierer et al., 1972; bode et al., 1986; holstein et al., 1991; bosch, 1998; hassel, 1998; galliot and schmid, 2002; steele, 2002; bode, 2003; holstein et al., 2003; bosch, 2007). this review focuses on recent studies investigating the innate immune system in h. vulgaris. ___________________________________________________________________________ corresponding author: boran altincicek interdisciplinary research center institute of phytopathology and applied zoology justus-liebig-university of giessen d-35392 giessen, germany e-mail: boran.altincicek@agrar.uni-giessen.de hydra vulgaris as model system to study innate immunity in higher animals, immunity is a complex and highly developed system of specialized cells and organs that protects an organism against bacterial, parasitic, fungal, and viral infections. besides the conserved innate immunity, the adaptive or acquired immunity is present in vertebrates representing a novel evolutionary acquirement which enables the somatic generation of highly variable t-cell receptors and b-cell derived antibodies by recombination-activating gene dependent genomic rearrangements (germain, 2004; akira et al., 2006; medzhitov, 2007). these molecules provide pathogen-specific antigen recognition and create the so-called immunological memory. invertebrate immunity shows many parallels to the innate immunity of vertebrates, which complements the adaptive immunity. it includes cellular phagocytosis, antiviral rna-interference, and killing of pathogens by antimicrobial peptides, lysozymes, and reactive oxygen species (boman, 2003; kim and ausubel, 2005; cherry and silverman, 2006; lemaitre and hoffmann, 2007) (fig. 1). both vertebrate and invertebrate innate immunity recognize invading pathogens by hereditary pattern recognition receptors including 106 mailto:boran.altincicek@agrar.uni-giessen.de fig. 1 origin and evolution of immunity in metazoa. many mechanisms of innate immunity such as toll-like receptors (tlr), rna interference (rnai), antimicrobial peptides and toxic compounds are conserved across all animals. however, some aspects such as acquired immunity have only been evolved in vertebrates. several animal species that we examined for immune inducible genes to further understanding of the evolution of innate immunity are written in gray (altincicek and vilcinskas, 2007a, b, c, 2008b, c; altincicek et al., 2008). nfκb, nuclear factor kappa b, myd88, myeloid differentiation primary response gene 88; ig-like, immunoglobulin-like; srcr, scavenger receptor cysteine rich; gnbp, gram-negative bacteria-binding protein; ppo, prophenoloxidase. toll receptors, peptidoglycan recognition proteins, and lectins which bind to microbial components such as bacterial lipopolysaccharides, peptidoglycans, or fungal ß-1,3 glucans (royet, 2004; akira et al., 2006; medzhitov, 2007). moreover, immune related signaling is achieved in animals ranging from sponges to humans mainly by myeloid differentiation primary response gene 88 (myd88), nuclear factor kappa b (nfκb)-like factors, and stress-activated protein kinases such as p38 and jnk (böhm et al., 2002; wiens et al., 2005; akira et al., 2006; wiens et al., 2007) (fig. 1). this suggests a shared root for invertebrate and vertebrate immune pathways conserved over hundreds of millions of years. to elucidate origin and evolution of animal innate immunity, immune defense reactions have been studied in basal animals such as poriferans and cnidarians. recently, methods like dsrnamediated gene silencing (lohmann et al., 1999; chera et al., 2006; miljkovic-licina et al., 2007) and of transgenesis allowing stable genetic manipulation have been established in h. vulgaris (wittlieb et al. 2006; khalturin et al., 2007). hence, h. vulgaris represents a complementary invertebrate model system to the fruit fly drosophila melanogaster and the nematode caenorhabditis elegans for studying important processes of life including the innate immune defense. moreover, whole genome sequences have become available for two cnidarians, the hydrozoan hydra magnipapillata and the anthozoan nematostella vectensis (technau et al., 2005). this allowed recent comparative investigations of gene sets regarding innate immunity proteins in these two basal eumetazoans (hemmrich et al., 2007; miller et al., 2007). regarding the use of h. vulgaris as model system to study innate immunity, it should be mentioned that numerous hydra species live in mutualistic symbiosis with algae (pardy and heacox, 1976). moreover, symbiosis with algae seems to represent a common feature of the hydra group since symbiosis can be experimentally induced in non-symbiotic hydra species (rahat and reich, 1984, 1985, 1986). a tight association also exists with bacterial symbionts or commensals (wilkerson, 1980; rahat and dimentman, 1982; fraune and bosch, 2007; fraune et al., 2009) indicating that the immune system of hydra polyps is capable to distinguish between beneficial and pathogenic microbes. theory predicts that hydra may induce sophistically regulated and probably specific immune responses, which control interacting microbes. yet, specific receptors or signaling pathways have not been identified, but it is obvious that the evolutionarily conserved programmed cell death (cikala et al., 1999; böttger and alexandrova, 2007) plays a major role in controlling mutualistic symbionts or pathogens probably similar to observations from other cnidarian species (dunn et al., 2002; dunn et al., 2004; ainsworth et al., 2007; dunn, 2009; dunn and weis, 2009). 107 fig. 2 early phase responses of h. vulgaris polyp to bisection: (a) h. vulgaris is organized as body column with a mouth and ring of tentacles at the apical pole and a foot process with a basal disk at the distal pole. buds are formed for asexual reproduction. (b) bisection of a polyp results in abrupt muscle contraction and subsequently in limited escape behavior. (c) the contraction reduces the area of injury to a minimum which is indicated by a circle. (d) within several minutes post bisection mucus is produced and motile cells appear at the wounding site which phagocytize debris form damaged cells. nc, nematocyst. bars = 200 µm (a, b); 20 µm (c, d). immunorecognition and effector molecules h. vulgaris polyps show many parallels to complex animals regarding to their innate immune reactions. bisection of h. vulgaris polyps, for example, leads to an abrupt muscle contraction and escape reaction (figs 2a, b). these behavioural reactions are accompanied by the massive production of mucus at the wounding site and the appearance of phagocytic cells eliminating cell debris and dying cells (figs 2c, d). significantly, homogenates obtained from bisected animals exhibited a significant higher level of antimicrobial activity than homogenates obtained from untreated animals as determined by an inhibition zone assay with live bacteria (fig. 3). this is in line with recent observations that the hydra innate immune system senses tissue injury itself or along with bacterial contamination from the environment to induce the massive production of antimicrobial peptides along 108 with further potential bactericidal or bacteriostatic molecules (altincicek and vilcinskas, 2008a; jung et al., 2008; bosch et al., 2009). the finding that the drosophila transmembrane protein toll, which is essential for the development of embryonic dorsoventral polarity in the fruit fly, also mediates immune responses to infection had a pioneering role in the identification of toll-like receptors as evolutionarily conserved and essential regulators of immunity (beutler, 2004). mammalian toll-like receptors recognize a highly divergent collection of ligands such as lipoproteins, bacterial lipopolysaccharide and peptidoglycans, fungal zymosan, plant diterpene paclitaxel, viral proteins, and endogenous molecules such as heat-shock proteins and nucleic acids, all of which have different structures (akira et al., 2006; karin et al., 2006). in nematostella, toll-like receptors and a nfκb homolog have been identified but, surprisingly, not in hydra suggesting that a substantial immune gene loss or significant gene diversification of these factors have occurred during hydra evolution (miller et al., 2007). consistent with this hypothesis, a recent study indicated that in hydra instead of typical toll-like receptors two related toll-il-1 receptor (tir) domain transmembrane proteins interact with leucine-rich repeat and epidermal-growth factor domain transmembrane proteins to induce the expression of antimicrobial peptides (bosch et al., 2009); however, a corresponding transcriptional activator of this signaling pathway has yet not been identified. our recent studies with h. vulgaris revealed several immune-relevant inducible genes including, for example, a potential antimicrobial peptide, major vault protein and p47phox protein or neutrophil cytosolic factor 1 (altincicek and vilcinskas, 2008a). in mammals, major vault protein has been demonstrated to be essential for optimal epithelial cell phagocytosis of the pathogenic bacterium pseudomonas aeruginosa (kowalski et al., 2007). phagocyte napdh-oxidase (phox) is responsible for the generation of superoxide with secondary production of other microbicidal reactive oxygen species (ros) in human neutrophils and macrophages. phox consists of the catalytic subunit gp91phox, along with the regulatory subunits p22phox, p67phox, p40phox, the small gtpase rac, and p47phox or neutrophil cytosolic factor 1 (el-benna et al., 2009). since both molecules, major vault protein and p47phox protein, are reported to be important in mammalian immunity to eliminate invading pathogens investigation of their counterparts in hydra immune responses may further our understanding of human immune defense reactions. injury induces immune responses and regeneration processes the ability to respond to injury and to repair tissue is a fundamental property of all multicellular organisms. injury of the vertebrate skin, for example, initiates a complex sequence of events involving inflammation as well as the formation and remodeling of new tissue (schäfer and werner, fig. 3 bisection induces expression of antimicrobial factors in h. vulgaris polyps. homogenates obtained from 10 animals 24 h post bisection exhibited a significant higher level of antimicrobial activity than homogenates obtained from 10 untreated animals. antibacterial activity was determined with live micrococcus luteus as described (altincicek and vilcinskas, 2008c). results represent mean values of three independent repetitions ±sd. statistical significance was determined by student’s t-test (p<0.01). 2007). at the wounding site, multiple biological pathways immediately become activated and are synchronized to respond. the mammalian immune system is triggered by both microbial pattern molecules and endogenously derived danger signals such as extracellular heat shock proteins, uric acid, and hmgb1, which are released during tissue injury (matzinger, 2002; akira et al., 2006; karin et al., 2006). similarly, hydra is capable in sensing microbial molecular pattern such as lipopolysaccharide (altincicek and vilcinskas, 2008a; bosch et al., 2009) and flagellin (bosch et al., 2009) as well as danger molecules derived from dying or damaged cells such as extracellular nucleic acids and uric acid to induce expression of antimicrobial peptides (bosch et al., 2009). in vertebrates, wound healing requires dickkopf and wnt/β-catenin signaling and, additionally, the action of tgf-β/bmp and matrix metalloproteinases (mmps) (stoick-cooper et al., 2007). homologs of all of these factors have also been described to regulate hydra regeneration processes (galliot and schmid, 2002; holstein et al., 2003). recent studies, for example, demonstrated that the wnt and tgf-β/bmp signaling pathway components are transcriptionally up-regulated early during regeneration in hydra (holstein et al., 2003) and that the hydra dickkopf-like protein expression that antagonizes wnt/β-catenin signaling is stimulated by the injury signal itself (holstein et al., 2003). it has furthermore been shown that hydra astacinclass metalloproteinases are crucial for both foot and head regeneration (yan et al., 2000a, b) and that 109 fig. 4 phylogenic analysis of cnidaria mmps. phylogenic reconstruction was performed with the software package mrbayes 3.1.2 similar as described (knorr et al., 2009). the model with the overall highest posterior probability was wag. the average standard deviation of split frequencies at 106 generations was 0.01 and therefore indicated that the two chains that were run converged on similar results. the tree was drawn with figtree (tree.bio.ed.ac.uk/software/figtree). posterior probabilities are plotted at the nodes, which can be interpreted as the probability that the tree or clade is correct. the scale bar represents the substitutions per site. accession numbers of investigated mmps are as follows: t. castaneum (tribolium1, xp_968822; tribolium2, xp_969495; tribolium3, xp_972146); d. melanogaster (drosophila1, np_523852; drosophila2, np_995788), homo sapiens (human19, q99542; human28, q9h239; human11, p24347; human21, q8n119; human23, o75900; human17, q9ulz9; human25, q9npa2; human14, p50281; human15, p51511; human16, p51512; human24, q9y5r2; human20, o60882; human12, p39900; human13, p45452; human1, p03956; human8, p22894; human3, p08254; human10, p09238; human7, p09237; human26, q9nre1; human2, p08253; human9, p14780); h. vulgaris mmp (aad45804); h. magnipapillata (hyd1, xp_002158607; hyd2, xp_002163047; hyd3, xp_002163794; hyd4, xp_002160477; hyd5, xp_002170324; hyd6, xp_002164979; hyd7, xp_002168433; hyd8, xp_002168462; hyd9, xp_002155089; hyd10, xp_002161129; hyd11, xp_002166835; hyd12, xp_002168334; hyd13, xp_002155205; hyd14, xp_002154985; hyd15, xp_002165116; hyd16, xp_002163948; hyd17, xp_002164594; hyd18, xp_002165203; hyd19, xp_002163814); n. vectensis (nema1, xp_001640669; nema2, xp_001640565; nema3, xp_001633230; nema4, xp_001629775; nema5, xp_001636910; nema6, xp_001635715; nema7, xp_001640426; nema8, xp_001638726; nema9, xp_001640819; nema10, xp_001630740; nema11, xp_001626901; nema12, xp_001640427; nema13, xp_001622031; nema14, xp_001630704; nema15, xp_001642037; nema16, xp_001635184; nema17, xp_001628710). 110 http://tree.bio.ed.ac.uk/software/figtree a hydra matrix metalloproteinase (mmp) represents a central effector molecule in extracellular matrix degradation, epithelial morphogenesis, and probably in cell differentiation (leontovich et al., 2000; sarras et al., 2002; shimizu et al., 2002). in humans, more than 20 different mmps have been identified which degrade virtually all extracellular matrix proteins and release or degrade bioactive fragments and growth factors (pagemccaw et al., 2007). they influence fundamental biological and pathological processes like embryonic development, angiogenesis, tissue homeostasis and morphogenesis, wound repair, inflammation, and tumor progression (page-mccaw et al., 2007). the recently investigated h. vulgaris mmp (leontovich et al., 2000; sarras et al., 2002; shimizu et al., 2002) has a well-conserved overall domain structure to that of their invertebrate and vertebrate counterparts. h. vulgaris mmp includes a propeptide sequence containing a conserved inhibitory cysteine switch sequence and the catalytic domain with the conserved hexxhxxgxxh sequence critical for binding the catalytically active zinc ion (nagase and woessner, 1999). interestingly, examination of the cnidarian whole genome sequences revealed that 19 h. magnipapillata and 17 n. vectensis mmp homologs exist which most of them clade together with the identified h. vulgaris mmp (fig. 4). these observations suggest that mmps fulfill complex and overlapping functions in cnidaria probably similar to mammalian mmps (page-mccaw et al., 2007). interestingly, two nematostella mmps grouped together with human mmp19, 21, 23, 28 and insect mmp1s indicating possible evolutionarily conservation of these mmps in n. vectensis and loss in h. magnipapillata (fig. 4). mmps have further been recognized as important modulators of immunity in both mammals (parks et al., 2004; vanlaere and libert, 2009) and insects (altincicek and vilcinskas, 2008c; knorr et al., 2009); hence, we believe that their investigation in hydra will help to unravel the complex interdependence of the immunity with regeneration processes. altincicek b, vilcinskas a. identification of immunerelated genes from an apterygote insect, the firebrat thermobia domestica. insect biochem. mol. biol. 37: 726-31, 2007c. future priorities in the field of studying hydra innate immunity goals in the field of studying innate immunity in the h. vulgaris model system will be the elucidation of both genetic mechanisms driving host-parasite as well as host-symbiont co-evolution and principles of epithelial immune mechanisms in an animal that lacks a coelomic cavity. examination of the hydra immunity may further help to understand interactions of reef-building cnidaria with their pathogens as well as their symbionts (bosch, 2008) helping to keep these ecosystems alive. in this context, however, it should also be noted that the depth of the split between the cnidarian classes anthozoa (nematostella) and hydrozoa (hydra) has been determined to be as great as that between protostomia and deuterostomia (miller and ball, 2008). this observation indicates that immune mechanisms may be highly derived within cnidaria as, for example, found for the toll and nfκb signaling as mentioned above. in conclusion, the investigation of innate immune defense of basal animals with h. vulgaris as a genetically tractable model system is an exciting field, which will give valuable insight into the evolution of animals. combining mechanistic understanding of immunity along with processes of regeneration and of development will be, without doubt, a fruitful union. acknowledgements i want to thank monika hassel (philipps university of marburg, germany) for kindly providing me with hydra vulgaris (zürich strain) and for helpful comments. i am grateful to andreas vilcinskas (justus-liebig-university of giessen, germany) and katja altincicek for critical reading of the manuscript. financial support by the german research foundation (deutsche forschungsgemeinschaft, dfg) with the heisenberg fellowship (al902/4-1) is greatly appreciated. references ainsworth td, kvennefors ec, blackall ll, fine m, hoegh-guldberg o. disease and cell death in white syndrome of acroporid corals on the great barrier reef. mar. biol. 151: 19-29, 2007. akira s, uematsu s, takeuchi o. pathogen recognition and innate immunity. cell 124: 783801, 2006. altincicek b, knorr e, vilcinskas a. beetle immunity: identification of immune-inducible genes from the model insect tribolium castaneum. dev. comp. immunol. 32: 585-595, 2008. altincicek b, vilcinskas a. analysis of the immuneinducible transcriptome from microbial stress resistant, rat-tailed maggots of the drone fly eristalis tenax. bmc genomics 8: 326, 2007a. altincicek b, vilcinskas a. analysis of the immunerelated transcriptome of a lophotrochozoan model, the marine annelid platynereis dumerilii. front. zool. 4: 18, 2007b. altincicek b, vilcinskas a. comparative analysis of septic injury-inducible genes in phylogenetically distant model organisms of regeneration and stem cell research, the planarian schmidtea mediterranea and the cnidarian hydra vulgaris. front. zool. 5: 6, 2008a. altincicek b, vilcinskas a. identification of immune inducible genes from the velvet worm epiperipatus biolleyi (onychophora). dev. comp. immunol. 32: 1416-1421, 2008b. altincicek b, vilcinskas a. identification of a lepidopteran matrix metalloproteinase with dual roles in metamorphosis and innate immunity. dev. comp. immunol. 32: 400-409, 2008c. bode hr. head regeneration in hydra. dev. dyn. 226: 225-236, 2003. bode h, dunne j, heimfeld s, huang l, javois l, koizumi o, et al. transdifferentiation occurs continuously in adult hydra. curr. topics dev. biol. 20: 257-280, 1986. 111 böhm m, gamulin v, schröder hc, müller we. evolution of osmosensing signal transduction in metazoa: stress-activated protein kinases p38 and jnk. cell tissue res. 308: 431-438, 2002. boman hg. antibacterial peptides: basic facts and emerging concepts. j. intern. med. 254: 197215, 2003. bosch tcg. the path less explored: innate immune reactions in cnidarians. in heine h (ed), innate immunity of plants, animals, and humans, berlin, springer-verlag, pp 27-42, 2008. bosch, tcg. in ferretti p, geraudie j (eds), cellular and molecular basis of regeneration: from invertebrates to humans, wiley, new york, pp 111-134, 1998. bosch tcg. why polyps regenerate and we don't: towards a cellular and molecular framework for hydra regeneration. dev. biol. 303: 421-433, 2007. bosch tcg, augustin r, anton-erxleben f, fraune s, hemmrich g, zill h, et al. uncovering the evolutionary history of innate immunity: the simple metazoan hydra uses epithelial cells for host defence. dev. comp. immunol. 33: 559569, 2009. böttger a, alexandrova o. programmed cell death in hydra. semin. cancer biol. 17: 134-146, 2007. campbell rd. tissue dynamics of steady state growth in hydra littoralis. ii. patterns of tissue movement. j. morph. 121: 19-28, 1967. chera s, de rosa r, miljkovic-licina m, dobretz k, ghila l, kaloulis k, et al. silencing of the hydra serine protease inhibitor kazal1 gene mimics the human spink1 pancreatic phenotype. j. cell sci. 119: 846-857, 2006. cherry s, silverman n. host-pathogen interactions in drosophila: new tricks from an old friend. nat. immunol. 7: 911-917, 2006. cikala m, wilm b, hobmayer e, böttger a, david cn: identification of caspases and apoptosis in the simple metazoan hydra. curr. biol. 9: 959962, 1999. dunn sr. immunorecognition and immunoreceptors in the cnidaria. inv. surv. j. 6: 7-14, 2009. dunn sr, bythell jc, le tissier mda, burnett wj, thomason jc. programmed cell death and cell necrosis activity during hyperthermic stressinduced bleaching of the symbiotic sea anemone aiptasia sp. j. exp. mar. biol. ecol. 272: 29-53, 2002. dunn sr, thomason jc, le tissier mda, bythell jc. heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration. cell death diff. 11: 1213-1222, 2004. dunn sr, weis vm. apoptosis as a post-phagocytic winnowing mechanism in a coral-dinoflagellate mutualism. environ. microbiol. 11: 268-276, 2009. el-benna j, dang pm, gougerot-pocidalo ma, marie jc, braut-boucher f. p47phox, the phagocyte nadph oxidase/nox2 organizer: structure, phosphorylation and implication in diseases. exp. mol. med. 41: 217-225, 2009. finnerty jr, pang k, burton p, paulson d, martindale mq. origins of bilateral symmetry: hox and dpp expression in a sea anemone. science 304: 1335-1337, 2004. fraune s, abe y, bosch tcg. disturbing epithelial homeostasis in the metazoan hydra leads to drastic changes in associated microbiota. environ. microbiol. online version ahead of print, 2009. fraune s, bosch tcg. long-term maintenance of species-specific bacterial microbiota in the basal metazoan hydra. proc. natl. acad. sci. usa 104: 13146-13151, 2007. galliot b, schmid v. cnidarians as a model system for understanding evolution and regeneration, int. j. dev. biol. 46: 39-48, 2002. germain rn. an innately interesting decade of research in immunology. nat. med. 10: 13071320, 2004. gierer a, berking s, bode h, david cn, flick k, hansmann g, et al. regeneration of hydra from reaggregated cells, nat. new biol. 239: 98-101, 1972. guder c, pinho s, nacak tg, schmidt ha, hobmayer b, niehrs c, et al. an ancient wntdickkopf antagonism in hydra. development 133: 901-911, 2006. hassel m. upregulation of a hydra vulgaris cpkc gene is tightly coupled to the differentiation of head structures. dev. genes evol. 207: 489501, 1998. hemmrich g, miller dj, bosch tc. the evolution of immunity: a low-life perspective. trends immunol. 28: 449-454, 2007. holstein tw, hobmayer e, technau u. cnidarians: an evolutionarily conserved model system for regeneration? dev. dyn. 226: 257-267, 2003. holstein tw, hobmayer e, david cn. pattern of epithelial cell cycling in hydra. dev. biol. 148: 602-611, 1991. jung s, dingley aj, augustin r, anton-erxleben f, stanisak m, gelhaus c et al. hydramacin-1; structure and antibacterial activity of a protein from the basal metazoan hydra. j. biol. chem. 284: 1896-1905, 2008. karin m, lawrence t, nizet v. innate immunity gone awry: linking microbial infections to chronic inflammation and cancer. cell 124: 823-835, 2006. khalturin k, anton-erxleben f, milde s, plötz c, wittlieb j, hemmrich g, et al. transgenic stem cells in hydra reveal an early evolutionary origin for key elements controlling self-renewal and differentiation. dev. biol. 309: 32-44, 2007. kim dh, ausubel fm. evolutionary perspectives on innate immunity from the study of caenorhabditis elegans. curr. opin. immunol. 17: 4-10, 2005. knorr e, schmidtberg h, vilcinskas a, altincicek b. mmps regulate both development and immunity in the tribolium model insect. plos one 4:e4751, 2009. kowalski mp, dubouix-bourandy a, bajmoczi m, golan de, zaidi t, coutinho-sledge ys, et al. host resistance to lung infection mediated by major vault protein in epithelial cells. science 317: 130-132, 2007. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. 112 leontovich aa, zhang j, shimokawa k, nagase h, sarras mp jr. a novel hydra matrix metalloproteinase (hmmp) functions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot process. development 127: 907-920, 2000. lohmann ju, endl i, bosch tcg. silencing of developmental genes in hydra. dev. biol. 214: 211-214, 1999. matzinger p. the danger model: a renewed sense of self. science 296: 301-305, 2002. medzhitov r. recognition of microorganisms and activation of the immune response. nature 449: 819-826, 2007. miljkovic-licina m, chera s, ghila l, galliot b. head regeneration in wild-type hydra requires de novo neurogenesis. development 134: 11911201, 2007. miller dj, ball ee. cryptic complexity captured: the nematostella genome reveals its secrets. trends genet. 24: 1-4, 2008. miller dj, hemmrich g, ball ee, hayward dc, khalturin k, funayama n, et al. the innate immune repertoire in cnidaria--ancestral complexity and stochastic gene loss. genome biol. 8: r59, 2007. nagase h, woessner jf. matrix metalloproteinases. j. biol. chem. 274: 21491-21494, 1999. page-mccaw a, ewald aj, werb z. matrix metalloproteinases and the regulation of tissue remodelling. nat. rev. mol. cell. biol. 8: 221233, 2007. pardy rl, heacox ae. growth of algal symbionts in regenerating hydra. nature 260: 809-810, 1976. parks wc, wilson cl, lopez-boado ys. matrix metalloproteinases as modulators of inflammation and innate immunity. nat. rev. immunol. 4: 617-629, 2004. rahat m, dimentman c. cultivation of bacteria free hydra viridis: missing budding factor in nonsymbiotic hydra. science 216: 67-68, 1982. rahat m, reich v. intracellular infection of aposymbiotic hydra viridis by a foreign freeliving chlorella sp.: initiation of a stable symbiosis. j. cell sci. 65: 265-277, 1984. rahat m, reich v. correlations between characteristics of some free-living chlorella sp. and their ability to form stable symbioses with hydra viridis. j. cell sci. 74: 257-266, 1985. rahat m, reich v. algal endosymbiosis in brown hydra: host/symbiont specificity. j. cell sci. 86: 273-286, 1986. royet j. infectious non-self recognition in invertebrates: lessons from drosophila and other insect models. mol. immunol. 41: 10631075, 2004. sarras mp jr, yan l, leontovich a, zhang js. structure, expression, and developmental function of early divergent forms of metalloproteinases in hydra. cell. res.12: 16376, 2002. schäfer m, werner s. transcriptional control of wound repair. annu. rev. cell. dev. biol. 23: 69-92, 2007. shimizu h, zhang x, zhang j, leontovich a, fei k, yan l, et al. epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases. development 129: 15211532, 2002. steele re. developmental signaling in hydra: what does it take to build a "simple" animal? dev. biol. 248: 199-219, 2002. stoick-cooper cl, moon rt, weidinger g. advances in signaling in vertebrate regeneration as a prelude to regenerative medicine. genes dev. 21: 1292-1315, 2007. technau u, rudd s, maxwell p, gordon pm, saina m, grasso lc, et al. maintenance of ancestral complexity and non-metazoan genes in two basal cnidarians. trends genet. 21: 633-639, 2005. trembley a. m´emoires pour servir `a l’histoire d’un genre de polypes d’eau douce, `a bras en forme de cornes, verbeck, leiden, 1744. vanlaere i, libert c. matrix metalloproteinases as drug targets in infections caused by gramnegative bacteria and in septic shock. clin. microbiol. rev. 22: 224-239, 2009. wiens m, korzhev m, krasko a, thakur nl, perović-ottstadt s, breter hj, et al. innate immune defense of the sponge suberites domuncula against bacteria involves a myd88dependent signaling pathway. induction of a perforin-like molecule. j. biol. chem. 280: 27949-27959, 2005. wiens m, korzhev m, perovic-ottstadt s, luthringer b, brandt d, klein s, et al. toll-like receptors are part of the innate immune defense system of sponges (demospongiae: porifera). mol. biol. evol. 24: 792-804, 2007. wilkerson fp. bacterial symbionts on green hydra and their effect on phosphate uptake. microb. ecol. 6: 85-92, 1980. wittlieb j, khalturin k, lohmann ju, anton-erxleben f, bosch tcg. transgenic hydra allow in vivo tracking of individual stem cells during morphogenesis. proc. natl. acad. sci. usa 103: 6208-6211, 2006. yan l, fei k, zhang j, dexter s, sarras mp jr. identification and characterization of hydra metalloproteinase 2 (hmp2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis. development 127: 129-141, 2000. yan l, leontovich a, fei k, sarras mp jr. hydra metalloproteinase 1: a secreted astacin metalloproteinase whose apical axis expression is differentially regulated during head regeneration. dev. biol. 219: 115-128, 2000. 113 ii incontro degli ascidiologi italiani isj 5: 83-96, 2008 issn 1824-307x report of meeting ii scientific meeting of the italian ascidiologists, 30 june – 1 july 2008, department of animal biology, university of palermo, palermo, italy organizers: n parrinello, v arizza, m cammarata, m vazzana, a vizzini marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy session 1. development and morphogenesis sensory organs in the oral siphon of pyrosoma p burighel, l manni f caicci department of biology, university of padua, padua, italy in previous papers we studied the sensory structures in the branchial siphons of ascidians evidencing the presence of sparse primary sensory cells and secondary sensory cells. the latter constitute an extended mechanosensory organ (the coronal organ) presents in the oral siphon of all the examined ascidians. for position, origin and ultrastructural features these sensory cells have several correspondences with the hair cells of vertebrates. thus, the possibility that they derive from embryonic territories sharing common aspects to the neural placodes of vertebrates was proposed. in order to verify whether the secondary sensory cells can be considered a common feature of all tunicates, we studied a representative of the pyrosomatida (thaliacea). pyrosomes are planktonic free-swimming tunicates forming tubular colonies. the individuals are embedded in the wall of the tube, with their oral siphon opened at the outer surface and their cloacal siphon opened into the common, central cavity. a water current passes each individual from the oral to the cloacal siphon. the oral siphon is rich in musculature and is covered by the epidermis also in its interior surface. a crown of tentacles run at the base of the siphon; in particular, a long ventral tentacle extends down toward the branchial cavity. with series of thick (1 µm) and thin sections we have evidenced that two kind of sensory cells are present in the oral siphon of pyrosomes. one is represented by the sensory cells of small, scattered cupular organs, which are formed of the central sensory cell accompanied by two supporting cells. the sensory cell is covered by a tunic-like cupula and its apical region has an extracellular canal containing a long cilium emerging among numerous long microvilli. the organisation of these cupular organs strictly recall the mechanoreceptor capsular organs of some ascidians. the second kind is represented by secondary mechanosensory, ciliated cells whose base contact neurites directed to (or arriving from) the brain. these cells run in a continuous row along the border of all the tentacles, forming a coronal organ corresponding to that of ascidians and resemble vertebrate hair cells for the presence of one-two cilia surrounded by a dozen of microvilli. the data suggest that the coronal organ is a common feature of tunicates. differentiation of sensory papillae in the embryo and larva of botryllus schlosseri f caicci, p burighel, l manni department of biology, university of padua, padua, italy in ascidians, the main class of the tunicates, the primary sensory cell constitutes the basic cellular element of most sensory structures, both in the freeswimming larva and in the sessile adult. the larva has sensory structures specialized for reception of gravitational, light, tactile and chemical stimuli. in particular, the sensory papillae are considered chemoreceptive structures, localized in rostral position of larval cephalenteron, which recognize and select the suitable substrate for adhesion at the onset of metamorphosis. by light and electron microscopy, we have studied the development and the structure of the sensory papillae in the embryo and larva of botryllus schlosseri. in this species, the papillae are three in a triradial arrangement and were defined in the past as “ganglionated type” for the presence of a ganglion to their base. the first rudiments of the papillae appear in early embryo in form of three distinct ectoderm evaginations, delimiting a small cavity in communication with the underlying mesenchymal space. in the successive stages, the cavity of each papilla enlarges to receive the papillary ganglion, which is constituted of primary sensory cells. these latter are bipolar neurons possessing receptor terminations in the tunic and a long axon extended to the central visceral ganglion. the papillary ganglion becomes recognizable under the epidermis owing to extension of the sensory neuron bodies into the papillary cavity. in the swimming larvae, the papillae are formed of three cell types: central sensory cells of papillary ganglion, peripheral sensory cells and cubical parietal cells. immediately before the adhesion to substrate, the papillae modify their shape: they elongate, the 83 ganglion becomes embedded in the papillary ectoderm, and the receptor terminations, crossing the tunic layer, are exposed to external side through fissures of the tunic. after adhesion, the papillae are retracted, while the central interpapillary area reaches the substrate; at the same time, the larva protrudes the blood ampullae for the definitive attach to the substrate. developmental expression and gene organization of protochordate synapsins: highlights on basic functions s candiani1, m parodi1, d oliveri2, f benfenati1, m pestarino1 1department of biology university of genoa, genoa, italy 2department of experimental medicine, university of genoa, genoa, italy synapsins belong to a family of neuron-specific phosphoproteins and are involved in several functions correlated with neurotransmitter release and synaptogenesis (reviewed in hilfiker et al. phil.trans. r. soc. lond. b 354: 269-279, 1999). the comprehension of the basal role of the synapsin family is hampered in vertebrates by the presence of multiple members. then, the study of homologous genes in protochordates, such as ascidians and amphioxus, lacking the whole genome duplications characteristic of vertebrates, could help to better understand the complex functions of synapsins. therefore, we have cloned and analyzed two synapsin genes, ci-syn in the ascidian ciona intestinalis and amphisyn in the amphioxus branchiostoma floridae. our results show the presence of a single synapsin gene in both organisms, and the occurrence of a second transcript by alternative splicing in b. floridae. our genomic analysis reveals an high homology degree with vertebrate synapsins, in particular with synapsin iii, the occurrence of three conserved domains a, c and e, and the presence of the classical nested organization of a timp (tissue inhibitor of metalloproteinase) gene in the intronic sequence. moreover, we have localized by in situ hybridization ci-syn and amphisyn transcripts exclusively at neuronal levels and in particular in a relevant portion of developing neurons, suggesting that the basic role of synapsins as regulators of neurotransmission and synaptogenesys has been conserved during evolution. sex hormone profiles in ciona intestinalis (ascidiacea urochordata) and their modulation by tributyltin (tbt) mv cangialosi1, e puccia1, a mazzola2, a arukwe3 1department of animal biology “g. reverberi”, university of palermo, palermo, italy 2department of ecology, university of palermo, palermo, italy 3department of biology, norwegian university of science and technology (ntnu), høgskoleringen 5, 7491 trondheim, norway regardless of species, steroid hormones are derived from cholesterol as a common precursor. in chordate, genome sequencing showed that cytochrome p450 (cyp) enzyme system play integral roles steroidogenic pathways and these processes are well conserved in many species. interestingly, in the protochordate ciona intestinalis, several rate-limiting steps involving cyp enzymes are lacking, although steroid hormones have been detected. the aim of this work is investigate the synthetic pathway of steroid hormones in c. intestinalis. given the lack of integral cyp enzyme genes, our hypothesis is that steroid hormone synthesis in c. intestinalis are probably mediated through an alternative pathway involving second messenger (camp) that activate protein kinases to regulate acute steroid production at level of substrate (cholesterol) availability. our preliminary data showed an increase in cholesterol and ca2+ concentrations in ovary of c. intestinalis after in vitro tbt treatment. based on these results, we started the investigation of steroid hormones levels in c. intestinalis after in vitro tbt treatment. analysis on testosterone, 17β-estradiol, aldosterone, progesterone and cortisol concentrations has shown appreciable variations in these hormones. thus, we are currently evaluating whether these responses could be activated or enhanced in the presence of camp or second messenger activator (forskolin). ion current and molecules involved in meiotic progression and fertilization of ciona intestinalis oocytes a cuomo1, f silvestre1, s bilotto1,3, gl russo2, e tosti1 1stazione zoologica “anton dohrn”, napoli, italy 2istituto di scienze dell’alimentazione, cnr, avellino, italy 3dipartimento di biologia, università di napoli “federico ii”, napoli, italy we performed an electrophysiological characterization of immature oocytes of the ciona intestinalis at different stages of growth and meiotic competence. oocytes showing a clear germinal vesicle (gv) have been classified on the basis of diameter and cytoplasm pigmentation as follows: stage a: <70μm diameter with a clear cytoplasm; stage b: 70-120μm diameter with a yellow cytoplasm; stage c: >120μm diameter with a brown cytoplasm. using the whole-cell voltage clamp technique on the oocytes collected from the ovary and deprived of accessory membranes, we showed for the first time a high l-type calcium currents activity at the stage a, these currents significantly decreased through meiosis up to the metaphase i (mi). oocytes at stage b showed the first appearance of sodium current activity that are still active at the mi phase. in vitro maturation experiments in calcium free sea water showed a significant reduction of maturation of oocytes at the stage c. intracellular calcium release was higher in mi than in previous stages; at last by using cyclic amp activators we showed a significant decrease of the germinal vesicle breakdown. fertilization current generated in sodium free sea water was significantly lower than the control, furthermore, oocytes fertilized in absence of sodium showed high development of the anomalous “rosette” embryos. 84 taken together these results imply: i) an involvement of external calcium in modulating oocytes growth, meiotic competence acquisition, prophase/metaphase transition and calcium stores refilling necessary for oocyte contraction at fertilization ii) a role of cyclicamp in maintaining the meiotic arrest as it happens in mammals; iii) a role of na+ currents during electrical events at fertilization and subsequent embryo development. a morpho-functional analysis of the adhesive papillae of ascidian larvae f de bernardi, g zega, s groppelli, c sotgia, r pennati dipartimento di biologia, università di milano, milano, italy most ascidian larvae adhere to the substrate by mucous secreted by the adhesive papillae, which in nearly all species are made of elongated secretory cells and primary sensory neurons. the exceptions are the papillae of the species of the genus botrylloides, which are considered to have only a sensorial function (grave, 1934) and that of genus clavelina, which are reported to be only secretory (turon, 1991). we analysed the adhesive papillae of botrylloides leachi, clavelina lepadiformis and diplosoma listerianum by histological analysis and by immunolocalization of serotonin and β-tubulin. we demonstrated that in all analysed species the adhesive papillae have both sensorial and secretory function. in particular, larvae of b. laechi have a lot of secretory cells gathered in the centre of a small area between the three adhesive papillae, forming a glandular organ. this organ could allow the adhesion of the larva to the substrate also by a sucker mechanism. the adhesive papillae of c. lepadiformis contain, in addition to the secretory cells, many primary neurons, the axons of which gathered to form papillary nerves. all analysed species contain two different types of neurons with different localisation and possibly a different function. the first type neurons, likely mechanoceptors or chemoceptors, emerge from the tunic at the apex of the papillae and play a role in the substrate choice. the second type neurons are localised in more lateral position in the papillae and do not contact the substrate until the papillae are retracting owing to a permanent adhesion. these neurons contain serotonin and they could play a role in triggering metamorphosis. on the basis of the role of neurotransmitters in ascidian metamorphosis, we hypothesize that serotonin released by the peripheral neurons after the permanent adhesion may acts as an internal signal triggering the metamorphosis process. the adult musculature in botryllus schlosseri: differentiation and gene expression v degasperi, f gasparini, l manni p, burighel department of biology, university of padua, padua, italy typically, ascidians have three types of muscles: skeletal in the larva, cardiac and smooth in the postmetamorphic sessile organism. the larval and cardiac muscles are striated, with vertebrate-like arrangement of myofilaments. instead, the smooth body-wall musculature has intermediate characters between smooth and striated muscle of vertebrates. we studied the musculature in the blastozooids of botryllus schlosseri analysing its organisation, differentiation and gene expression. we isolated and characterised two transctipts resulted homologous to muscle genes of other adult ascidians: a muscle-type actin (bsma2) and troponin t (bstnt-a); moreover, we obtained also the genomic sequence coding for bsma2. phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle genes. the bsma2 genomic sequence was compared in the exon-intron organization with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. our analysis showed that intron positions are conserved in ascidian and in the other deuterostomes. we detected the expression of the two genes by in situ hybridization (ish), in order to follow the muscle development throughout the blastogenetic cycle of b. schlosseri. the ish, in parallel with phalloidin staining experiments, showed that the first diffuse signal of muscle differentiation appears in the intersiphonal epidermis of young buds. then, the muscle fibers differentiate into the body-wall, while an intense expression of bsma2 marks the heart myocardium just when it begins contractions. the ultrastructure of smooth muscle cells was also investigated during differentiation from mesenchymal cells in the bud, in the adult, and in the fibers contraction during regression of zooids. preliminary data on antiproliferative effects in haemocyte extracts of the ascidian styela plicata (stolidobranchiata, styelidae) ma di bella1, mc carbone1, r alessandro1, e fattorusso2, m menna2, g de leo1 1dipartimento di biopatologia e metodologie biomediche, università degli studi di palermo, palermo, italy 2dipartimento di chimica delle sostanze naturali, università degli studi di napoli “federico ii”, napoli, italy several marine invertebrate are among the most important sources of biomedically relevant natural products. a recent expansion of the studies of marine natural products involves the search for compounds with antitumor properties. in particular, among the compounds that have been isolated from sponges, bryozoans, and tunicates, several have been widely described (discodermolide, bryostatin-1, et-743), and have progressed to pre-clinical or clinical-trial phases. tunicates, organisms having a wide geographic distribution, have increasingly become the target of natural products research and have been reported to harbour metabolites with a wide range of biological activities such as metal accumulation, sclerotization of their extracellular matrices, and antimicrobial activity. some of these activities in marine invertebrate are also carried out by the circulating hemolymph that presents different types of haemocytes embedded in the liquid plasma and contains biologically active substances that contribute to the innate immune mechanisms. recent researches have shown that the extracts of the total haemocytes population from different ascidians 85 contain several compounds with an antimicrobial activity such as antibacterial, antifungal and antiviral ability. the aims of the present study was to extend the knowledge on the biological activities of these compounds testing the antiproliferative potential of the haemocyte extracts from the solitary ascidian st. plicata on leukaemia cell lines. comparative ultrastructural investigation on the adhesive papillae of the swimming larvae of three ascidiae species g dolcemascolo1, r pennati2, f de bernardi2, f damiani2, m gianguzza1 1dipartimento di biopatologia e metodologie biomediche, sez. di biologia e genetica, università degli studi di palermo, palermo, italy 2dipartimento di biologia, università degli studi di milano, milano, italy ascidian swimming larvae bear three peculiar organs of ectodermic origin, named “palps” or adhesive papillae, located in the anterior region of cephalenteron. term “adhesive” is correlated to one of the function of these structure based on secretion of an adhesive substance which enables swimming larvae to adhere to a substratum. recently a sensory function has also been described in some phlebobranchia papillae with a simple morpho-functional organization. there are few ultrastructural investigations in literature, sometimes disputed, able to make clear papillae cells functions. to clarify this problem, a comparative investigation has been carried out, just in this work, about ultrastructural and morpho-functional organizations of adhesive papillae of three ascidian swimming larvae, ascidia malaca, phallusia mammillata and ciona intestinalis. the investigations has been carried out by transmission electronic microscopy and confocal microscopy. papillae of above-mentionated ascidian swimming larvae, are three in number and they are located at the vertices of a triangular field. according to a recent classification scheme by burighel and cloney, they are coniform and non-eversible simple type papillae. in the apex of each papilla there is an hyaline cap with an electron-dense substance made of proteoglycan component. it’s quite certain that this substance is used by ascidian larvae to adhere to the substratum. ultrastructural investigations carried out on the adhesive papillae of a. malaca and p. mammillata have not emphasize significant ultrastructural differences in the papillae cells body. a. malaca and p. mammillata papillae are made of three types of cells that characterize their functions: a) collocytes, b) axial columnar cells, c) sensory cells. collocytes, whose ultrastructure is typical of cells with secretory activity, are certainly deputed to form adhesive secretion. they lie in a median or semilateral side of the papillar body. axial columnar cells that show an elongated shape, lie in the mid-central papillae region and they are characterized by the presence, in their apical part of long microvilli that run along the whole of hyaline cap length. above a structural supporting role, it is supposed a possible sensory function concerning these cells. sensory cells presents an ovoidal and elongated shape holding on tight at their base like a funnel lying a lateral and marginal position in papillar body. these type of cells have been described in a. malaca which can be classified as primary sensory neurons. these are characterized by the presence of a single cilium in the apical end and by an axonic process at their base extending over papilla. ultrastructural investigations on the adhesive papillae of c. intestinalis confirmed, even if showing some differences, ultrastructural likeness the two above-mentioned species of adhesive papillae. collocytes with secretory activity, sensory cells and columnar axial cells come into sight also in c. intestinalis papillae. the columnar axial cells ultrastructure is different from the a. malaca and p. mammillata papillae. in the apical part of these cells there are not microvilli but digitiform protusions with numerous microtubules inside running paralleling along longitudinal cell axis. the ultrastructure of these cells suggest their lengthening ability to allow digitiform protrusions to put into contact with the cuticular layer of hyaline cap apex, during step before the adhesion. ultrastructural characteristics suggest that they can also perform a sensorial duty but their mechanism is still unclear. the observation was carried out by confocal microscopy on the ascidian larvae just before or at the beginning of adhesion to the substratum (6-7 hours from hatching). the use of anti-β-tubulin antibody, have shown an extensive nervous network in all three ascidiae species starting from the papillar base and converging into only one nerve extending up to the cerebral vescicle. these data confirm previous observations carried out with confocal microscopy on a. malaca and p. mammillata. larvae. in c. intestinalis adhesive papillae marked with anti-β-tubulin antibody, the fluorescence is more bright than p. mammillata and a. malaca. probably this result is correlated with apical protrusion rich in microtubule. ultrastructure observations of some neurons being in the anterior region of cephalenteron, extending from the base of papilla to the cerebral vescicle, have been emphasized in this work. these neurons are in the same position of the ones described like rten (rostral trunk epidermal neuron) by imai and meinertzhagen and they are formed by cellular ovoidal body extending into a long axon. ultrastructural of these neurons have pointed out likeness with vertebrate neurons. concluding, ultrastructural investigations have emphasize only some difference between papillar cells of a. malaca, p. mammillata and c. intestinalis larvae, confirming adhesive and sensory functions. evolution of anterior hox regulatory elements among chordates a locascio1, m manzanares2, ml chiusano3, a amoroso1, e d’aniello1, r krumlauf4, m branno1 1laboratory of biochemistry and molecular biology, stazione zoologica “anton dohrn”, villa comunale, naples, italy 2department of cardiovascular developmental biology, centro nacional de investigaciones cardiovascularescnic, madrid, spain 3department of structural and functional biology, università degli studi di napoli “federico ii”, monte s. angelo, naples, italy 4stowers institute for medical research, kansas city, mo 64110, usa hox genes determine ap identities from insects to 86 vertebrates in all the animal species where they have been studied. their organization in cluster and spatiotemporal colinearity of expression have usually been considered their more significant characteristics. the sequencing of the genomes of even more species has helped to understand various aspects of hox gene organization. among chordates, particularly interesting are the anterior groups of hox genes since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed. hox genes exert a fundamental role in vertebrate hindbrain formation and segmentation. both ascidians and amphioxus lack a segmented hindbrain but at the same time show restricted expression patterns of anterior hox genes like their vertebrate counterparts. changes in gene regulatory regions are considered a driving force for the evolution of more complex body plan structures. to investigate how hox gene regulation changed and evolved in the chordate lineage, we have analysed and compared various hox regulatory regions of three chordate species, amphioxus, ascidian ciona intestinalis and mouse. in particular, we focused our attention on elements controlling anterior hox genes expression and active in the anterior nervous system. we have studied the ability of amphioxus and mouse regulatory elements to function in ciona embryos and to reproduce the segmental expression pattern typical of hox genes. conversely, we have also analysed the ability of ciona nervous specific enhancers to be recognized by the mouse regulatory mechanisms. anatomy of ciona intestinalis: larval, metamorphic and juvenile phases l manni, f gasparini, p burighel department of biology, university of padua, padua, italy the solitary ascidian ciona intestinalis is a model organism frequently exploited for comparative investigations of chordate development and for unravelling the molecular mechanisms underlying morphogenesis and cell fate specification. this species has an indirect development comprising the embryonic phase, free swimming larval stage, metamorphosis to form a sessile juvenile and, ultimately, the adult with the mature reproductive organs. some of the first developmental stages have been described in detail using different classical and modern techniques. thank to these lines of study, cell lineages in ascidian embryo were thoroughly described from fertilized egg until the larval stage. recently, also successive developmental stages, such as the metamorphosis and the juvenile differentiation, has begun to be investigated, although less extensively. all these studies concur to improve our knowledge on ciona development, but several questions remain to be solved, because each technique has intrinsic advantages and limits. in this light, the authors discuss recent data from other laboratories examining also results obtaining recently by confocal microscopy. despite this technique permits the three dimensional reconstruction of the main developmental events in young embryos, it remain unsatisfactory in resolving precise aspects of interaction and differentiation between cells and/or tissues in successive stages. this is particularly relevant for late larvae and during metamorphosis, when the complex organogenesis of new rudiments and body reorganization occur accompanied by tissue shrinkage and disaggregation of embryonic structures,. the authors exemplify, analysing by means accurate reconstruction of specimens with histology and ultrastructure, critical stages of ciona development. the data show that also today the classical histology and the ultrastructure are complementary and essential techniques for the understanding of the anatomical organization of tissues, organs and the organism as a whole, and can represent fundamental reference for interpretations of sections in which tissues are partially labelled, as in in situ-hybridization or in immunocytochemistry. distribution of neural phenotypes in the larva of ciona intestinalis: comparison with cephalochordata and vertebrata r pennati1, g zega1, s candiani2, s groppelli1, m pestarino2, f de bernardi1 1department of biology, university of milan, milan, italy 2department of biology, university of genoa, genoa, italy the complex organisation of vertebrate nervous system is not only the product of an ontogenetic process, but it is the result of a long evolutionary history. a disclosure of this history can help to understand i) which developmental processes are more ancient and constitute the basis of vertebrate nervous system formation; ii) which processes evolved and/or superimposed in a second time. ascidians belong to tunicata subphylum are a very good model for these studies, because they are chordata and, according to recent studies, may be considered the group phylogenetically closest to vertebrates. ascidians and vertebrates share a tripartite organisation of the central nervous system, including an anterior region, domain of otx genes, a central region which express pax 2/5/8 and a posterior region regulated by the expression of hox genes. within this structural regionalization, during differentiation single neurons acquire an important phenotypic character given by the neurotransmitter released to excite or inhibit the target cells. we identified the serotonergic, gabaergic and dopaminergic neurons in developing central nervous system of ciona intestinalis by studying the expression pattern of genes codifying the neurotransmitter synthesis enzymes. we showed that some functional regions are conserved among the cns of ascidian larva, of the amphioxus and of vertebrates. this allowed us to hypothesize that these regions were already present in the ancestral chordate. citcf and pigment organs formation during ciona embryogenesis e riano, p d’ambrosio, a spagnuolo laboratory of biochemistry and molecular biology, stazione zoologica “anton dohrn”, villa comunale, naples, italy 87 the wnt signaling cascade plays an important role during embryonic patterning and cell fate determination and is highly conserved throughout evolution. factors of the tcf/lef hmg domain family (tcfs) are the downstream effectors of this signal transduction pathway. to study the function of tcf during ascidian embryogenesis, we attempted to isolate the ciona counterpart(s). our data indicate that ciona genome contains a single tcf gene, citcf (as confirmed by the analysis of the annotated genome) compared to the mammalian genome that harbours four tcf family members. in situ hybridization experiments indicate that citcf is broadly expressed at the early stages of development; from early neurula stage citcf signal becomes localized to the two pigment cell precursors. block of citcf function, by morpholino-antisense injection, strongly suggests its involvement in pigment cells formation; on the other end, transcriptional regulation analysis, by electroporation method, demonstrates that a 2.0 kb citcf promoter region, upstream from the tata box, is able to drive the tissue specific spatial expression of reporter gene. by a comparative analysis with ciona savigny we have identified a shared element of about 0.3 kb, upstream from the ci/cstcf genes, able to reproduce the pattern obtained with the 2.0 kb region. deletion constructs are currently under investigation, in order to further restrict the 0.3 kb minimal promoter fragment and to identify the core elements, controlling ci-tcf expression, and then the transcription factor(s) able to bind to this sequence. the homologous ciona factors will then be searched, by blast analysis against the annotated genome, fished in the cdna collection, present in our laboratory, and tried both in vitro and in vivo for their ability to interact with ci-tcf promoter and transactivate the reporter gene angiogenic-like mechanism in the colonial circulatory system of botryllus schlosseri g zaniolo, f gasparini department of biology, university of padua, padua, italy the colonial circulatory system (ccs) of the ascidian botryllus schlosseri runs in the common tunic and forms an anastomized network of vessels, defined by simple epithelium, connected to the open circulatory system of the zooids. the ccs originates from epidermal evagination, grows and increases its network accompanying colony propagation by means of mechanisms of tubular sprouting. we evidenced that the regeneration of experimentally ablated areas of ccs occurs by the same sprouting mechanism. in the two cases (normal growth and regeneration, the same histogenetic mechanism and homologous factors and receptors are shared. strong similarities in organization (e.g., simple cubic epithelium) and cell structure (e.g., extension of filopodia) in the apexes of sprouting vessels during normal growth and regeneration were observed. immunohistological responses to anti-pcna a marker of cell proliferationand antibodies against vertebrate angiogenic growth factors (vegf, egf, fgf-2) and receptors (vegfr-1, vegfr-2, egfr), reveal that cell proliferation and the same angiogenic signals take part in corresponding sprouting regions of ccs during normal development and regeneration. sprouting is the most common and best-known mechanism of vertebrate angiogenesis, but it is also found in other developing structures, such as nerves, and in drosophila tracheas. all our observations show that correspondences exist between the ccs sprouting modality of b. schlosseri and angiogenic sprouting in vertebrates, during both normal development and regeneration, and support the idea that this morphogenetic mechanism was co-opted during the evolution of various developmental processes in different taxa. effects of paraquat on the development and metamorphosis of phallusia mammillata and ciona intestinalis (ascidiacea, tunicata) g zega, s groppelli, f de bernardi, r pennati department of biology, university of milan, milan, italy paraquat (1,1’dimethyl-4,4’-bipyridylium) is an herbicide largely employed in agriculture, the use of which has been authorized in 120 countries. several toxicological studies demonstrated that this substance has neurotoxic effects on numerous animal models and causes symptoms similar to those observed in patients with parkinson’s disease. we exposed phallusia mammillata embryos at different concentrations of paraquat until they reached the swimming larva stage, then we immunolabeled their nervous system with anti β tubulin antibody, in order to analyzed the malformations eventually induced by the treatments. treated larvae showed dosedependent alterations of the ocellus and of the fibres that innervate the otolith. the gravity of the malformations decreased when the embryos were treated with both paraquat and ascorbic acid, suggesting that oxidative stress is involved in the onset of the observed malformations. moreover, we observed that treated larvae survived for more than four days, but they could not metamorphose. exposure to paraquat had similar effects on ciona intestinalis embryos. we characterized paraquat induced phenotype in c. intestinalis larvae by in situ hybridization with marker genes of different neural populations in order to evaluate the specificity of action of this neurotoxic agent. treated larvae showed a drastic reduction of gabaergic neurons, while the number of dopaminergic neurons was not affected by the treatments. these results are in contrast with the supposed effects of paraquat on humans. in fact this herbicide is suspected to be involved in the etiology of parkinson’s disease, a pathology characterized by progressive loss of dopaminergic neurons of the substantia nigra. role of nitric oxide during ciona intestinalis development a palumbo, d d’esposito, e ercolesi, g fiore, a locascio, m branno laboratory of biochemistry and molecular biology, stazione zoologica “anton dohrn”, villa comunale, naples, italy 88 nitric oxide (no) plays an important role in fertilization and development of some marine organisms, including sea urchins, marine snail and ascidians. we have recently reported that in ciona intestinalis no is involved in larval development and metamorphosis. the spatial patterns of nitric oxide synthase expression, as well as no detection, during larval development are very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system, tail and juvenile digestive organs, thus suggesting the involvement of no in many processes. in particular, no regulates tail regression acting on caspase-dependent apoptosis. to further investigate the role of no during c. intestinalis development we focused our attention on no signalling during metamorphosis. experiments have been carried out to examine how the modulation of this signalling affects tail resorption and subsequent juvenile development. parallel experiments have been also performed to investigate the possible involvement of no in the embryonic development. to this aim we have examined the spatial expression patterns of nitric oxide synthase and no detection in embryos at different stages as well as the effect of endogenous no levels modulation on development mathematical models for excitability of egg cells pg reas department of mathematics and applications, university of palermo, palermo, italy the excitable systems play a very important role in biology and medicine. phenomena such as transmission of impulses between neurons, the cardiac arrhythmia he aggregation of amoebas, the appearance of organized structures in the cortex of egg cells, all derive from the activity of excitable media. in the first part of this work a general definitions of excitable system is given; we then analyze some cases of excitability, distinguishing between electrical ,chemical and mechanical excitability and comparing experimental observations with simulations carried out by appropriate mathematical models. such models are almost always formulated by partial differential equations of “reaction-diffusion” type and they have the characteristic to describe propagations of electrical waves or chemical and mechanical waves (propagation of ca waves and mechanical waves in the endoplasmic reticulum). the aim is to put in evidence that the biological systems can show not only excitability of electrical type, but also excitability of chemical and mechanical nature, which can be observed in the first steps of development of egg cells. session 2. phylogenesis and microevolution the fast evolutionary dynamics of ascidian mitochondrial genome: an exception to the general deuterostome evolutionary trend c gissi1, f iannelli1, f griggio2, g pesole1 1dipartimento di scienze biomolecolari e biotecnologie, università di milano, milano, italy 2dipartimento di biochimica e biologia molecolare “e. quagliariello”, università di bari, bari, italy the availability of more than one thousand sequences of mitochondrial genomes (mtdna) of metazoa provides an almost unique opportunity to decode the mechanisms of evolution of an entire genome in a phylogenetic framework. we have compared several structural features of the ascidian mtdna (gene content, genome size and architecture, and gene strand asymmetry) to that of other metazoans, focusing also on comparisons at congeneric level, as analyses at short evolutionary distances reduce the risk of saturation effects in the observed genomic changes. the current data show that ascidians exhibit a degree of mtdna variability very high and comparable to that of non-deuterostome groups. indeed, a trend toward stabilization of the mt genomic features has occurred in most deuterostomes and has been exacerbated in vertebrates, where gene content, genome architecture and gene strand asymmetry are almost invariant. on the contrary, ascidians show a gene strand asymmetry and a variability in mtdna architecture similar to nonbilaterians and lophotrochozoans, suggesting that these features are primitive, rather than a derived traits of the mtdna. in addition, our data highlight that the high degree of rearrangements in mtdna architecture found in ascidians, as well as in enoplean nematodes, is due to translocations of both rna and proteincoding genes, while changes in genome architecture are mostly due to the variation of number/position of trnas in taxa with moderate/low rearrangements. the variability observed in congeneric species significantly recapitulates the overall mtdna evolutionary dynamics observed at higher taxonomic ranks, especially in taxa with high genome plasticity and/or fast nucleotide substitution rate. moreover, congeneric comparisons appear quite promising to investigate in detail mtdna evolutionary mechanisms. one ring to divide them all: mitochondrial genomics unveils two cryptic species in ciona intestinalis f iannelli1, g pesole2, p sordino3, c gissi1 1dipartimento di scienze biomolecolari e biotecnologie, università di milano, milano, italy 2dipartimento di biochimica e biologia molecolare “e. quagliariello”, università di bari, bari, italy 3laboratory of biochemistry and molecular biology, stazione zoologica “a. dohrn”, naples, italy the circular mitochondrial genome (mtdna) of metazoans represents a rich source of genetic markers for phylogenetic analyses at many taxonomic levels. both sequences and genome-level features, such as gene arrangement, have been used to resolve deeplevel phylogenetic relationships, whereas single mitochondrial genes or regions are commonly analysed in population genetic studies. we used a mitogenomic approach, based on the comparison of several genome-level mitochondrial features, to unambiguously demonstrate the existence of two cryptic species in the ascidian ciona intestinalis, a model chordate whose taxonomic status of a single species has been recently questioned. a comprehensive comparative analysis between the mtdna of the two putative cryptic species revealed significant differences in gene order, size and number of non-coding regions, 89 compositional features, and evolutionary rate of protein-coding genes. these mitochondrial features are clearly incompatible with intra-species variability, and strongly suggest the existence of the two cryptic species. furthermore, our approach allowed to set two pcr-based diagnostic tests for the discrimination of the cryptic species without recourse to morphological analyses, demonstrating that mtdna represents an accessible and powerful tool to be used both in routine analyses and in high-throughput screenings. phylogenetic conservation of csf-related genes in the ascidian ciona intestinalis gl russo1,2, s bilotto1,2,3, g ciarcia3, e tosti1 1stazione zoologica ‘anton dohrn’, napoli, italy 2institute of food sciences, national research council, avellino, italy 3department of biology, section of zoology, university of naples, italy in all vertebrates, mature oocytes arrest at the metaphase of the ii meiotic division, while some invertebrates arrest at metaphase i, others at pronucleus stage. fertilization induces completion of meiosis and entry into the first mitotic division. how the different mechanisms underlying meiotic regulation evolved is very far from being clarified. in the past decades, several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (csf) and the maturation promoting factor (mpf) in metaphase i or ii. some of these questions remain elusive and can be approached by the introduction of new experimental models. in the recent past, we proposed the oocytes of ascidian ciona intestinalis as a new model to study the meiotic division both at the physiological and molecular level. here, taking advantage of the recent publication of a draft copy of c. intestinalis genome, we present a phylogenetic analysis of key genes involved in the control of meiotic completion after fertilization, such as cdc2/cyclin b and mapk/mos components of mpf and csf, respectively. the presence of c-mos homolog in c. intestinalis genome suggests a regulation of metaphase arrest in ascidians different than in other invertebrates, where this gene is not conserved. we further investigated the regulation of csf by demonstrating that both csf and mpf inactivation, at the exit of metaphase i, are independent from protein synthesis. in fact, oocytes loaded with emetin completed meiosis i after fertilization, similarly to control oocytes, as demonstrated by mpf decrease and extrusion of the first polar body. this result indicates the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. in addition, antibody raised against the xenopus homolog of mos are able to abolish c. intestinalis csf activity. finally, mapk enzymatic activity sharply decreases after fertilization, as confirmed by a mapk specific antibody that was able to recognize the active, phosphorylated form of the kinase. the results obtained indicate that meiotic regulation in c. intestinalis resembles that of vertebrates, such as xenopus, more than those of other invertebrates. advancing forward genetics in ciona intestinalis sp. a. p sordino, n andreakis, l caputi laboratory of biochemistry and molecular biology, stazione zoologica “a. dohrn”, villa comunale, naples, italy our aim is to elucidate the cellular, molecular and evolutionary basis of pattern and differentiation in animal body plans. we combine classical and modern techniques for understanding gene functions during embryonic development by centering our activity on ciona intestinalis (ascidiacea). herein, we report about the implementation of forward genetics approaches. the primary objective of forward genetics is a low-cost resource of mutants with interesting phenotypes to be rapidly mapped and identified at the molecular level, a concept which applies to a limited number of model species in which mutagenesis techniques allow to compare phenotype and genotype in great detail. we are currently analyzing several aspects that are essential for the establishment of a phenotype-driven methodology. among them are the existence of two cryptic species, as shown by independent species concepts, the identification of a source of mutants, the construction of high-resolution genetic linkage map, the generation and culturing of inbred or semi-inbred strains, a protocol of sperm cryopreservation for the optimization of line management, and the characterization of genetic and morphological variation in natural populations. also, we are studying the phylogeographic structure of c. intestinalis at global and local scale by using different molecular markers, with the aim to examine how historical, geographical and environmental factors influence the distribution of morphological and genetic polymorphism in a model chordate. shuttling and rrna processing of pre-ribosomal subunits in the ascidian ciona intestinalis r barbieri dipartimento di biologia cellulare e dello sviluppo, università di palermo, palermo, italy ribosome biogenesis in eukaryotes is one of the most challenging topics in molecular and cellular biology of the last ten years. although different steps of pre-ribosome maturation has been elucidated in the last years, some intriguing facets remains still unresolved. among these questions, the different fates of the two sub-units during ribosome biogenesis, and the reason why different maturation steps of these particles occur in different cellular compartments. it is generally acknowledged for all the eukaryotes that the two different ribosomal pre-subunits (pre-40s and pre-60s) undergo to different fates during their late maturation steps. the smaller one (containing a 21s rrna) is exported to the cytoplasm where its maturation is completed, whereas the pre-60s particle (containing a 28s rrna) completes its maturation in the nucleus, and successively is exported in the cytoplasm as a mature particle. to the contrary of the ribosome maturation pattern up to now proposed, we found in the ascidian ciona intestinalis, as well as in sea urchin and human cells, that pre-ribosomal subunits maturation is a concerted, parallel processing 90 and shuttling mechanism involving both the two preribosomal particles; this mechanism includes a cytoplasmic rrna processing event never described before. primer extension experiment on total rna extracted separately from nuclei and cytoplasm of ciona intestinalis 4/8 blastomeres embryos, reveals the presence of the same “large” rrna precursors both in the nucleus and in the cytoplasm of these cells. the comparison with what occurs in the sea urchin p. lividus (left panel) and in human white blood cells (not shown), allowed us to demonstrate both that the shuttling/processing model is substantially different with respect to the one proposed, and that the shuttling/processing events we demonstrated work probably in all the eukaryotic species. frank homologies between invertebrate and vertebrate chordates revealed through the neurophysiology of swimming in ciona tadpoles brown er1, piscopo s1, nishino a2, okamura y3 1 laboratorio di fisiologia animale ed evoluzione, stazione zoologica anton dohrn, villa comunale, 80121 napoli, italy 2 department of biological sciences, graduate school of science, osaka university, machikaneyama 1-1, toyonaka, osaka 560-0043, japan 3 department of integrative physiology, graduate school of medicine, osaka university, yamada-oka 2-2, suita, osaka, 565-0871, japan with the recent sequencing of the genome of ciona intestinalis there has been an increase in interest in ascidians as models to study the evolution of chordates. one way to do this is to look at the similarities and differences between genes and families of genes at the genomic and functional level. however deeper homologies may be revealed by looking at (in addition) the function of proteins in a given physiological system which may shed light on their evolutionary pathways. here we report progress in our understanding of chordate locomotion and physiology through study of the neurophysiology of swimming in c. intestinalis. although ciona has a sessile adult form, the tadpole-like larva swims with rapidly alternating tail beats that superficially resemble vertebrate swimming. in vertebrates, such alternating activity during swimming is generated in spinal segments and is controlled by a combination of glutamatergic excitation of motorneurones and inhibitory glycinergic interneurons which provide contralateral inhibition. modulation of the period of swimming is achieved through gabaergic inhibition. these networks of excitatory and inhibitory neurons are known as central pattern generators (cpgs). we show that: 1) a cpg exists in the visceral ganglion and nerve chord and is excited by glutamate, 2) precise alternation of tailbeats is controlled by a glycinergic system consisting of interneurons and glycine receptors (ciglyr), 3) a gabaergic system modulates swimming but does not control alternating movements. we conclude that the ‘spinal-like’ cpg and the ‘use’ of glycinergic and gabaergic inhibition for the control and modulation of swimming were ancestral chordate innovations. session 3. immunity stem cells and chimerism in colonial ascidian a voskoboynik institute of stem cell biology and regenerative medicine, department of pathology, stanford university school of medicine, stanford, ca 94305, usa, and department of developmental biology, stanford university hopkins marine station, pacific grove, ca 93950 usa natural chimerism is the coexistence of cells of two genetically distinct organisms in one individual. it is a common phenomenon, which can be detected in a wide variety of multicellular organisms, including vertebrates. in mammals, natural chimerism is usually established during pregnancy between the mother and the fetus or between fetuses in multiple embryos pregnancy. colonial marine ascidians, like botryllus schlosseri, may serve as evolutionary model system to vertebrates chimeras. in these organisms, pairs of allogeneic colonies can establish a natural chimerism upon physical contact. the ability to create a chimeric entity between these colonies is determined by a single, highly polymorphic, fusion / histocompatibility locus (fu/hc). colonies that share at least one allele in their fu/hc locus (mainly kin under in situ conditions) would fuse upon contact. a pair that does not share any fu/hc allele would not. following fusion, cells transmigrate between colonies and, in some cases, replace the germline and/or the somatic tissues of the host (termed as germline and somatic cell parasitism respectfully). the replacement of host tissues by a donor genotype is pre-determined genetically and follows hierarchies of “winner strains” replacing “loser strains” tissues. in both mammals and ascidians, natural creation of a chimera entity is restricted to kin; longterm chimerism can be established by stem cells; and tolerance or intolerance state to donor tissues can be mediated by chimerism. while several studies and observations across different species, tissues and systems link chimerism to tolerance, its actual role in tolerance induction or maintenance is yet unknown. here i’ll review the chimerism phenomenon in mammals and ascidians, discuss the possible role of stem cells as mediators of chimerism and the possible role of cells chimerism as mediators of tolerance. future studies, which monitor the dynamics of chimeric donor cells within the host, identify the factors that support or inhibit survival and proliferation of donor cells and its affects on tolerance or intolerance should shed new light on this widespread natural phenomenon. morula cells, phenoloxidase and dopacontaining proteins in the compound ascidian botryllus schlosseri l ballarin1, s scippa2, f cima1 1department of biology, university of padua, padua, italy 91 2department of biological sciences, section of genetics and molecular biology, university of naples, ”federico ii”, naples, italy morula cells (mcs) represent the most abundant circulating haemocyte type in the compound ascidian botryllus schlosseri. they are involved in defence reactions as they: i) can recognise alien substances and cells and induce cytotoxicity; ii) are the effectors of the cytotoxic rejection reaction which occurs between contacting, genetically incompatible colonies. a main role in mc-related cytotoxicity is exerted by the enzyme phenoloxidase (po) which converts polyphenol substrata to quinones; the latter, in turn, polymerise to form melanins. in the present research, we carried out new spectrophotometrical and cytochemical analysis to investigate further the behaviour of po and the nature of its substrates. results confirm that po is located inside mc vacuoles. in addition, immunocytochemical analysis indicate that mcs contain quinones which probably represent ready-to-use cytotoxic molecules, likely deriving from the oxidation, by po, of dopacontaining proteins. in addition, small dopacontaining peptides, called tunichromes, are likely present inside mcs. vcbp genes in ciona intestinalis. i. structural analysis s giacomelli1, r de santis1, w litmang2, d melillo1, mr pinto1, i zucchetti1 1laboratory of cell biology, stazione zoologica “anton dohrn”, napoli, italy 2department of pediatrics, university of south florida, saint petersburg, florida, usa the pivotal genes encoding for adaptive immunity molecules, such as the major histocompatibility complex (mhc) class i and ii, t-cell receptors, or dimeric immunoglobulins, have not been identified in jawless vertebrates, protochordates and in nonchordate invertebrates. however, in an attempt to identify adaptive immune-like genes, a secretionsignal-peptide-selection-based approach in the amphioxus branchiostoma floridae, allowed the identification of five gene families encoding secreted proteins characterized by a pair of n-terminal immunoglobulin variable domains (v) and a single cterminal chitin-binding domain. these families are distinguished by sequence differences in their v regions. the high degree of germline polymorphism, the chimeric immunoglobulin-lectin structure of vcbps and their tissue-specific expression, are all consistent with involvement of these molecules in immune recognition. vcbps may reflect structural characteristics of an important transition between nonrearranging innate pattern-recognition molecules and the conventional adaptive immune receptors. in an attempt to add further evidences for the presence of alternative solutions for both innate and adaptive immunity along the phylogenetic tree, a search for vcbp genes has been carried out in ciona intestinalis genome and est libraries. this resulted in the identification of three vcbp-like genes, ci-vcbp1, ci-vcbp 2 and ci-vcbp 3, encoding three proteins of 349 (ci-vcbp1) and 338 (ci-vcbp2 and ci-vcbp3) aminoacids. the analysis of the deduced aminoacid sequences indicates that ci-vcbp2 and 3 exhibit 72 % identity, which is about 29 % when ci-vcbp1 is compared with ci-vcbp2 or ci-vcbp3. the domain structure analysis of the three gene products revealed the presence of a signal peptide sequence, two immunoglobulin domains and a chitin-binding domain. a very preliminary analysis carried out by pcr with degenerate oligonucleotides, designed on the alignment of the three nucleotide sequences, allows us to exclude the presence of other paralogous genes in c. intestinalis genome. furthermore, pcr analysis performed with cdna from different individuals evidenced the presence of limited allelic polymorphism. molecular events triggered by the interaction between the ciona intestinalis anaphylatoxin and its specific receptor d melillo, r de santis, s giacomelli, mr pinto laboratory of cell biology, stazione zoologica “anton dohrn”, naples, italy many molecules belonging to the arms of the innate immune system have been found scattered in invertebrates at different levels of the phylogenetic tree. in this context, a major breakthrough has been the identification in the echinoderms, in the urochordates and in the cephalochordates of gene homologs of c3, a multifunctional protein that plays a central role in the complement system. in mammals, c3 activation produces two bioactive proteolytic fragments: c3b responsible of the opsonic pathway, and the anaphylatoxin c3a, a potent mediator of inflammatory reactions. we have demonstrated that in ciona intestinalis, like in mammals, the bioactive fragment c3a, released from c3 during complement activation, promotes hemocyte chemotaxis, thus suggesting an important role for this molecule in inflammatory processes. cic3a exerts its functional activity through the specific binding to a cell surface g protein-coupled seven-transmembrane receptor (cic3ar), present on granular and hyaline amoebocytes, which has been recently identified and sequenced for the first time in an invertebrate species by our group. in an attempt to better characterize the cellular and molecular events triggered by the ligand-receptor (cic3a-cic3ar) interaction, we have focused our attention on the receptor internalization process, which is an important control mechanism described for g protein coupled receptors. we have analyzed by confocal microscopy the dynamic process of internalization of the cic3ar induced by the binding of cic3a in the form of the c-terminal eighteen aminoacid peptide (cic3a59-76) of the cic3a anaphylatoxin. the kinetic of the overall process has been monitored by fixing hemocyte samples at intervals between 0 and 30 minutes, and by immunostaining, performed with an anti-cic3ar antibody and an anti-rabbit igg fitcconjugated secondary antibody. preliminary results indicate that cic3ar, following the cic3a59-76 stimulus, is internalized in few minutes, and, in 30 minutes, recycled to the cell surface, made again available for cic3a binding. immunomodulatory soluble factors in the compound ascidian botryllus schlosseri 92 a menin, l ballarin department of biology, university of padua, padua, italy cytokines are proteins with immunomodulatory activity acting in a paracrine or autocrine way. most of them are produced and secreted by activated immunocytes after the recognition of non-self and are involved in several immunological processes such as inflammation, apoptosis, clearance of effete cells and corpes, cytotoxicity and phagocytosis. today the characterization of invertebrate cytokines represents one of the major topics of interest in immunobiology and, although the presence of molecules sharing homologies with vertebrate cytokines is still a matter of great debate, the presence of endogenous immunomodulatory molecules was indirectly demonstrated in various invertebrate taxa such as molluscs, anellids, echinoderms and tunicates. in tunicates, the presence of molecules able to influence the behaviour of immunocytes has been reported in both solitary and colonial ascidians. in the compound ascidian botryllus schlosseri, the presence of soluble opsonins released by phagocytes as well as the presence of molecules able to enhance yeast phagocytosis, modulate cytotoxicity and cross-reacting with antibodies raised against mammalian proinflammatory cytokines such as tnf-α and il-1α has already been described. in the present report, we investigated the presence and the behaviour, under various conditions, of molecules recognised by antibodies raised against mammalian cytokines. results confirm the presence of soluble peptides, upon the recognition of foreign molecules, able to increase the phagocytic index of b. schlosseri and recognised by antibodies antitnf-α and il-1α. the rise of phagocytosis is significantly decreased in presence of specific sugars such as galactose, rhamnose and sorbitol. the same behaviour was observed in haemoagglutination assays where the presence of glucose and rhamnose lowered the haemoagglutination titre. this suggests that lectins, able to modulate the immune responses, are secreted by stimulated immunocytes. vcbp genes in ciona intestinalis. ii. gene expression analysis i zucchetti1, r de santis1, s giacomelli1, gw litman2, d melillo1, mr pinto1 1laboratory of cell biology, stazione zoologica “anton dohrn”, naples, italy 2department of pediatrics, university of south florida, saint petersburg, florida, usa it has been found that vcbp genes of branchiostoma floridae are selectively expressed in scattered cells of the intestine, a digestive tract continuously exposed to a diverse range of potential pathogens. the overall size of these gene families together with their extensive polymorphism may be associated with specific v-directed recognition of foreign antigens. furthermore, it has been reported that some chitin-binding proteins found in invertebrates have an antimicrobial and antifungal role. hence, vcbps could be considered bifuctional molecules with features of the adaptive immune receptors combined with innate immune functions. in this context, due to its phylogenetic position, the study of ciona intestinalis vcbps may provide interesting indications. this, together with the possibility of the easy handling of both embryonic stages and adult tissues in c. intestinalis could allow a more extensive analysis of the gene expression and function. whole mount in situ hybridization experiments of ci-vcbp1, ci-vcbp2 and ci-vcbp3 carried out on young adults have demonstrated that the genes are exclusively expressed in the stomach. in situ hybridization on stomach sections confirms and extends these results providing also a more exhaustive analysis at cellular level. ci-vcbps expression is enhanced by challenging the animals, or blood cells, with bacterial lipopolysaccharides. preliminary whole mount in situ hybridization experiments carried out on larva stage indicate that ci-vcbps expression is localized in defined regions of the endoderm. infammatory responses of the ascidian ciona intestinalis n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy innate immune system responses include acute inflammatory and granulation tissue production phases. cell migration, phagocytosis, encapsulation, tissue injury, matrix production, endothelial and epidermis activity, coordinate hemocyte stimulation. and wound repair have been described in ciona intestinalis following body wall challenge with lps. several reports suggest that in invertebrates immune system, cell proliferation, phagocytosis and chemotaxis are regulated by cytophilic humoral molecules with functional similarities to vertebrate pro-inflammatory cytokines (il1, il6, tnf) and galectins. here we show that like in mammals this inflammatory agent promptly challenges several cellular and molecular responses including enhanced serum lectins with il-1 epitopes, tnf, and type ix-like collagen. galectin-like molecules (ca2+-independent, specific for d-galactose and β-galactosides) with opsonic property can be enhanced as a response to a body wall wound and their release further stimulated by lps. the western blot pattern and immunohistochemical methods display oligomers with hril1α epitopes expressed by hemocytes, endothelial tissue and present in hemopoietic sites. these lectins can be promptly released by hemocytes challenged in vitro with lps; both cell lysate supernatants and hemocyte culture medium displayed hemagglutinating and opsonic activities inhibited by β-galactosides, and anti-hril1 antibodies. although a percoll density gradient separation method showed that several hemocyte types contain and release β-galactosidespecific molecules, assay of enriched hemocyte populations suggest that amoebocytes are the primary source of these molecules. for the first time we show citnfα to be constitutively expressed in the ascidian inflamed body wall and hemolymph, and upregulated by in vivo lps inoculation. cdna and deduced aminoacid sequence as well as lps challenged gene expression demonstrate unequivocally the involvement of soluble and cell bound citnfα forms in the ascidian 93 inflammatory response. sequence similarities with vertebrate tnfs include this cytokine into the tnf family. a prompt (2-4 h) enhanced citnf gene expression in the inflamed body is shown: in situ hybridization assays, cytometry and immunohistochemical methods support the involvement of pharynx and circulating hemocytes in the inflammatory response. immunoblotting assay with anti-citnf specific or anti-hrtnf antibodies revealed that hemocytes contains a 43 kda citnf whereas a 15 kda tnf is released into the serum hemolymph suggesting that, like in mammals and fish, citnfα cell bound homotrimer may be processed to a monomeric soluble form. densitometry analysis confirmed that citnfα protein expression is modulated by the lps challenge. enhanced transcript of a collagen with facit structural features (ci-typeix-col), in hemocytes, epidermis and migrating cells is also shown, while flow cytometry with specific antibodies, raised against an opportunely chosen ci-typeix-col synthetic peptide, displays fibroblast-property of hemocytes challenged in vitro with lps (at 4 hrs). in situ hybridation assay and immunocytochemistry identified collagen expressing hemocytes and disclosed epidermis gene expression in the time-course of the inflammatory reaction presumably involved in the inflammatory granulation phase. finally, the expression of a phenoloxidase component appears to be stimulated by inflammatory stimuli. in conclusion, the inflammatory response of c. intestinalis is characterized by components homologous (tnf, c3-like, collagens) and analogous (galectin-like) to the vertebrate ones. tunicate immunocytes can be cytotoxic toward foreign cells v arizza, ft giaramita, d parrinello , m vazzana, a vizzini, g salerno, m cammarata, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy tunicate immunocytes can be cytotoxic toward foreign cells, and cytolytic molecules (“lysins”) have been revealed in vitro by using erythrocyte targets. in ciona intestinalis the hemocyte cytotoxic activity has been examined towards mammalian erythrocytes in a medium isosmotic to the hemolymph containing 10 mm ca2+ (tbs). unilocular refractile hemocytes (urgs) release cytotoxic factors inhibited by sphingomyelin in a plaque-forming assay. to separate the lysinreleasing cells from the hemolymph and characterize lysins, a discontinuous percoll gradient was performed and hemocyte populations were separated in 6 bands. urgs cytotoxic for re were enriched (~40 %) in the band 5 (b5) and then lysed to obtain the supernatant. the b5-lysate supernatant (b5-hls) showed lytic activity (~84 %) specific for re and k562 cells whereas a lower level of cytotoxicity was found against sheep erythrocytes (se). such an activity was ca2+dependent and thermostable at 56 °c, b5-hls also showed a ca2+-independent hemagglutinating activity against trypsinized re (ht 4.3) but not toward trypsinized se. inhibition experiments displayed that lysins and lectins could be inhibited by carbohydrates (galactose, thio-digalactoside, lactose, lactulose) whereas sphingomyelin (2.5 µg/ml) only inhibited the lytic activity, suggesting that lectins may be involved in membrane sphingomielyn-lysin interactions. to check for the enzymatic nature of the lysins, phospholypase a2 inhibitors such as dibucain and quinacrine were assayed. the experiments showed that both the molecules were inhibitors of b5-hls cytotoxic activity suggesting the involvement of a ca-dependent phospholypase a2 activity. a cytotoxic mechanism based on phospholypase a2 activation due to lectinsugar interactions is discussed as a model. lysins against re and k562 cells with the same properties including sugar and phospholypase a2 inhibition, can be promptly (within 3 h) released in vitro by urgs in a culture medium suggesting that activated cells could participate in the defence response exerting a cytotoxic role. the prophenoloxidase system is activated during the tunic inflammatory reaction of ciona intestinalis m cammarata, v arizza, c cianciolo, d parrinello, m vazzana, a vizzini, g salerno, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy in invertebrates, the "prophenoloxidase activating system" involved in immune responses, is challenged by ß-1,3 glucans or lipopolysaccharides through a limited proteolysis due to serine proteases. phenoloxidase (po) is usually synthesized as proenzyme (prophenoloxidase, propo) and, upon activation, it plays a key role in humoral immune response and melanization processes. po activity in the tunic tissue of ciona intestinalis following lps intratunic injection was examined. tunic homogenate supernatant (ths), assayed with dopa-mbth reaction, displayed ca2+-independent po activity that was raised by lps and further enhanced by proteases. specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. assay with soybean trypsin inhibitor suggests that, in the tunic, proteases diverse from serine proteases could also be involved in the activation pathway. in vivo experiments were carried out by injecting isosmotic medium or lps, and ths assayed for its po activity. anova analysis of the time course profiles showed that lps was more effective in activating propo. to disclose the po response at the injured site, an assay with dopa-mbth was performed in vitro. quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. microscopy observations and immunohistochemistry with anti-cinpo-2 antibodies showed granulocytes and unilocular refractile granulocytes containing po, whereas rare morula cells were stained. in ths zymograms (sds-page), po activity linked to 90 and 120 kda bands was observed as an effect of lps injection, whereas the density of the 170 kda po was weak. in addition a third presumptive po enzyme (cinpo-3) containing the cinpo-2 peptide was identified in the recent ciona genome version. the possible involvement of a presumptive cinpo-3 similar to cinpo-2, as predicted by in silico analysis with blast (pam 30 scoring matrix) of ciona genome sequences (jgi v2), is 94 discussed. presumably, lps stimulated the production and dimerization (120 kda) of cinpo-3 (66 kda). in conclusion, the activated propo system includes several pos distinguishable in their size contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars. isolation, characterization and expression analysis of a collectin in tunicate ciona intestinalis a bonura1, a vizzini2, g salerno2, d parrinello2, n parrinello2, v longo1, p colombo1 1institute of biomedicine and molecular immunology "a. monroy". the national research council (cnr) palermo, italy 2department of animal biology, university of palermo, palermo, italy collectins are a family of calcium-dependent (ctype) lectins characterized by four functional domains: a short amino terminal ‘tail’ domain, a collagen-like domain that is typified by its repeating pattern of glycine-x-y (gly-x-y) amino acid triplets, a distinct neck region and the carboxy-terminal c-type carbohydrate recognition domain (crd). six distinct classes have been identified in vertebrates: mannose binding lectins (mbl), lung surfactant proteins a and d (sp-a and sp-d), conglutinins, serum collectin-43 (cl43) and serum collectin-46 (cl-46). mbl can recognize carbohydrates on the surface of pathogens and then activate the central complement component, via a mannose binding lectin-associated serine protease (masp), during acute phase responses to infection. in order to study molecules with differential activation of gene expression during the immune response, an injection of lipopolysaccharide (lps) into the tunic tissue at the median body region of ciona intestinalis was performed. one hour following injection animal was sacrified and rna was extracted and used for subtractive hybridization. rna was reverse transcribed and cloned using e. coli. sequencing analysis identified a cdna of 863 nucleotides encoding a protein containing 221 amino acids for a molecular weight of 24426 daltons. the sequence of this protein showed structural domains typical of collectin and a high degree of similarity with the mbl of gallus gallus and human mbl 2. in addition, studies of gene expression conducted through analysis of real time and in situ hybridization, have highlighted the increase of expression of this mrna compared to control animals that an increase of cells that express the mrna collectin near the site of lps inoculation. morphological characterization and acetylcholinesterase activity in ciona intestinalis hemocytes v mansueto, v arizza, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy the ascidian hemocytes have been reported to be involved in various functions including coagulation, nutrition, defense, infiammatory-like reaction, allogeneic reaction, tunic, gonad and germ cells formation. in addition, some of them are known to be involved in vanadium accumulation. hemocytes have been classified as stem cells, pigment cells, hyaline amoebocytes, granular amoebocytes, unilocular refractile granulocytes (urg), compartment cells, signet-ring cells and morula cells. cell lineage analysis showed that hemocytes may originate from a pair of a7.6 blastomeres of a 64-cell embryo, which give rise to trunk lateral cells (tlcs). our previous researches showed an acetylcholinesterase (ache) activity in the tlcs of ciona intestinalis swimming larva. in mammals, this enzyme has a non-classical control role in stem cell differentiation, apoptosis, defense responses and haematopoiesis. several c. intestinalis circulating hemocytes were characterized by means of different staining methods, and ache activity was identified in morula and signet ring cells. in c. intestinalis , this enzyme could function as an inhibitor of stem cells proliferation also related to cell differentiation. we show once again that several hemocyte forms circulate in c. intestinalis hemolymph some of them presumably due to differentiation steps from stem cells. session 4. enviromental stress tributyltin-induced effects on mapk signaling in ascidian embryos f damiani, m gianguzza, g dolcemascolo dipartimento di biopatologia e metodologie biomediche, sez. di biologia e genetica, università degli studi di palermo, palermo, italy among the class of organotin compounds, the most well known is tributyltin (tbt). organotin have many applications, which include use in pvc, as catalyst in chemical reactions, agricultural pesticides and antifungal treatments for textile polymers. in particular tbt is used in marine antifoulant paints to prevent the growth of organisms such as barnacles on the hull of ships. extensive use in antifouling paints led to the widespread distribution of tbt and its breakdown products in the global marine, sediment and biota. high levels of tbt in the waters were found to have impaired reproduction, by inhibiting embryogenesis and larval development in a variety of marine organisms. symptoms of the exposure to high levels of tbt in some invertebrates includes the development of male sexual characteristics as a penis and vas deferens by females (imposex). ascidians are a good model for the study of embryonal development. they are also sensitive bioindicators of habitat degradation. the effects of tributyltin (iv) chloride (tbt chloride) solutions on ascidian embryos of ciona intestinalis at different stages of development have been described. previously, we carried out observations with both the light and the electron microscope on ciona intestinalis embryos and larvae incubated in tbt solutions. this studies showed morphological and ultrastructural modifications of the embryos and larvae after incubation in tbt chloride at different concentrations. to understand molecular effects of tbt-induced on ascidians embryogenesis we have set out to study the effects of tbt at different concentrations, testing the activity of some protein with a basic role in embryonic development. in ascidian 95 embryos, a fibroblast growth factor (fgf)-like signal has been proposed to be involved in induction of notochord and mesoderm formation. a main pathway is a protein kinase transduction pathway, which includes ras, raf, mitogen-activated protein (map) kinase and extracellular signal-regulated kinase/map kinase (erk). the aim of this work in progress is to understand whether the tbt exposure on ascidian embryos at different stage of development cause alterations in tyrosine phosphorylation pattern and in mapk activity. tyrosine phosphorylation promotes cell growth, differentiation and apoptosis, due to activity of receptor tyrosine kinases and furthermore different stressors are known to stimulate tyrosine kinase activity. at first we focused our attention on tyrosine phosphorylation pattern after ascidian embryos to different stage of development tbt treatment. phosphorylated proteins pattern is evaluated by sdspage electrophoresis and western blotting on protein extract of ascidian embryos incubated with tbt, using anti-phosphotyrosine-antibody directed against mammalian phosphotyrosine. preliminary results showed a different pattern on protein phosphorylation in response to the incubation with tbt in µm range. since mapks play a key role in animal responses to a wide variety of environmental stresses, we have thought to test the role of mapk pathway proteins such as mapk p38 (thr 180 and tyr 182), p44/42 (thr 202/tyr 204) and c-jun n-terminal kinases (jnk) after tbt treatment. effects of cadmium on the functionality of haemocytes from the compound ascidian botryllus schlosseri n franchi, m di silvestro, l ballarin department of biology, university of padua, padua, italy ascidians share a variety of circulating haemocytes differing in their morphology and involvement in various biological functions. most of them are immunocytes, able to mount defence reactions against foreign, potentially dangerous, cells or molecules. metallothioneins are ubiquitary, cysteines-rich proteins which exert fundamental roles in the detoxification of trace metals such as ag, hg, cd, cu and zn, some of which are required for normal cell metabolism. up to now, no molecular data are available in both solitary and compound ascidians, included the species botryllus schlosseri, an important model organism for a wide variety of studies. in the attempt to study the involvement and the role of metallothioneins in botryllus immunobiology, we investigated the effects of acute exposure of haemocytes to cd on cell functionality. preliminary results indicate a dose-dependent negative effect on the ability to adhere of haemocytes and a decrease of the capability of phagocytes to spread on the substrate and to trigger a respiratory burst when matched with yeast cells. a cdna library from cd-exposed haemocytes is now under construction from which we expect to find metallothionein sequences toxic effect of methylmercury on ascidian (styela plicata) immunocyte responses mg parisi, m cammarata, g benenati, v arizza, t cillari, d piazzese, a gianguzza, m vazzana, a vizzini, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy pollution by heavy metals is one of the major risk in aquatic ecosystem, where high concentration cause adverse biological effects, including changes in immune function of invertebrate and vertebrate species. in marine environment, although mercury concentration in water column and sediments may be low, filter feeding invertebrates highly accumulate this metal in their tissues. in addition, biological processes mediate the mercury methylation transforming the metal in methylmercury which is the most toxic form due to the methyl group that facilitates cell penetration and interaction with proteins interfering with their synthesis and leading to lipid peroxidation. this study shows that high methylmercury concentrations are cytotoxic for styela plicata haemocytes, whereas subletal concentrations promptly affect immunocyte responses. moreover, haemocytes exposed to the xenobiotic present a significantly enhanced phenoloxidase activity as revealed in the haemocyte lysate supernatant compared to the control. although the cytotoxic activity of s. plicata haemocytes toward rabbit erythrocytes is a po-dependent cell-target reaction due to quinone products, it was significantly decreased by suitable methylmercury concentrations in the medium. the same xenobiotic concentrations decreased the haemocyte phagocytic activity toward yeast. in both the responses cell-target contacts could be affected by methylmercury, whereas the releasing capacity appeared to be unchanged as indicated by haemocyte po-release in the medium. finally, changes in haemocyte shape and spreading capacity were shown. effects on cytoskeleton could be responsible of changes in the haemocyte morphology and spreading capacity as revealed by the microplate assay we performed. on the basis of the present results, s. plicata could be an additional sentinel species for heavy metal environmental pollution by using immunotoxicology tests and a microplate method that reveals cell morphological changes and spreading capacity. similar results on the cells were obtained by assaying polluted sea water from different sicilian coastal sites. 96 isj102.pdf 91 isj 2: 91-104, 2005 issn 1824-307x review the point about oxidative stress in molluscs h manduzio, b rocher, f durand, c galap, f leboulenger laboratory of ecotoxicology (lema), upres ea 3222, ifrmp 23, ufr of sciences and techniques, university of le havre, france accepted july 8, 2005 abstract in the normal metabolism of the aerobic cell, oxygen is used for various biochemical reactions. because of its two lone electrons of parallel spins, the molecular oxygen is stable. however, oxygen generates reactive oxygenated species or ros by successive transfer of electrons. the ros have a strong reactivity and can potentially interact with all other cellular components (lipids, proteins, dna). they are at the origin of oxidations in chain by creating radicals. the cell has antioxidant systems which limit the effects of the ros. these systems are composed of enzymes such as glutathione reductase, glutathione peroxidase, etc., and molecules of nonenzymatic nature like the reduced glutathione or vitamins. the production and the destruction of the radicals of oxygen coexist in a weak balance. if this balance is broken in favour of the ros, an oxidative stress is generated. xenobiotics could influence this balance by catalysing production of ros. key words: oxidative stress; molluscs; ros; antioxidant; xenobiotics introduction face to chemical stress, each type of organisms, and for any species has a capacity of adaptation, based on regulating processes. these processes make it possible to maintain physiological homeostasis and the integrity of the individual, structural or functional deteriorations remainder entirely reversible or reparable. corresponding author: hélène manduzio, laboratory of ecotoxicology (lema), upres-ea 3222, ifrmp 23, ufr of sciences and techniques, university of le havre, 25 rue philippe lebon – bp 540, 76058 le havre cedex, france e-mail: helene.manduzio@univ-lehavre.fr list of abbreviations: cat: catalase; dt-d: dt-diaphorase; gpx: glutathione peroxydase; grd: glutathione reductase; gsh: reduced glutathione ; gst: glutathione s-transferase; mda: malondialdehyde; pah: polyaromatic hydrocarbons; h202: hydrogen peroxide; mda: lipoperoxydation; pcb: polychlorinated biphenyls; pco: peroxidation of proteins; segpx: glutathione peroxydase seleno-dependent; sod: superoxide dismutase passed the threshold of toxicity, the more marked irreversible attacks lead to a pathological state which results in a significant deterioration of the individual performances and later lead to death of the organism. now, many xenobiotics are recognized like exerting their harmfulness by catalysing production of oxygenated radicals (winston and di giulio, 1991). so the impact study of the toxic effects of the contaminants rejected into the environment requires a preliminary knowledge of the normal physiological mechanisms of adaptation (ecophysiology) and the comprehension of deteriorations of these processes induced by the contaminants (ecotoxicology). oxygen holds a capital place in the diversification of the species and their occupation of a majority of ecosystems. being at the base many biochemical processes of the metabolism of the aerobic organisms, oxygen is an essential molecule. however, its oxidizing capacities make of it a potentially aggressive element for the majority of the bio-molecules. in order to limit its harmful effect, the antioxidant mechanisms were set up which make it possible the organism to maintain the rate of radicals on a low basal level. in the event of oxidative stress, the antioxidant systems can be exceeded, then causing the oxidation of different molecules and leading to cellular dysfunctions. 92 in a context of a multiple contamination in particular in water ecosystem, the study of oxidative stress is very used in biomonitoring. the molluscs are especially used in this type of study on account of their characteristics. we will try to resume the data about oxidative stress in these organisms. nature and origin of the reactive oxygenated species (ros) the free radicals are atoms or molecules unstable presenting one or more lone electrons. to reach a better level of stability, they will yield or tear off electrons from molecules met. ros create new radical species thus, causing oxidations in chain. all the bio-molecules of the cell (nucleic acids, lipids, proteins, polysaccharides) are potential substrates of ros. a significant criterion in the characterization of the radicals is their diffusion capacity, which reflects the level of stability of the ros. a little reactive form tends to act far from its site of production and thus has a significant diffusion. on the contrary, a very reactive species acts very quickly and its diffusion is so limited. molecular oxygen o2 can be regarded as a radical species since it has two lone electrons; however, this molecule has a significant stability, the simultaneous addition of two electrons being difficult. in order to carry out this reaction, enzymes will create intermediates, which constitute the ros in particular during respiration and photosynthesis. the superoxide anion radical o2 -· is produced during endergonic reduction of molecular oxygen by capture of an electron. this reaction can be spontaneous in aerobic medium. o2 -· is then generated primarily in membranes because of the high solubility of oxygen in hydrophobic medium (gutteridge and halliwell, 1993). however, it is produced during various reactions. metals of transition such iron and enzymes are implied in its formation. the flavoenzymes and the xanthine oxidase activated by ischemia produce it (fukai et al., 2002). in addition, the phagocytic cells generate some for the degradation of the immune complexes by the means of four enzymes: nadph oxidase, superoxide dismutase, nitric oxide synthase and myeloperoxydase (babior, 1978a, 1978b). the superoxide anion radical is characterized by a low reactivity. moreover, it does not have the capacity to pass the membranes; it remains limited to the compartment where it was produced. but it is at the origin of the oxidation of lipids. in fact, the deterioration of the membrane structures is carried out by nucleophilic attack between fatty acids and glycerol of phospholipids. o2 -· can act at the same time like an oxidant and a reducer. in the presence of some metals (manganese or vanadium), it catalyses reactions of oxidation in chain for example oxidation of many molecules of nad(p)h (liochev and fridovich, 1989). on the contrary, within the framework of metals of transition (iron or copper) present at the active sites of enzymes, it presents a reducing behaviour (liochev and fridovich, 1989). hydroxyl radical oh· can be produced during the thermal reactions or under the effect of ionizing radiations. it can also be generated during a reaction implying iron and which is translated by the fenton’s reaction. the hydroxyl radical can be also produced by homolytic fission of the h2o2. this reaction of haberweiss is catalysed by metals of transition. the first stage is the reduction of the superoxide anion radical and the second corresponds to the reaction of fenton. the haber-weiss reaction can be inhibited by chelating of metals, in particular the desferrioxamine in the case of iron. the hydroxyl radical is very reactive and thus its diffusion is limited. it will interfere with the first molecules met, generally on the level of the site of production. it acts, either by addition or by wrenching of hydrogen from the target molecule, or by transfer of electrons. it is at the origin of the lipid peroxidation. the h2o2 is produced by the dismutation of the superoxide anion radical. this anion leads spontaneously to h2o2 under the conditions of physiological ph. this reaction can be accelerated by action of superoxide dismutases (fridovich, 1975). the h2o2 is moderately reactive but its diffusion is high, having the capacity to cross the membranes. its intracellular concentration is very weak between 0.001 and 0.1 µm (sies, 1991). however, in mitochondria and peroxysomes, these concentrations can reach higher levels (boveries et al., 1972). the h2o2 holds a significant place among the ros because it plays the role of intermediate in the production of other reactive radicals. by the means of metals of transition (copper, iron), it gives rise to the hydroxyl radical. moreover, the h2o2 is an intracellular signalling factor (sundaresan et al., 1995). the nitric oxide no· is not always regarded as a ros. indeed, it plays at the same time a role in the destruction and the production of radicals. moreover, it is not very reactive with the cellular components and reacts with radicals generating of less reactive species. it is thus able to inhibit lipid peroxidation (rubbo et al., 2000). conversely, the nitric oxide combined with the superoxide anion radical involves the formation of peroxynitrite, highly toxic radical (beckman and koppenol, 1996). the no is produced from endogenous or exogenous no donors or thanks to a reaction of oxidation of the arginine in the presence of oxygen and nadph. this reaction is produced in particular on the level of the phagocytic cells (marletta, 1994). the nitric oxide and the radicals that result from this are gathered under the term of reactive nitrogenized species rns. the hydroperoxyl (roo· ) and alcoxyl (ro· ) radicals rise from the peroxidation of the lipids. these radicals allow the propagation gradually of the lipid peroxidation. after degradation, they lead to aldehyde formation organized in three major groups: 2acetaldehydes (e.g. 2-hexenal), 4-hydroxy-2acetaldehydes (e.g. 4-hydroxynonenal) and ketoaldehydes (e.g. malondialdehyde) (uchida, 2003). origin and role of ros the major role of the endogenous production of ros is an activity of regulation. indeed, the radicals can interact directly with the molecules containing sulfhydryl groups and thus change their conformation. this type of regulation can in particular affect molecules implied in the mechanisms of signal transduction like protein kinase c (dalton et al., 1999). 93 table 1. compounds generating ros and biomarkers of oxidative stress measured in bivalves contaminants organisms measures authors menadione mytilus edulis sod, gpx, cat (livingstone et al., 1990) dt-d, mda mytilus edulis mda, gsh (ribera et al., 1991) b(a)p mytilus edulis sod; gpx, (livingstone et al., 1990) cat, dt-d, mda paraquat geukensia demissa sod, cat, (wenning et al., 1988) gsh, mda thiram unio tumidus gpx, grd, sod (doyotte et al., 1997) cat, gsh, mda nitrofurantoine mytilus edulis ros (martinez et al., 1995) h2o2 mytilus cat, sod, (cavaletto et al., 2000) galloprovincialis gpx, gsh, mda aroclor 1254 chamaelea gallina cat, grd, (rodriguez-ariza et al., 2003) gpx, gsh table 2. metals generating ros and biomarkers of oxidative stress measured in bivalves contaminants organisms measures authors copper mytilus edulis mda, gsh (viarengo et al., 1989) mytilus edulis sod (manduzio et al., 2003) mytilus sod, grd, gpx (regoli and principato, 1995) galloprovincialis cat, gsh mytilus mda, gsh (viarengo et al., 1990) galloprovincialis lysosomal stability mytilus gsh (canesi et al., 1999) galloprovincialis unio tumidus gpx, grd, sod (doyotte et al., 1997) cat, gsh, mda ruditapes sod, cat, segpx (geret et al., 2002) decussatus gpx, mda cadmium mytilus mda, gsh (viarengo et al., 1990) galloprovincialis lysosomal stability zinc mytilus mda, gsh (viarengo et al., 1990) galloprovincialis lysosomal stability mercury anadara granosa grd, gsh (patel et al., 1990) mytilus gsh (canesi et al., 1999) galloprovincialis selenium anadara granosa grd, gsh (patel et al., 1990) complex mytilus gpx, cat, sod, (regoli and principato, 1995) contamination galloprovincialis grd, gsh 94 table 3. in situ contamination studies and biomarkers of oxidative stress measured in bivalves contaminants organisms measures authors hap perna viridis sod, cat, gpx (cheung et al., 2001) dt-d, gsh, mda mytilus edulis sod, cat (eertman et al., 1995) saccostrea cucullata cat, sod (nyogi et al., 2001b) gpx, dt-d hap + pcb mytilus sod, segpx, cat (solé et al., 1995) galloprovincialis unio tumidus grd, segpx, gpx, (cossu et al., 1997), gsh, cat, sod, mda (cossu et al., 2000) mytilus cat, sod (porte et al., 1991) galloprovincialis gpx, dt-d complex crassostrea virginica gsh, mda (ringwood et al., 1999) contamination lysosomal stability mytilus gsh, grd, gpx (regoli and principato, 1995) galloprovincialis cat, sod dreissena mda, dna (de lafontaine et al., 2000) polymorpha mytilus cat, dt-d, sod (livingstone et al., 1995) galloprovincialis mytilus edulis sod, gpx, gst (manduzio et al., 2004) metals mytilus grd, segpx, gpx (regoli and winston, 1998) galloprovincialis cat, sod, gsh mytilus lysosomal stability (domouhtsidou galloprovincialis mda and dimitriadis, 2001) they allow the regulation of many other molecules (babior et al., 1997). studies showed that the h2o2 can replace insulin in its role of growth promoter (allen and tresini, 2000). thereafter, other experiments showed the stimulation of the production of h2o2 by insulin and nerve growth factor (mukherjee et al., 1978; mukherjee and mukherjee, 1982). the nitric oxide plays itself a role in the vasodilatation and the neurotransmission by activation of enzymes (allen and tresini, 2000). the radicals also play of the roles of control of various factors of transcription (sen and packer, 1996). a significant source of free oxygenated radicals comes from the redox cycles and of the oxidation catalysed by cytochrome p450 monooxigenases. these enzymes allow the addition of a functional group to the exogenous compounds. the redox cycles pass by reactions of oxidation, reduction and hydrolysis, each mediated by transfers of electrons. at the time of each reaction, ros are formed. many exogenous compounds can stimulate the production of ros (tables 1-3). several modes of action were described. many xenobiotics catalyse the microsomal transfer coming from the nad(p)h towards oxygen of electrons and then involving ros formation. it is the case of the nitroaromatic compounds (e.g. nitrofurantoin), quinones (e.g. menadione), and derived from the bypiridium (e.g. paraquat). various studies showed a production nad(p)h-dependent of ros stimulated by contaminants (lemaire et al., 1994; peters et al., 1996; lemaire and livingstone, 1997; livingstone et al., 2000). a number of compounds will involve the formation of active species of oxygen after metabolisation during which they become themselves of the radicals as quinones. in this case, xenobiotic is first reduced by a nadph-dependent reductase during the reaction on phase i producing a radical. this last can then transfer an electron to oxygen. a superoxide anion radical is generated. in each cycle, two potentially harmful compounds can thus be produced (winston and di giulio, 1991; goeptar et al., 1995). a deficiency in metal can also lead to an oxidative stress. indeed, several metals are integrated into proteins; it is the case of the copper in cu/zn-sod (l’abbe and fischer, 1984; taylor et al., 1988). in the rat, a feeding without coppers leads to a rise of the quantity of oxidized proteins and to a fall of the activity of cu/zn-sod in erythrocytes (sukalski et al., 1997). however, in excess, copper can increase the rate of malondialdehyde, marker of the lipid peroxidation, and induce a reduction in the rate of glutathione. indeed, copper is suitable for catalyse the production of hydroxyl radicals via the reaction of haber-weiss (kadiiska et al., 1993; bremmer, 1998). 95 the ionizing and ultraviolet rays are also of significant sources of radicals of oxygen by break of the molecules. origin of ros in molluscs in ecotoxicology, many compounds such as pah, pcb and metals were implicated in the induction of the production of radicals at the laboratory like in situ (tables 1-3). however, it has a lack of data about the exact mechanism of action of these compounds. the measurement of the oxidative stress is generally carried out by the follow-up of the modifications of the activity levels of enzymes (cat, sod, etc.) and of the rates of the molecules (gsh, etc.) implied in the antioxidant defence in two main tissues, digestive gland and gills. these studies involved a hierarchical organization of the cellular answers and more specifically of the antioxidant enzymes. thus, cat is regarded as an enzyme presenting a clear and early response to contamination (wenning et al., 1988). the induction of the gpx is generally noted in a concomitant way to that of the cat and sometimes to that of sod (rodriguez-ariza et al., 1993). in molluscs, as in the mussel mytilus galloprovincialis, sod seems to be a stable enzyme, seldom presenting variations of activity (livingstone et al., 1995). the various studies can however show contradictory results. thus, the sod is sometimes described like presenting a modification of activity in a concomitant way at the cat as in geukensia demissa after 12 h of exposure to the paraquat (wenning et al., 1988). géret et al. (2002) describe a reduction in the levels of seleno-dependent and total gpx activities in ruditapes decussatus exposed to copper (0.5; 2.5 and 25 µg.l-1) after one day of exposure. at the reverse, the carp cyprinus carpio morpha presents an increase in the gpx activity after 1 day of exposure to copper (5, 10, 25 and 50 µg.l-1) (radi and matkovics, 1988). other molecules implied in antioxidant defences are measured, most current being gsh. the reduction in the rate of reduced gsh was observed at bivalves m. galloprovincialis and unio tumidus in correlation with the presence of pah and pcb in the medium (regoli and principato, 1995; doyotte et al., 1997; cossu et al., 1997, 2000). the modification of the rate of reduced gsh as well as balance between the rates of reduced and oxidized glutathione (gsh/gssg) can be correlated with the variation of grd activity. patel et al. (1990) observed a reduction in the grd activity and an increase in the rate of oxidized glutathione during the exposure of bivalves (anadara granosa) to mercury. in this context, the grd activity also constitutes an interesting enzyme in the study of the oxidative stress. the reduction in the rate of reduced gsh was connected to the induction of the lipid peroxidation in particular in the mussel exposed to metal contaminants (viarengo et al., 1988). nevertheless, the lipid peroxidation is generally correlated with the reduction in the whole of the antioxidative enzymes (doyotte et al., 1997; cossu et al., 1997, 2000; géret et al., 2002). the environmental parameters are also suitable for induce a variation of pro-oxidant/antioxidant balance. many studies showed seasonal variations of the antioxidant activities at marine species (viarengo et al., 1991; orbea et al., 2002). these variations are due to the fluctuations of temperature, salinity, the oxygen rate and the quantity of food available (viarengo et al., 1991). changes of seasonal nature were also studied in order to understand their implication in the answers of the antioxidant systems to the contaminants. this kind of studies allows establishing the link between ecophysiologic parameters and ecotoxicological reactions (niyogi et al., 2001a, 2001b). effects of ros and diseases ros can act on the whole of the cellular components. the variations of level of these radicals thus have significant effects on cellular functions. the ros influence in particular the thiol groups of proteins, leading to the formation of intraor inter-molecular disulphide bridges. the most studied action of ros is the lipid peroxidation. this reaction is mainly carried out by the hydroxyl radical (stegeman et al., 1992a; steinberg, 1997). this process corresponds to reactions in chain. after rearrangement and addition of oxygen, peroxyl (roo· ) and alcoxyl (ro· ) radicals are generated. oxidation is propagated thereafter with other unsaturated lipids and can even reach proteins. the oxidation of phospholipids membranes involves disturbances of these structures. as a first consequence, we can observe a reduction in fluidity of the membranes and the inactivation of the receptors and enzymes located at their level (snell and mullock, 1987). in a second time, this oxidation and particularly oxidized products increase the permeability of the membranes, in particular with the calcium ions leading to cellular death (gutteridge and halliwell, 1990). on the level of the mitochondria like lysosomes, the lipid peroxidation results in the lysis of these organelles and the release of enzymes. these enzymes then catalyse the decomposition of proteins, nucleic acids and cellular polysaccharides (horton and fairhurst, 1987; snell and mullock, 1987; pre, 1991). as example, the oxidation of the polyinsaturated lipids can induce the appearance of cardiovascular diseases (wattanapitayakul and bauer, 2001). in a general way, during this reaction, various compounds are produced such malondialdehyde (mda) and 4-hydroxynonenal (hne), both able to bind to proteins and to form adducts. indeed, these compounds react in a spontaneous way with cysteines of proteins and with glutathione. the 4hydroxynonenal can inhibit the synthesis of the nucleic acids and proteins, and block the cellular proliferation (benedetti et al., 1982, 1986; esterbauer and cheeseman, 1990; esterbauer, 1993). another action of the ros relates to proteins. the oxidation of proteins derives from direct action of ros or indirect interaction with the alcoxyl (ro· ) or peroxyl (roo· ) radicals generated at the time of the lipid peroxidation. the amino acids most sensitive are those including sulfhydryl groups such methionine and tryptophan. this oxidation can involve of: (i) change of protein conformation by modification of some amino acids; (ii) generate bridges between proteins and proteins and lipids; (iii) cuts (levine et al., 1994). the structural modifications induce functional changes in 96 particular cellular metabolism (shacter et al., 1994). indeed, the oxidation of proteins can result in a disturbance of ionic transport, enzymatic activities and calcium homeostasis. the damaged proteins are then more sensitive to the proteases action, and thus destroyed more quickly, this being able to induce tissue degradation (rice-evans et al., 1991). the nucleic acids are also targets for the free oxygenated radicals. the damage is not specific: simple or double cuts, formation of abasic sites, covalent bonds between dna or dna and proteins, and modifications of the bases, the most reached being the deoxyguanosine oxidized in 8-hydroxy-2'deoxyguanosine (8-ohdg) (meneghini, 1988; dizdaroglu, 1991; spencer et al., 1996). the dna damages are mainly caused by the hydroxyl radical (oh· ). superoxide anion radical can cause also cuts of dna and lesions of the bases. if guanine is the majority target, each base can undergo these attacks (halliwell and dizdaroglu, 1992). finally, the glucid oxidations in presence of metals involve protein cuts. this reaction is initiated by the hydroxyl radical, which tears off a hydrogen atom to the one of carbons close to the glycoprotein. other radicals are produced such as the peroxyl radical. the cytotoxicity of the radicals of oxygen takes part in the development of much pathology. the oxidative stress is thus implied in the disease of alzheimer (bowling and beal, 1995; ihara et al., 1997). the damage caused by the radicals of oxygen among parkinsonian patients was shown and would be related to a deficiency of the system of defence in brain, in particular in sods activity (radunovic et al., 1997). conversely, an overproduction of radicals of oxygen is implicated in the development of some diseases. at the time of the respiratory syndrome of distress, an infiltration of fluid is observed in the air cells resulting from damage of the endothelium of the capillaries. at the people reached of this syndrome, the lungs contain a significant number of neutrophils (weiland et al., 1986). the production of ros is also implied in rheumatoid arthritis. the therapies against this pathology include antioxidant components (reglinski et al., 1997). balance between pro-oxidants and antioxidants systems would be also implied in the phenomenon of cellular ageing (sagar et al., 1992). the oxidative stress would increase during cellular differentiation and ageing (sohal et al., 1990). however, the implication of the radicals in cellular ageing is not cleared up. indeed, the studies do not show all the same variations according to the age (e.g. mizuno and ohta, 1986; sohal et al., 1990; hussain et al., 1995; sahoo and chainy, 1997; kim et al., 2002). effects in molluscs at the marine molluscs, the physiological and morphological modifications in response to the chemical stress are not much studied. indeed, the appearance of pathologies is generally synonymous with irreversible damage. the major observations related to hepatic pathologies such as the increase in the occurrence of parasitic infections, ignition and necrosis in the fish (vethaak, 1992). however, in molluscs, the majority of the studies evaluate the impact of pollution by the appearance of neoplasia (malins et al., 1988; kinae et al., 1990). in molluscs, the observation of pathologies does not correspond to a major axis of ecotoxicological studies. nevertheless, in situ works bring back the observation of a blood neoplasm, haematopoietic neoplasm, disseminated neoplasia, hemic neoplasia, leukaemia or proliferate cellular disorder (krishnakumar et al., 1999). this syndrome was observed overall on 15 species of bivalves including 4 of oysters, 6 of clams and 5 species of moulds (re-examined of elston et al., 1992). this disease is characterized by the proliferation of circulating haemocytes. they present a significant core of lobed form which compared fills the major part of the cell to the cytoplasm. moreover, one or more micronuclei are observed as well as a high frequency of mitoses (farley, 1969; mix, 1983). the origins of this neoplasia remain discussed. indeed, some authors advance a potential implication of carcinogenic compounds (lowe and moore, 1978; farley et al., 1991). other studies connect on the contrary, the appearance of this pathology to a retrovirus or to a genetic disposal of the individuals (couch and harshbarger, 1985; elston et al., 1988). nevertheless, lowe and moore (1978) showed the appearance of neoplasms in the mussel mytilus edulis subjected to a pollution of domestic and industrial nature including hydrocarbons. other authors blame these same xenobiotics in the appearance of these tumours in different bivalves, in particular of oysters, subjected to the pollution generated by the shipwreck of amoco cadiz in france (balouet et al., 1986; barry and yevich, 1975; yevich and parszcz, 1977). conversely, mix and schaffer (1983) do not note any incidence of pah on the frequency of appearance of neoplasia in m. edulis. in addition, farley et al. (1991) showed a linear correlation between the appearance of neoplasms and the tissue concentration in chlordane. the exposures in laboratory lead in the same way to contradictory results, with either inductions of tumours or no incidence following treatments with the pah or other xenobiotics (khudoley and syrenko, 1978; rasmussen et al., 1983a, b, 1985; winstead and couch, 1988; krishnakumar et al., 1999). however, the implication of the oxidative stress is however not proven in a sure way (elston et al., 1988; moore et al., 1991; krishnakumar et al., 1994, 1999). moreover, in m. edulis and mya arenaria, an increase in the frequency of appearance of the neoplasms is observed during the coldest months of the year. these observations can be related to the reduction in the activities of the antioxidant enzymes at low temperatures (elston et al., 1992). defences of the organisms the production and the action of the ros must be controlled in order to limit the cellular damage. this limitation is carried out initially by sequestration, even the destruction, of the systems pro-oxidants such as the complexation of free metals by metallothioneines. the antioxidant systems also include enzymes whose activity involves the destruction of the reactive oxygenated species. these enzymes can act by the 97 means of metals of transition. a last means of fight against the oxidative stress is the stop of oxidations in chain of the cellular components. the antioxidant capacities are variable from one species to another. moreover, it is allowed that these activities vary according to the seasons. lastly, another adaptation of the organisms to the increase in the production of ros is the induction of the synthesis of antioxidant molecules. two categories of antioxidant systems are generally defined: antioxidant enzymes and molecules without enzymatic activity. three major enzymes act jointly for the destruction of the ros in the cell: sods, cat and gpxs. the grd can be added to these enzymes even if it does not present a direct role in the catabolism of the oxygenated radicals. sods (sod; ec. 1.15.1.1) will allow thereafter the destruction of the superoxide anion radical by dismutation out of h2o2 dealt by cat. the two enzymes, sod and cat, have the same principal localization in the cell, the peroxysomes. isoenzymes of the sod are found in the various compartments of the cell, but their active site has a tertiary structure overall good preserved, made of a hydrophobic well where the superoxide anion radical fits. the reaction of dismutation is catalysed by a metal from which nature makes it possible to distinguish three types of isoenzymes. during the reaction, the metal ion captures an electron of the superoxide anion radical. sods seem to be very significant enzymes because of their ubiquity and of their localization at the same time intraand extra-cellular (stegeman et al., 1992b). cu/zn-sod (35 kda) was identified for the first time in 1968, in bovines erythrocytes by mccord and fridovitch (mccord and fridovich, 1969). in addition to its localization in the cytoplasm, its presence was also shown on the external face of the endothelial cells and in the blood plasma. later on, it was also detected in the peroxysomes, the lysosomes and the core of the eukaryotes cells (beyer et al., 1991). this isoenzyme is made up of two identical subunits from approximately 15 000 da each one, to which two metal atoms are added: copper and zinc. the function of destruction of the superoxide anion is provided by copper whereas zinc would have only one structural role. cu/zn-sod was described at the vertebrate ones, the aquatic and terrestrial invertebrates, like at the plants on the chloroplastic and cytosolic level. more recently, an extra-cellular form noted ec-sod, was characterized at the vertebrate ones, then at the invertebrates, and more precisely the nematode caenorhabditis elegans (hjalmarsson et al., 1987; wilson et al., 1994; folz et al., 1997). indeed, this extracellular copper/zinc-sod was detected in the fluids circulating like plasma, the lymph and the synovial liquid (marklund, 1982; fridovich, 1995). however, it would be mainly related to the proteoglycanes of the cellular membrane and only less than 1 % would be present in circulating form (karlsson and marklund, 1987; karlsson et al., 1988; adachi et al., 1995). the mitochondrial matrix contains mn-sod in eukaryotes and bacteria and fe-sod in plants and bacteria. these two isoenzymes are also present in lysosomes, peroxysomes and nuclear compartment. fe-sod is localised in the chloroplasts of plants and constitutes for those the most significant form. these two shapes of sods present analogies of structure; however, it is allowed that the mn-sod is inducible by the superoxide anion radical, whereas it would not be the case of fe-sod. various substances are able to inhibit the sod with for some, specificity with respect to an isoenzyme. thus the cu/zn form is inhibited by cyanide (weisiger and fridovich, 1973) and mn-sod by a treatment to sodium dodecyl sulfate. the h2o2 inactivates fe-sod (hodgson and fridovich, 1975). however, yim et al. (1990) bring back an inhibition at the same time of cu/zn-sod and fe-sod by h2o2. all these enzymes are also inhibited by elimination of their metal of transition, by chelators. however, this inhibition is reversible. lastly, the activity of mn-sod is inhibited with ph 9 whereas that of cu/zn-sod is not influenced by the ph. deficiency in sods or their inhibition increases the sensitivity of the organisms to oxidants. in this general context, it was shown in particular that the mitochondrial mn-sod is essential for the life. indeed, mn-sod deficiency was implicated in the appearance of serious pathologies, the production of superoxide anion becoming very significant. thus, the knockout mice for mn-sod die after the birth or suffer from neuro-degenerative diseases (melov et al., 1998). the form of mn-sod, contrary to that of cu/zn-sod, would be controlled by the superoxide anion and in a general way by the radicals of oxygen (liu et al., 2000). in the bacteria, this induction brings into play a locus soxr which controls the transcription of nine genes implied in the synthesis of enzymes for the production of nadph, the repair of the dna, the protein synthesis and the membrane permeability (harris, 1992). the form of mn-sod is thus inducible by cytokines in various cellular types and by ionizing radiations (masuda et al., 1988). studies showed that the tumoral necrosis factor tnf-á could induce the expression of the manganese-sod (wong et al., 1989, 1995). in the same way, otieno et al. (2000) showed the transcriptional regulation of mn-sod by the chemoprotective 3h-1,2-dithiol-3-thiol. conversely, the rates of mrna of cu/zn-sod do not vary following this treatment. in fact, cu/zn-sod cytosolic appears less significant in the limitation of the oxidative stress. indeed, the transgenic animals not expressing this enzyme present a normal phenotype (ohlemiller et al., 1999). cat (cat; ec. 1.11.1.6) is present primarily at peroxisomial level. this inducible enzyme allows the destruction of h2o2 out of water and oxygen. it is a hemoprotein including an iron atom per unit, the number of units varying according to species. it catalyses a two stages reaction corresponding to a catalasic activity. however, the cat can also present a peroxidasic activity (leguille-cossu, 1996). the cat has other functions during the normal function of the cell. thus, this enzyme catalyses the detoxication of substrates such alcohols and phenols in connection with the reaction of reduction of hydrogen peroxide (akyilmaz and dinckaya, 2003). however, generally, this enzyme is regarded as being able to catalyse only the destruction of hydrogen peroxide (stegeman et al., 1992b). it was shown in the rat, the possibility of a transcriptional induction of cat under treatment by a chemoprotective, 3h-1,2-dithiole-3-thione (otieno et al., 2000). its localization makes it possible this 98 enzyme to carry on an activity complementary to the gpx. in the bacteria, its activity is induced by h2o2 on the level of the locus oxyr (harris, 1992). one second way of destruction of h2o2 utilizes the gpxs (gpx; ec.1.11.1.9). the enzymatic activity is coupled with the oxidation of the gsh and generates alcohols. the gpxs are also able to reduce other peroxides. these enzymes are localised in the cytoplasm and the mitochondrial matrix of the cells. they include two categories: the segpx and a seindependent form, which corresponds in fact to a glutathione s-transferase with a peroxidasic activity. this last form is a dimer of 50 kda localised mainly in the microsomes and catalyses only the reduction of organic peroxides. the segpx is a tetrameric metalloenzyme (80 kda) of which each subunit comprises a selenium atom in the form of a selenocysteine residue and incorporated in the active site thanks to a selenocysteine-specific rna (spallholz and boylan, 1991). a selenium deficiency thus will involve an inhibition of this enzyme. there are other inhibitors of gpx whose metals by a thioloprive action (cadmium and lead) and in a general way the detergents (triton, etc.). the catalytic mechanism proposed for the reduction of peroxides by gpx passes by the oxidation of the active site in selenic acid (seoh). the seoh is transformed by adjunction of a molecule of reduced gsh. the addition of one second molecule of gsh regenerates the active site of the enzyme and the oxidized glutathione (gssg). moreover, ursini et al. (1982) described another form of se-dependent gpx in the liver on pig, the phospholipid hydroperoxide peroxidase (plgpx; ec. 1.11.1.12). this enzyme is a monomer (22 kda) and is implied in the protection of the liposomes and membranes against the oxidative damage. contrary to the preceding enzyme, its selenium requirement is less strict (spallholz and boylan, 1991). the grd (grd; ec. 1.8.1.7) is not always recognized as an antioxidant enzyme. it can nevertheless be included in this category because it makes it possible to reduce the oxidized glutathione (gssg) according to a nadph-dependent process, and it is thus at the base of the regeneration of reduced gsh necessary to the operation of the gpxs and of many other enzymes of the cell. balance between gssg and gsh is capital in the maintenance of cellular homeostasis (winston and di giulio, 1991). in the cell, all these enzymes will intervene in concert, each one according to a specific cellular under-localization, in order to control the quantity of free radicals. other enzymes are regarded as having an antioxidant action. it is the case of dt-d and the glutathione s-transferase. indeed, the glutathione stransferase presents a peroxidasic activity with respect to organic peroxides and belongs to the group of the se-independent gpx. dt-d, also indicated as nad(p)h oxidoreductase 1 (nqo1, ec. 1.6.99.2) catalyses the reduction of quinones by addition of two electrons, thus generating hydroquinones which are more easily excreted after conjugation with sulfates groups or glucuronide (cadenas, 1995). this enzyme thus makes it possible to produce a quinoline stable form without passage by radicals intermediates. in this direction, dt-d can be regarded as an antioxidant enzyme. however, hydroquinones are also able to generate radical species of oxygen, or to react directly with the dna in reactions of alkylation (cadenas, 1995). in this last case, dt-d constitutes an enzyme of bioactivation. more recently, a new family of antioxidant enzymes, the peroxiredoxines, was described in some procaryotic organisms and mammals (chae et al., 1994). these proteins have homologies of sequence with the thioredoxine peroxidase of yeast and they have a peroxidasic function. six groups were defined according to their sequences and their immunological properties (kang et al., 1998; chae et al., 1999; seo et al., 2000). in human, they are expressed in the brain, each of the six groups presenting of the particular localizations (kang et al., 1998; chae et al., 1999; seo et al., 2000). their differential expression was connected to neurodegenerative disorders such as the disease of alzheimer (krapfenbauer et al., 2003). antioxidants of nonenzymatic nature exist too. an antioxidant capacity is conferred on the gsh (l-γglutamyl-l-cysteinyl glycin) by the presence of the thiol group carried by the cysteinic residue. the gsh is a tripeptide of glutamate (l-glu), cysteine (l-cys) and glycin (gly), the glutamate and cysteine being connected by γ-peptide connection. it is synthesized by the consecutive action of two enzymes, γglutamylcysteine synthetase and the gsh synthetase. in the cells, the gsh is present mainly in its reduced form gsh and represents the most significant thiol in eukaryotes cells (0,2 to 10 mm). an increase in the proportion of oxidized form (gssg) translates an oxidative stress. the gsh exerts many functions in the cell. it intervenes in the processes of reduction such as the synthesis and the degradation of proteins, the formation of deoxyribonucleotides, the regulation of the enzymes and the protection of the cells against the free radicals of oxygen. the gsh also plays the role of co-enzyme for various reactions and it is combined with compounds either endogenous (oestrogens, prostaglandins and leucotrienes) or exogenous (drugs and xenobiotics), thus taking part in their metabolism. the gsh thus indirectly supports the detoxication of the radical compounds by its function of co-substrate of the antioxidant enzymes such as the gpxs. moreover, it is the co-substrate of a significant enzyme in the process of detoxication: the gst. the gsh is thus regarded as a central element of antioxidant defences. indeed, a depletion in gsh induces an increase in the sensitivity of the organisms to xenobiotics or overall, with generating processes of radicals (jones et al., 1995; conners, 1998). other compounds known as low-weight molecular have an antioxidant role. thus, the lipoic acid in reduced form can reduce the gsh and the peroxyl radicals. it also has a chelating capacity of metals, quenching of free radicals (kagan et al., 1992) and of regeneration of others antioxidants like the ascorbic acid and the vitamin e (packer et al., 1995). other protective elements are brought by the food: vitamin e, vitamin c and pigments such carotenoids. these antioxidant systems make it possible to stop the chain reactions, in particular those of the lipid peroxidation. indeed, these substances are localised on the level of the membranes and destroy the free radicals by collecting the lone electron. other natural compounds 99 also have an antioxidant character: urea, thiourea, mannitol and dimethyl sulfoxide. in substitution for iron, zinc exerts also an antioxidant action. metals constitute however a particular case because they can at the same time generate radicals and destroy them. antioxidants in molluscs the antioxidant systems known in the mammals are found in the marine organisms. all in all, the antioxidant activities are lower at the aquatic species compared to those of the mammals. in particular, the mn-sod is little expressed in tissues of m. edulis, cu/zn-sod being the main form (livingstone et al., 1992; manduzio et al., 2003). furthermore, the expression of the cu/zn-sod is modulated by xenobiotics. manduzio et al. (2003) described the induction of expression of a cu/zn-sod isoform in mussel m. edulis exposed to contaminants in field and laboratory. however, the bivalve molluscs have levels of activity in digestive gland of the same order as those measured in the liver of fish. at these organisms, the antioxidant activities however are influenced by various factors: (i) an anaerobic respiration gives rise to a reduction in the enzymatic activities and lipid peroxidation, levels returning to normal when oxygenation is restored (viarengo et al., 1989); (ii) the laying involves an increase in the antioxidant activities in march-april, followed by a progressive reduction at spring whereas the availability in food and the temperature increase (solé et al., 1995); (iii) the age sensitizes with the oxidizing effects by reduction of the antioxidant capacities what results in an increase in the rates of lipid peroxidation (viarengo et al., 1991). in the same way, the seasonal fall in antioxidant enzymes is concomitant to an increase in the rate of lipid peroxidation. however, this decrease could be compensating by an augmentation of gst activity in gills of mussels (power and sheehan, 1996; sheehan and power, 1999; manduzio et al., 2004). this observation is all the more pronounced since the water is polluted as described in the harbour of le havre, which is characterized by a general contamination by various compounds such as pahs, pcbs and heavy metals (manduzio et al., 2004). conclusion in spite of many studies about oxidative stress in molluscs, there still exists many questions. this could be explaining sometimes contradictory data. it would be interesting to study thoroughly physiological natural factors which could induce modification between prooxidant and antioxidant systems. in particular, among these factors, the phenomena of hypoxia/anoxia could have an important impact. moreover, the disappearance of environment being able to be considered as free from pollution and so being able to constitute a reference is limiting. it is all the more significant to develop rapidly reliable tools of diagnostic of environmental safety that the levels of pollution increase and new xenobiotics are synthesize each year. references adachi t, yamada h, futenma a, kato k, hirano k. heparin-induced release of extracellularsuperoxide dismutase form (v) to plasma. j. biochem. 117: 586-590, 1995. akyilmaz e, dinckaya e. development of a catalase based biosensor for alcohol determination in beer samples. talanta 61: 113-118, 2003. allen rg, tresini m. oxidative stress and gene regulation. free radic. biol. med. 28: 463-499, 2000. babior bm. oxygen dependent microbial killing by phagocytes. 1. n. engl. j. med. 298: 659-668, 1978a. babior, b. m. oxygen dependent microbial killing by phagocytes. 2. n. engl. j. med. 298, 721-725, 1978b. babior bm, el benna j, chanock sj, smith rm. oxidative stress and the molecular biology of antioxidant defences, cold spring harbor laboratory press, cold spring harbor, 1997. balouet g, poder m, cahour a, auffret m. proliferative hemocytic condition in european flat oysters (ostrea edulis) from breton coasts: a 6-year survey. j. invertebr. pathol. 48: 208-215, 1986. barry m, yevich pp. the ecological, chemical and histopathological evaluation of an oils spill site. iii. histopathological studies. mar. pollut. bull. 6: 171-173, 1975. beckman js, koppenol wh. nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. am. j. physiol. 271: c1424-c1437, 1996. benedetti a, fulceri r, ferrali m, ciccolo l, esterbauer h, comporti m. detection of carbonyl functions in phospholipids of liver microsomes in ccl4and brccl3-poisoned rats. biochem. biophys. acta 712: 628-638, 1982. benedetti a, pompella a, fulceri r, romani a, comporti c. detection of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice. bioch. biophys. acta 876: 658-666, 1986. beyer w, imlay j, fridovich i. superoxide dismutases. prog. nucl. acid res. mol. biol. 40: 221-253, 1991. boveries a, oshino n, chance b. the cellular production of hydrogen peroxide. biochem. j. 128: 617-630, 1972. bowling ac, beal mf. bioenergetic and oxidative stress in neurodegenerative diseases. life sci. 56: 1151-1171, 1995. bremmer i. manifestations of copper excess. am. j. clin. nutr. 67: 1069-1073. 1998. cadenas e. antioxidant and prooxidant functions of dt-diaphorase in quinine metabolism. biochem. pharmacol. 49: 127-140, 1995. canesi l, viarengo a, leonzio c, filippelli m, gallo g. heavy metals and glutathione metabolism in mussel tissues. aquat. toxicol. 46: 67-76, 1999. cavaletto m, ghezzi a, burleto b, ceratto n, evangelisti v, viarengo a. effect of hydrogen peroxide on oxidative stress and metallothionein level in mytilus galloprovincialis. comp. biochem. physiol. 126a: 24, 2000. chae hz, robinson k, poole lb, church g, storz g, rhee sg. cloning and sequencing of thiolspecific antioxidants from mammalian brain: alkyl 100 hydroperoxide reductase and thiol-specific antioxidants define a large family of antioxidant enzymes. proc. natl. acad. sci. usa 91: 70177021, 1994. chae hz, kang sw, rhee sg. isoforms of mammalian peroxiredoxin that reduce peroxides in presence of thioredoxin. methods enzymol. 300: 219-226, 1999. cheung ccc, zheng gj, li amy, richardson bj, lam pks. relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, perna viridis. aquat. toxicol. 52: 189-203, 2001. conners de. the toxicological significance of glutathione depletion in oysters crassostrea virginica, exposed to copper under laboratory and field conditions. medical university of south carolina, charleston, 1998. cossu c, doyotte a, babut m, exinger a, vasseur p. antioxidant biomarkers in freshwater bivalves, unio tumidus, in response to different contamination profiles of aquatic sediments. ecotoxicol. environ. saf. 45: 106-121, 2000. cossu c, doyotte a, jacquin mc, babut m, exinger a, vasseur p. glutathione reductase, seleniumdependent glutathione peroxidase, glutathione levels, and lipid peroxidation in freshwater bivalves, unio tumidus, as biomarkers of aquatic contamination in field studies. ecotoxicol. environ. saf. 38: 122-131, 1997. couch ja, harshbarger jc. effects of carcinogenic agents on aquatic animals: an environmental overview. environ. carcinog. rev. 3: 63-105, 1985. dalton tp, shertzer hg, puga a. regulation of gene expression by reactive oxygen. ann. rev. pharmacol. toxicol. 39: 67-101, 1999. de lafontaine y, gagné f, blaise c, costan g, gagnon p, chan hm. biomarkers in zebra mussel (dreissena polymorpha) for the assessment and monitoring of watre quality of the st lawrence river (canada). aquatic toxicol. 50: 51-71, 2000. dizdaroglu m. chesmitry of free radical damage to dna and nucleoproteins. in: halliwell b, aruoma oa (eds), dna and free radicals, ellis horwood ltd., cichester, pp 19-40, 1991. domouhtsidou gp, dimitriadis vk. lysosomal and lipid alterations in the digestive gland of mussels, mytilus galloprovincialis (l.) as biomarkers of environmental stress. environ. pollut. 115: 123137, 2001. doyotte a, cossu c, jacquin m-c, babut m, vasseur p. antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive gland of the freshwater bivalve unio tumidus. aquat. toxicol. 39: 93-110, 1997. eertman rhm, groenink clfmg, setee b, hummel h. response of the blue mussel mytilus edulis l. following exposure to pahs or contaminated sediment. mar. environ. res. 39: 169-173, 1995. elston ra, kent ml, drum as. progession, lethality and remission of hemic neoplasia in the bay mussel mytilus edulis. dis. aquat. org. 4: 135142. 1988. elston ra, moore jd, brooks k. disseminated neoplasia of bivalve molluscs. rev. aquat. sci. 6: 405-466, 1992. esterbauer h. cytotoxicity and genotoxicity of lipidoxidation products. am. j. clin. nutr. 57: 779-786, 1993. esterbauer h, cheeseman kh. determination of aldehydic lipid peroxidation products: malondialdehyde and 4-hydroxynonenal. methods enzymol. 186: 407-421, 1990. farley ca. probable neoplastic disease of the hematopoietic system in oysters, crassostrea virginica and crassostrea gigas. natl. cancer inst. monogr. 31: 541-555, 1969. farley ca, plutschak dl, scott rf. epizzotiology and distribution of transmissible sarcoma in marylet softshell chams, mya arenaria 1984-1988. environ. health perspect. 90: 35-41. 1991. folz rj, guan j, seldin mf, oury td, enghild jj, crapo jd. mouse extracellular superoxide dismutase: primary structure, tissue-specific gene expression, chromosomal localization, and lung in situ hybridization. am. j. respir. cell mol. biol. 17: 393-403, 1997. fridovich i. superoxide dismutases. ann. rev. biochem. 44: 147-159, 1975. fridovich i. superoxide radical and superoxide dismutases. ann. rev. biochem. 64: 97-112, 1995. fukai t, folz rj, landmesser u, harrison dg. extracellular superoxide dismutase and cardiovascular disease. cardiovasc. res. 55: 239-249, 2002. géret f, jouan a, turpin v, bebianno mj, cosson rp. influence of metal exposure on metallothionein synthesis and lipid peroxidation in two bivalve mollusks: the oyster (crassostrea gigas) and the mussel (mytilus edulis). aquat. living resour. 15: 61-66, 2002. goeptar ar, scheerens h, vermeulen npe. oxygen reductase and substrate reductase activity of cytochrom p450. crit. rev. toxicol. 25: 25-65, 1995. gutteridge jmc, halliwell b. the measurement and mechanism of lipid peroxidation in biological systems. trends biol. sci. 15: 129-135, 1990. gutteridge jmc, halliwell b. transition metal ions and antioxidant proteins in extracellular fluids. in: scott g (ed.), atmospheric oxidation and antioxidants, elsevier publisher, uk, 1993. halliwell b, dizdaroglu, m. the measurement of oxidative damage to dna by hplc and gc/ms techniques. free radic. res. 16, 75-87, 1992. harris ed. regulation of antioxidant enzymes. faseb j. 6: 2675-2682, 1992. hjalmarsson k, marklund sl, engstrom a, edlund t. isolation and sequence of complementary dna encoding human extracellular superoxide dismutase. proc. natl. acad. sci. usa 84: 63406344, 1987. hodgson ek, fridovich i. the interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. biochemistry 14: 5294-5299, 1975. horton aa, fairhurst s. lipid peroxidation and mechanisms of toxicity. crit. rev. toxicol. 18: 1779, 1987. 101 hussain s, slikker jw, ali sf. age-related changes in antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione in different regions of mouse brain. int. j. dev. neurosci. 13: 811-817, 1995. ihara y, hayabara t, sasaki k, fujisawa y, kawada r, yamamoto t, nakashima y, yoshimune s, kawai m, kibata m, kuroda s. free radicals and superoxide dismutase in blood of patients with alzheimer's disease and vascular dementia. j. neurol. sci. 153: 76-81, 1997. jones dp, brown ls, sternberg p. variability in glutathione-dependent detoxification in vivo and its relevance to detoxification of chemical mixtures. toxicology 105: 267-274, 1995. kadiiska mb, hanna pm, jordan sj, mason rp. electron spin resonance evidence for free radical generation in copper-treated vitamin eand selenium-deficient rats: in vivo spin-trapping investigation. mol. pharmacol. 44: 222-227, 1993. kagan ve, shvedova a, serbinova e, khan s, swanson c, powell r, packer l. dihydrolipoic acid--a universal antioxidant both in the membrane and in the aqueous phase: reduction of peroxyl, ascorbyl and chromanoxyl radicals. biochem. pharmacol. 44: 1637-1649, 1992. kang sw, bainess lc, rhee sg. characterization of mammalian peroxiredoxin that contain one conserved cysteine. j. biol. chem. 273: 63036311, 1998. karlsson k, lindahl u, marklund sl. binding of human extracellular superoxide dismutase c to sulphated glycosaminoglycans. biochem. j. 256: 29-33, 1988. karlsson k, marklund sl. heparin-induced release of extracellular superoxide dismutase to human blood plasma. biochem. j. 242: 55-59, 1987. khudoley vv, syrenko oa. tumor induction by nnitroso compounds in bivalve mollusks unio pictorum. cancer lett. 4: 349-354, 1978. kim jw, no jk, ikeno y, yu bp, choi js, yokozawa t, chung hy. age-related changes in redox status of rat serum. arch. gerontol. geriatr. 34: 917, 2002. kinae n, yamashita m, tomita i, kimura i, ishida h, kumai h, nakamura ga. possible correlation between environmental chemicals and pigment cell neoplasia in fish. sc. tot. environ. 94: 143153, 1990. krapfenbauer k, engidawork e, cairns n, fountoulakis m, lubec g. aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders. brain res. 967: 152-160, 2003. krishnakumar pk, casillas e, varanasi u. effect of environmental contaminants on the health of mytilus edulis from puget sound, washington, usa. i. cytochemical measures of lysosomal responses in the digestive cells using automatic image analysis. mar. ecol. prog. ser. 106: 249261, 1994. krishnakumar pk, casillas e, snider rg, kagley an, varanasi u. environmental contaminants and the prevalence of hemic neoplasia (leukemia) in the common mussel (mytilus edulis complex) from puget sound, washington, usa. j. invertebr. pathol. 73: 135-146, 1999. l'abbe mr, fischer pwf. the effects of high dietary zinc and copper deficiency on the activity of copper-requiring metalloenzymes in the growing rat. j. nutr. 114: 813-822, 1984. leguille-cossu c. activités des systèmes antioxydants chez unio tumidus, bivalve dulçaquicole en conditions physiologiques et de stress chimique. university of metz, centre des sciences de l'environnement, metz, 1996. lemaire p, livingstone dr. aromatic hydrocarbon quinone-mediated reactive oxygen species production in hepatic microsomes of the flounder (platichthys flesus l.). comp. biochem. physiol. 117c: 131-139, 1997. lemaire p, matthews a, foerlin l, livingstone dr. stimulation of oxyradical production of hepatic microsomes of flounder (platichthys flesus) and perch (perca fluviatilis) by model and pollutant xenobiotics. arch. environ. contam. toxicol. 26: 191-200, 1994. levine a, tenhaken r, dixon r, lamb c. h2o2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. cell 79: 583-593, 1994. livingstone dr, garcia-martinez p, michel x, narbonne jf, o’hara s, ribera d, winston gw. oxyradical production as a pollution-mediated mechanism of toxicity in the common mussel, mytilus edulis l., and other molluscs. funct. ecol. 4: 415-424, 1990. livingstone dr, lemaire p, matthews aa, peters l, porte c, fitzpatrick pj, förlin, l, nasci c, fossato v, wooton n, goldfarb p. assessment of the impact of organic pollutants on goby (zostericessor ophiocephalus) and mussel (mytilus galloprovincialis) from the venice lagoon, italy: biochemical studies. mar. environ. res. 39 : 235-240, 1995. livingstone d, lips f, martnez p, pipe r. antioxidant enzymes in the digestive gland of the common mussel (mytilus edulis l.). mar. biol. 112: 265276, 1992. livingstone dr, mitchelmore cl, o'hara scm, lemaire p, sturve j, forlin l. increased potential for nad(p)h-dependent reactive oxygen species production of hepatic subcellular fractions of fish species with in vivo exposure to contaminants. mar. environ. res. 50: 57-60, 2000. liochev si, fridovich i. vanadate-stimulated oxidation of nad(p)h. free radic. biol. med. 6: 617-622, 1989. liu h, zhu h, eggers dk, nersissian am, faull kf, goto, jj, ai j, seters-loehr j. gralla eb, valentine js. copper (2+) binding to the surface residue cysteine 111 of his46arg human copper, zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant. biochemistry 39: 81258132, 2000. lowe dm, moore mn. cytology and quantitative cytochemistry of a proliferative atypical hemocytic condition in mytilus edulis (bivalvia, mollusca). j. natl. cancer inst. 60: 1455-1459, 1978. malins dc, mccain bb, letahl jt, myers ms, krahn mm, brown dw, chan s-l, roubal wt. neoplastic and other diseases in fish in relation to toxic chemicals: an overview. aquat. toxicol. 11: 43-67, 1988. 102 manduzio h, monsinjon t, rocher b, leboulenger f, galap c. characterization of an inducible isoform of the cu/zn superoxide dismutase in the blue mussel mytilus edulis. aquat. toxicol. 64: 73-83, 2003. manduzio h, monsinjon t, galap c, leboulenger f, rocher b. seasonal variations in antioxidant defences in blue mussels mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoforme of cu/zn-superoxide dismutase and of glutathione s-transferase. aquat. toxicol. 70: 83-93, 2004. marklund sl. human copper-containing superoxide dismutase of high molecular weight. proc. natl. acad. sci. usa 79: 7634-7638, 1982. marletta ma. nitric oxide synthase: aspects concerning structure and catalysis. cell 78: 927930, 1994. martinez pg, winston gw, metashdickey c, ohara scm, livingstone dr. nitrofurantoin-stimulated reactive oxygen species production and genotoxicity in digestive gland microsomes and cytosol of the common mussel (mytilus edulis l.). toxicol. appl. pharmacol. 131: 332-341, 1995. mccord jm, fridovich i. superoxide dismutase: an enzymatic function for erythrocuprein. j biol. chem. 244: 6049-6055, 1969. melov s, schneider ja, day bj, hinerfeld d, coskun p, mirra ss, crapo jd, wallace dc. a novel neurological phenotype in mice lacking mitochondrial manganese superoxide dismutase. nat. genet. 18: 159-163, 1998. meneghini r. genotoxicity of active oxygen species in mammalian cells. mutat. res. 195: 215-230, 1988. mix mc. hemic neoplasm of bay mussels mytilus edulis from oregon: occurence, prevalence, seasonality and histological progression. j. fish. dis. 6: 239-248, 1983. mix mc, schaffer rl. concentration of unsubstituted polynuclear aromatic hydrocarbons in bay mussels (mytilus edulis) from oregon, usa. mar. environ. res. 9: 193-209, 1983. moore jd, elston ra, drum as, wilkinson mt. alternate pathogenesis of systemic neoplasia in the bivalve mollusc mytilus. j. invertebr. pathol. 58: 231-243, 1991. mukherjee sp, lane rh, lynn ws. endogenous hydrogen peroxide and peroxidative metabolism in adipocytes in response to insulin and sulfhydryl reagents. biochem. pharmacol. 27: 2589-2594, 1978. mukherjee sp, mukherjee c. similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: transmembrane signalling relative to insulin-mimicking cellular effects. biochem. pharmacol. 31: 3163-3172, 1982. niyogi s, biswas s, sarker s, datta ag. seasonal variation of antioxidant and biotransformation enzymes in barnacle, balanus balanoides, and their relation with polyaromatic hydrocarbons. mar. environ. res. 52: 13-26, 2001a. niyogi s, biswas s, sarker s, datta ag. antioxidant enzymes in brackishwater oyster saccostrea cucullata as potential biomarkers of polyaromatic hydrocarbon pollution in hooghly estuary (india): seasonality and its consequences. sci. tot. environ. 281: 237-246, 2001b. ohlemiller kk, mcfadden sl, ding dl, flood dg, reaume ag, hoffman ek, scott rw, wright js, putcha gv, salvi rj. targeted deletion of the cytosolic cu/zn-superoxide dismutase gene (sod1) increases susceptibility to noise-induced hearing loss. audiol. neurootol. 4: 237-246, 1999. orbea a, ortiz-zarragoitia m, solé m, porte c, cajaraville mp. antioxidant enzymes and peroxisome proliferation in relation to contaminant body burdens of pahs and pcbs in bivalve molluscs, crabs and fish from the urdaibai and plentzia estuaries (bay of biscay). aquat. toxicol. 58: 75-98, 2002. otieno ma, kensler tw, guyton kz. chemoprotective 3h-1,2-dithiole-3-thione induces antioxidant genes in vivo. free radic. biol. med. 28: 944-952, 2000. packer l, witt eh, tritschler hj. alpha-lipoic acid as a biological antioxidant. free radic. biol. med. 19: 227-250, 1995. patel b, chety jp, patel s. effect of mercury, selenium et glutathione on sulphydryl levels and glutathione reductase in blood clam anadara granosa (l.). ind. j. mar. sci. 19: 187-190, 1990. peters ld, o'hara scm, livingstone dr. benzo[a]pyrene metabolism et xenobioticstimulated reactive oxygen species generation by subcellular fraction of larvae of turbot (scophthalmus maximus l.) 1. comp. biochem. physiol. 114c: 221-227, 1996. porte c, solé m, albaigés j, livingstone dr. responses of mixed-function oxygenase and antioxidase enzyme system of mytilus sp. to organic pollution. comp. biochem. physiol. 100c: 183-186, 1991. power a, sheehan d. seasonal variation in the antioxidant defence systems of gill and digestive gland of the blue mussel, mytilus edulis. comp. biochem. physiol 114c: 99-103, 1996. pré j. la lipopéroxydation. pathol. biol. 39: 716-736, 1991. radi aar, matkovics b. effects of metal ions on the antioxidant enzyme activities, protein contents and lipid peroxidation of carp tissues. comp. biochem. physiol. 90c: 69-72, 1988. radunovic a, porto wg, zeman s, leigh pn. increased mitochondrial superoxide dismutase activity in parkinson's disease but not amyotrophic lateral sclerosis motor cortex. neurosci. lett. 239: 105-108, 1997. rasmussen lpd, hage e, karlog o. light and electron microscopic studies of the acute and chronic toxic effects of n-nitroso compounds on the marine mussel, mytilus edulis (l.). i. nnitrosodimethylamine. aquat. toxicol. 3: 285-299, 1983a. rasmussen lpd, hage e, karlog o. light and electron microscopic studies of the acute and chronic toxic effects of n-nitroso compounds on the marine mussel, mytilus edulis (l.). ii. nmethyl-n-nitro-n-nitrosoquanidine. aquat. toxicol. 3: 301-311, 1983b. rasmussen lpd, hage e, karlog o. light and electron microscopic studies of the acute and 103 long-term toxic effects of n-nitrosodipropylamine and n-methylnitrourea on the marine mussel mytilus edulis. mar. biol. 85: 55-65, 1985. regoli f, principato g. glutathione, glutathionedependent and antioxidant enzymes in mussel, mytilus galloprovincialis, exposed to metals under field and laboratory conditions: implications for the use of biochemical biomarkers. aquat. toxicol. 31: 143-164, 1995. regoli, f, winston gw. application of a new method for measuring the total oxyradical scavenging capacity in marine invertebrates. mar. environ. res. 46: 439-442, 1998. reglinski j, paterson de, latimer s, campbell jm, wilson r, porter d, sturrock rd, smith we. myocrisin-mediated oxidative stress. clin. chim. acta 268: 85-99, 1997. ribera d, narbonne jf, michel x, livingstone dr, o’hara s. response of antioxidants and lipid peroxidation in mussels to oxidative damage exposure. comp. biochem. physiol. 100c: 177181, 1991. rice-evans ca, diplock at, symons mcr. techniques in free radical research. in: burton rh, van knippenberg ph (eds), laboratory techniques in biochemistry and molecular biology, elsevier science publishers bv, amsterdam, 1991. ringwood ah, conners de, keppler cj, dinovo aa. biomarker studies with juvenile oysters (crassostrea virginica) deployed in-situ. biomarkers. 4: 400-414, 1999. rodriguez-ariza a, martinez-lara e, pascual p, pedrajas jr, abril n, dorado g, toribio f, barcena ja, peinado j, pueyo c, et al. biochemical and genetic indices of marine pollution in spanish littoral. sci. total environ. (suppl. pt 1) : 109-116, 1993. rubbo h, radi r, anselmi d, kirk m, barnes s, butler j, eiserich jp, freeman ba. nitric oxide reaction with lipid peroxyl radicals spares [alpha]tocopherol during lipid peroxidation. greater oxidant protection from the pair nitric oxide/[alpha]-tocopherol than [alpha]tocopherol/ascorbate. j. biol. chem. 275: 1081210818, 2000. sagar s, kallo ij, kaul n, ganguly nk, sharma bk. oxygen free radicals in essential hypertension. mol. cell biochem. 111: 103-108, 1992. sen ck, packer l. antioxidant and redox regulation of gene transcription. fed. am. soc. exp. biol. j. 10: 709-720, 1996. seo mk, kang sw, kim k, baines ic, lee th, rhee sg. identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. j. biol. chem. 275: 20346-20354, 2000. shacter e, williams ja, lim m, levine rl. differential susceptibility of plasma proteins to oxidative modification: examination by western blot immunoassay. free radic. biol. med. 17: 429437, 1994. sheehan d, power a. effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs. comp. biochem. physiol. 123c: 193-199, 1999. sies h. oxidative stress: introduction. in: sies h ( ed), oxidative stress, oxidants et antioxidants, academic press publisher, san diego, pp 1-15, 1991. snell k, mullock b. biochemical toxicology. a practical approach, irl press, oxford university press, oxford, 1987. sohal rs, arnold la, sohal bh. age-related changes in antioxidant enzymes and prooxidant generation in tissues of rat with special reference to parameters in two insect species. free radic. biol. med. 10: 495-500, 1990. solé m, porte c, albaiges j. seasonal variation in the mixed-function oxidase system and antioxidant enzymes of the mussel mytilus galloprovincialis. environ. toxicol. chem. 14: 157-164, 1995. spallholz je, boylan lm. glutathione peroxidase: the two selenium enzymes. in peroxidases in chemistry and biology, pp. 259-272, 1991. spencer jpe, jenner a, aruoma oi, cross ce, wu r, halliwell b. oxidative dna damage in human respiratory tract epithelial cells. time course in relation to dna stret breakage. biochem. biophys. res. commun. 224: 17-22, 1996. stegeman jj, brouwer m, di giulio rt, förlin l, fowler ba, seters bm, van veld pa. molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure et effect. in: huggett rj, kimerle ra, mehrle jpm, bergman hl (eds), biomarkers biochemical, physiological, and histological markers of anthropogenic stress, lewis publishers, chelsea, pp 235-335, 1992a. stegeman jj, brouwer m, digiulio rt, forlin l, fowler ba, seters bm, van veld pa. enzyme and protein synthesis as indicators of contaminant exposure, in biomarkers. in: kimerle rj, kimerle a, merle jpm, bergmans hl (eds), biochemical, physiological and histological markers of anthropogenic stress, lewis publishers, boca raton, pp 235-271, 1992b. steinberg d. low density lipoprotein oxidation and its pathobiological significance. j. biol. chem. 272: 20963-20966, 1997. sukalski ka, laberge tp, johnson wt. in vivo oxidative modification of erythrocyte membrane proteins in copper deficiency. free radic. biol. med. 22: 835-842, 1997. sundaresan m, yu zx, ferrans vj, irani k, finkel t. requirement for generation of h2o2 for plateletderived growth factor signal transduction. science 270: 296-299, 1995. taylor cg, bettger wj, bray tm. effect of dietary zinc or copper deficiency on the primary free radical defence system in rats. j. nutr. 118: 613-621, 1988. uchida k. 4-hydroxy-2-nonenal: a product and mediator of oxidative stress. progr. lipid res. 42: 318-343, 2003. ursini f, maiorino m, valente m, oregolin c. purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity onphosphatidylcholine hydroperoxides. biochim. biophys. acta 710: 197, 1982. 104 vethaak ad. diseases of flounder (platichthys flesus l.) in the dutch wadden sea, and their relation to stress factors. netherlets j. sea res. 29: 257271, 1992. viarengo a, canesi l, pertica m, livingstone dr. seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels. comp. biochem. physiol. 100c: 187-190, 1991. viarengo a, canesi l, pertica m, poli g, moore mn, orenesu m. heavy metal effects on lipid peroxidation in the tissues of mytilus galloprovincialis lam. comp. biochem. physiol. 97c: 37-42, 1990. viarengo a, pertica mc, biasi f, cecchini j, orunesu m. effects of heavy metals on lipid peroxidation in mussel tissues. mar. environ. res. 24: 354, 1988. viarengo a, pertica m, canesi l, accometo r, mancinelli g, orunesu m. lipid peroxidation and level of antioxidant compounds (gsh, vitamin e) in the digestive glets of mussels of three different age groups exposed to anaerobic and aerobic conditions. mar. environ. res. 28: 291-295, 1989. wattanapitayakul sk, bauer ja. oxidative pathways in cardiovascular disease: roles, mechanisms, and therapeutic implications. pharmacol. ther. 89: 187-206, 2001. wenning rj, di giulio rt, gallaghe, ep. oxidant mediated biochemical effects of paraquat on the ribbed mussel, geukensia demissa. aquat. toxicol. 12: 157-170, 1988. weiland je, davis wb, holter jf, mohammed jr, dorinsky pm, gadek je. lung neutrophils in the adult respiratory distress syndrome. clinical and pathophysiologic significance. am. rev. respir. dis. 133: 218-225, 1986. weisiger ra, fridovich i. mitochondrial superoxide dismutase. site of synthesis and intramitochondrial localization. j. biol. chem. 248: 4793-4796, 1973. wilson r, ainscough r, eterson k, baynes c, berks m, bonfield j, burton j, connell, m, copsey t. cooper 2.2 mb of contiguous nucleotide sequence from chromosome iii of c. elegans. nature 368: 32-38, 1994. winstead jt, couch ja. enhancement of protozoan pathogen (perkinsus marinus) infections in american oysters, crassostrea virginica, exposed to the chemical carcinogen n-nitrosodiethylamine (dena). dis. aquat. org. 5: 205-213, 1988. winston gw, di giulio rt. prooxidant and antioxidant mechanisms in aquatic organisms. aquat. toxicol. 19: 137-161, 1991. wong pc, pardo ca, borchelt dr, lee mk, copelet ng, jenkins na, sisodia ss, clevelet dw, price dl. an adverse property of a familial als-linked sod1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. neuron 14: 1105-1116, 1995. wong gh, elwell jh, oberley lw, goeddel dv. manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. cell 58: 923-931, 1989. yevich pp, parszcz ca. neoplasia in soft shell clams (mya arenaria) collected from oil impacted sites. ann. n.y. acad. sci. 298: 409-426, 1977. yim mb, chock pb. stadtman copper-zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxyde. proc. natl. acad. sci. usa 87: 5006-5010, 1990. isj 4: x-y, 2007 isj 4: 18-23, 2007 issn 1824-307x research report identification of a putative rnase iii (dicer homolog) gene in silkworm bombyx mori km ponnuvel, b mohana sundari, r saravana kumar, rk sinha, ck kamble biotechnology laboratory, central sericultural germplasm resources centre pb no 44, thally road, hosur 635 109, india accepted february 26, 2007 abstract like other invertebrates, silkworms also encounter a problem from microbial infection including from rna viruses. in insects, rna interference acts as a natural anitiviral response to rna virus infection. especially in drosophila, it is proved that dicer mediated rna interference directs innate immunity against rna viruses. this information prompted us to identify similar rnase iii (dicer) gene in mulberry silkworm bombyx mori. the drosophila dicer gene was blast-searched with b. mori genome and single contig (genbank accession n° aadk01001038) showed maximum homology with dicer gene, through which the rnase iii gene sequence was identified in the genome of silkworm b. mori. the rnase iii domain was present in the three regions with the length of 278 bp, 277 bp and 185 bp in the contig, possibly these three regions form exons. the primers were designed for three b. mori rnase iii regions and amplified through pcr. the region i was amplified only in pure mysore silkworm strain whereas all three regions were amplified in daizo strain. the pcr product sequences were translated and showed rnase iii domain with in the amplified product. the predicted b. mori rnase iii domain had phylogenetic relationship with other insect dicer genes. we presume that this rnase iii (dicer) would protect b. mori larvae from invading rna viruses, which exists in the other insects. key words: bombyx mori; rnase iii; dicer; domain __________________________________________________________________________________________ introduction mulberry silkworm bombyx mori, is highly susceptible to various diseases which adversely affects the silk production. among silkworm diseases, viral diseases are very severe in tropical countries due to unfavorable climatic conditions coupled with improper hygiene in rearing environment (subbarao et al., 1991). b. mori nuclear polyhedrovirus (bmnpv) is a major viral pathogen responsible for grasserie disease followed by other viral pathogens like cytoplasmic polyhedrovirus (cpv) and infectious flacherie virus (ifv) which are known to equally contribute to silkworm crop loss (samson et al., 1990). there are many reports on b. mori immune response against invading microbial pathogens especially against bacterial pathogens (ponnuvel and yamakawa, 2002). ___________________________________________________________________________ corresponding author: km ponnuvel biotechnology laboratory central sericulture germplasm resources centre pb no 44, thally road hosur 635 109, tamil nadu, india email: kmpvel@yahoo.com however, the information on viral disease resistance remains scanty. the red florescence protein, lipases and serine protease are involved in antiviral response especially against bmnpv, a dna virus (ponnuvel et al., 2003). at present, the molecular immune response existing in b. mori against rna viruses has not been reported. among silkworm viral pathogens, cytoplasmic polyhedrovirus (cpv) and infectious flacherie virus (ifv) are rna viruses. in cpv, the genome is a segmented double strand rna where as ifv is a positive single strand rna virus and both viruses do not have dna stage in their replication cycle. the ifv belongs to the picorna virus and rna dependent rna polymerase (rdrp) of the virus helps viral replication that converts genomic single strand rna into a double stranded rna (isawa et al., 1997). these two viruses possess the double stranded rna in their genome or in the replication stage. rnase iii is a double stranded rna-specific endonuclease. in prokaryotes, rnase iii is important in post-transcriptional control of mrna 18 stability and translational efficiency. it is involved in the processing of ribosomal rna precursors. prokaryotic rnase iii also plays a role in the maturation of trna precursors and in the processing of phage and plasmid transcripts (kharrat et al., 1995; nicholson 1999). while, the eukaryotic rnase iii's participate (through direct cleavage) in rrna processing, in processing of small nucleolar rnas (snornas) and snrnas (components of the spliceosome). in eukaryotes, rnase iii enzyme such as dicer is involved in rnai (rna interference) and mirna (micro-rna) gene silencing (hannon, 2002). eukaryotes have evolved many different systems to resist virus infection. identification of specific virus encoded molecules or recognition of nucleic acid structures that are present only in infected cells could induce antiviral responses (lichner et al., 2003). as long double-stranded (ds) rnas do not occur in the cytoplasm of eukaryotic cells, the accumulation of ds replicative intermediates of rna viruses, like cytoplasmic polyhedrovirus (cpv) and infectious flacherie virus (ifv) could activates antiviral responses as rna interference (rnai) or translation inhibition and apoptosis. rnai is an ancient defence mechanism that degrades dsrnas and cognate mrnas in a sequence-specific manner (bernstein et al., 2001, zamore et al., 2000). viral dsrnas are first processed by an rnase iii-like nuclease (dicer) into 21–26 nt dsrnas (sirnas) that guide another nuclease complex (risc) to cleave homologous single-stranded (ss) viral rnas (ding, 2000). sirnas also serve as guides for an rna-dependent rna polymerase to transform the target ssrna into dsrna (fire et al., 1998; vance and vauchert, 2001). rnai was shown to act as an efficient antiviral system in plant and insect cells (adelman et al., 2001; li et al., 2002) and might also play an antiviral role in mammalian cells. cell-autonomous rnai generates an unidentified mobile signal, thereby directing sequence-specific rna degradation in distant tissues (baulcombe, 1999). to inhibit the antiviral effect of rnai, plant (anandalakshmi et al, 1998) and insect (li et al., 2002) viruses express different rnai suppressor proteins. the above findings clearly indicate existence of host virus interaction and sequence specific antiviral defence mechanism against rna viruses in eukaryotic organisms. numerous experiments have already proved rnai activity in silkworm b. mori, whereas the characterization and sequence analysis of dicer gene is yet to be studied. the present paper describes identification and genomic organization of a putative rnase iii (dicer) gene in silkworm b. mori. materials and methods silkworm strains selected two multivoltine silkworms namely, pure mysore and daizo were selected in the present work. these two strains are polyvoltine breeds and showed tolerant response to different silkworm diseases. identification of dicer gene homolog in drosophila the dicer gene was already identified and their domains were well predicted (lee et al., 2004). the dicer gene sequence was blast (tblastn) searched with b. mori genomic dna database for identification homologous sequence of dicer gene. the genomic dna sequence showing homologous sequence to drosophila rnase iii gene was identified and subsequently translated to determine putative amino acid sequence. the amino acid sequence was further analysed through conserved domain search for the presence of the rnase iii domain in dicer protein. selection of primers the up and down gene specific primers were designed for all three rnase iii domains existing in the b. mori genomic contig using the software programme of primer3 (http://frodo.wi.mit.edu/cgibin/ primer3). based on the software programme the primer binding site and the pcr product size were determined. the three different regions located in the genomic contig were amplified by pcr using genomic dna as template. the forward primer in rnaseregion i was (5’-tgtttgaaggttgggacagc–3’) and reverse primer was (5’-cgaaaataggac acgcgaaa–3’). similarly in rnase iii, the region ii forward primer used was (5’-ggagaaagcacggt ttgataa-3’) and reverse primer was (5’-cgcg ttcaaacaaacaaact-3’), while in region iii forward primer was (5’-cgaactgttgatgctaag acca) and reverse primer was (5’-cctcagcgc gtaacagtaca-3’). the pcr products were then cloned into the ta cloning vector with m13 primer sequence in both 5’ and 3’ ends. pcr and analysis of amplified product the genomic dna was isolated from silk moths using standard protocols and used as template dna in pcr reactions (nagaraja and nagaraju 1995). the reaction was done in an mj research thermal cycler, ptc200, using 20 μl reaction mixture containing 50-100 ng of genomic dna as template, 2.0 μl of 10xpcr buffer, 0.2 mm dntps, 1.5 mm mgcl2, 66 ng of forward and reverse primer each and 0.3 u of taq dna polymerase (mbi fermentas). the pcr schedule was 94 °c for 3min followed by 30 cycles of 94 °c for 30 s, 50 °c for 30 s, 72 °c for 2 min and a final extension of 7 min at 72 °c. the pcr products were resolved on 1.5 % agarose gel in tris-acetic acid/edta buffer (1xtae) and electrophoresis was carried out with a constant voltage of 80 v in parallel with molecular weight markers. gel was stained with ethidium bromide (0.5 μg/ml) and photographed with gel documentation. dna sequencing the pcr amplified products were purified through gel-spin column (bangalore genei). in sequencing reaction, the gene specific positive sense primer was used. 19 table 1 rnase iii (dicer) gene biological significance results and discussion in invertebrates, receptors of the innate immune system recognize pathogen-associated molecular patterns in order to activate early defense mechanisms (hoffmann, 2003). in insects, innate immune recognition plays an important role in clearing the majority of invading pathogens through activation of the toll and immune deficiency (imd) pathways. although the b. mori immune system has been well studied in the context of antibacterial response, there is no information at the cellular or molecular level regarding the immune response directed against rna viruses. keene et al. (2004) hypothesized that rnai may also act as an antagonist to alphavirus replication in anopheles gambiae because rna viruses form dsrna during replication and they proved that rnai is a mechanism to protect mosquitoes from viral infection. silencing agago2 expression would make a. gambiae mosquitoes more permissive to virus infection. the findings gave direct evidence that rnai is an antagonist of o’nyong-nyong virus replication in a. gambiae. in india, flacherie disease of the silkworm b. mori is a major factor causing serious loss in cocoon production to sericulture farmers. based on pathological symptoms, the causative agent of this disease is an infectious flacherie virus (ifv), which is an insect picorna virus (isawa et al., 1997). the target of ifv is the goblet cells of the midgut epithelium, and the virus multiplies in the cytoplasm. the complexity of the biological properties of dsrna in vivo became more apparent with the discovery of dsrna mediated post-transcriptional gene silencing (ptgs or rna interference [rnai]). the phenomenon has been described in both plants and animals, including nematodes, insects, and mammals (hannon, 2002). the sequence-specific effects of dsrna that result in endogenous rna degradation are widely conserved and probably present in most invertebrates including b. mori. wang et al (2006) selected drosophila model for studying the innate immunity against rna viruses in animals. they demonstrated that a rna interference pathway protects adult flies from infection by two evolutionarily diverse viruses. their findings describes a molecular framework for the viral immunity, in which viral double stranded rna produced during infection acts as the pathogen trigger, whereas drosophila dicer-2 and argonaute2 act as host sensor and effector respectively (lee et al., 2004). our paper reports that b. mori genome possess rnase iii (dicer) gene sequence and has maximum homology to drosophila dicer. further, the rnase iii domain of b. mori is conserved from plant to animal and it indicates that antiviral immunity in insects shares some of the molecular features of vertebrate antiviral responses. initially the drosophila sequence was compared with genomic dna database of silkworm b. mori. it was found that a single genomic contig (genbank accession n° aadk01001038) possessing the dicer gene homologous sequence (table 1). further, the genomic contig was analyzed for the presence of rnase iii domain using conserved domain search. using the program, distinct functional and structural unit of the translated protein was identified (http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb/cgi). the protein query sequence submitted to ncbi's protein blast search service was scanned for conserved domain signatures and results showed fig. 1 putative exons and introns of rnase iii gene present in silkworm genome (genomic contig accession no: aadk01001038). the arrow mark indicate forward and reverse primer binding site target genes nature of protein biological activity expressed in contig genbank accession n° dicer rnase iii development, genome organization, viral and transposon defense ubiquitous aadk01001038 20 table 2 details of genomic contig and amplicon size of dicer gene in silkworm bombyx mori target genes primer sequence (5’-3’) primer binding location in genomic contig. amplicon size rnase iii domain region corresponding dna contig accession number forward primer tgtttgaaggttgggacagc 451 bp rnaseregion i reverse primer cgaaaataggacacgcgaaa 859 bp 398 bp 523 bp to 801 bp ef117689 forward primer ggagaaagcacggtttgataa 19214 bp rnaseregion ii reverse primer cgcgttcaaacaaacaaact 19613 bp 400 bp 19335 bp to 19562 bp ef117690 forward primer cgaactgttgatgctaagacca 20797 bp rnaseregion iii reverse primer cctcagcgcgtaacagtaca 21176 bp 358 bp 20936 bp to 21121 bp ef117691 the rnase iii domain was present in the translated protein sequence. it was found that rnase iii domain was present in the contig in the three different regions, which were designated as region i, region ii and region iii. the first region was present from 523 bp to 801 bp whereas other two regions were at 19335 bp to 19562 bp and 20936 bp to 21121 bp (fig. 1, table 2). we believe that all three domains are partial and are scattered in the different exons and after splicing, it may form a dicer enzyme with single domain of rnase iii. in order to confirm the splicing the all three domains were joined together and analyzed for the conserved domain search and it was found that it formed a single protein with rnase iii domain (fig. 2). further, the primers were designed for the putative rnase iii regions based on the sequence available in the all three regions present in the single contig (genbank accession n° aadk01001038). in order to amplify rnase iii domain present in the genomic contig, the pcr was performed with three sets of primers, which were binding site at region i (451f – 859r), region ii (19214f – 19613r) and region iii (20797f – 21176r) (fig. 1). the primers designed in region i yield the pcr product of 398 bp, region ii amplified pcr product size is 400 bp, while region iii pcr product showed molecular weight of 358 bp (table 2). the first region was amplified in pure mysore strain, while the remaining two regions were not amplified due to lack of primer binding sequence in pure mysore. this is due to the fact that genomic dna database was derived from the silkworm strain of daizo and pure mysore strain sequence may not have homologous sequence with daizo. hence, the genomic dna of daizo was used as template and as a result all three regions were amplified in silkworm daizo strain (fig. 3). this finding indicates that the identified rnase iii is located in specified manner in genomic dna, presumably in a single chromosome. uhlirova et al. (2003) used a recombinant sindbis virus as a tool to silence brc expression in the silkmoth bombyx mori. the virus expressing a br-c antisense rna fragment reduced endogenous br-c mrna levels in infected tissues (adult wing and leg primordia) via rna interference (rnai). their findings support that the b. mori also have the rnase iii enzyme which degrade double stranded target rna in a sequence specific manner. the amplified pcr fragment was further sequenced to confirm the presence of rnase iii domains. the sequence was translated and also analyzed for the presence of rnase iii domain in the translated region. it clearly indicates that rnase iii domains exist in the genome of silkworm strains of pure mysore and daizo. the putative amino acid sequence was further analysed for phylogenetic relationship with other eukaryotic organisms. the rna silencing is rna guided gene regulatory mechanism that include post transcriptional gene silencing (pgts) and it is conserved from fission in yeast, plants to animals (ding et al., 2004). phylogenetic analysis was performed using clustalw and mega3 programmes and results showed that the bombyx rnase iii is strongly aligned with rnase iii sequences of insects, especially in tribolium castaneum, a red flour beetle (bucher et al., 2002). robaino et al. (2004) demonstrated that in invertebrate immune system, fig. 2 pcr products amplified in all three regions were joined together, analyzed for the conserved domain in translated amino acid sequence and formed a single protein with rnase iii domain of bombyx mori 21 fig. 3 pcr amplification of rnase iii gene in silkworm bombyx mori like its vertebrate counterparts, could recognize dsrna as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response. interestingly, all the vertebrate rnase iii formed a major group and plant rnase iii gene is more close to bacterial rnase iii. down regulation of dicer makes insects vulnerable to get viral infection (wang et al., 2006). all these findings clearly indicate that dicer enzymes are involved in disease resistant mechanism of insects, especially against rna viruses. silkworms are also known to get infection from rna viruses like cpv and ifv and it is reasonable to expect rnai mediated disease resistant mechanism may also prevail in b. mori. in future studies, the disease resistant mechanism of b. mori against the rna viruses will be deciphered by designing dsrna against b. mori dicer and other proteins involved in risc complex. nucleotide sequence accession number the nucleotide sequences of rnase iii regions have been deposited with ncbi, ddbj and embl libraries under accession n° ef117689, ef117690 and ef117691. acknowledgment this research work was supported by grants from department of biotechnology, ministry of science and technology, government of india (bt/pr4503/pbd/19/099/2003 dated 26th march 2004). references adelman zn, blair cd, carlson jo, beaty bj, olson ke. sindbis virus induced silencing of dengue viruses in mosquitoes. insect mol. biol. 10: 265273, 2001. anandalakshmi r, pruss gj, ge x, marathe r, mallory ac, smith th, et al. a viral suppressor of gene silencing in plants. proc. natl. acad. sci. usa 95: 13079-13084, 1998. baulcombe d. viruses and gene silencing in plants. arch. virol. suppl. 15: 189201,1999. bernstein e, caudy aa, hammond sm, hannon gj. role for a bidentate ribonuclease in the initiation step of rna interference. nature 409: 363-366, 2001. bucher g, scholten j, klingler m. parental rnai in tribolium (coleoptera). curr. biol. 12: 85-86, 2002. ding s w. rna silencing. curr. opin. biotechnol. 1: 152-156, 2000. ding sw, li h, lu r, li f, li wx. rna silencing: a conserved antiviral immunity of plants and animals. science 102: 109-115, 2004. fire a, xu sq, montgomery mk, kostas sa, driver se, mello cc. potent and specific genetic interference by double-stranded rna in caenorhabditis elegans. nature 391: 806-811, 1998. hannon gj, rna interference. nature, 418: 224251, 2002. hoffmann ja. the immune response of drosophila. nature 426: 33-38, 2003. isawa h, asano s, sahara k, iizuka t, bando h. analysis of genetic information of an insect picorna-like virus, infectious flacherie virus of silkworm: evidence for evolutionary relationships among insect, mammalian and plant picorna (-like) viruses. arch. virol.143: 127-143, 1997. keene km, foy bd, vargas is, beaty bj, blair cd, olson ke. rna interference acts as a natural antiviral response to o’ nyong-nyong virus infection of anopheles gambiae. proc. natl. acad. sci. 101: 17240-17245, 2004. kharrat a, macias mj, gibson tj, nilges m, pastore a. structure of the dsrna binding domain of e. coli rnase iii. embo j. 14: 3572-3584 1995 lee ys, nakahara k, pham jw, kim k, zhengying he, sontheimer ej, et al. distinct roles for drosophila dicer-1 and dicer-2 in the sirna/mirna silencing pathways. cell 117: 6981, 2004. li hw, li wx, ding sw. induction and suppression of rna silencing by an animal virus. science 296: 1319-1321, 2002. lichner z, daniel s, burgyan j. double-stranded rna-binding proteins could suppress rna interference-mediated antiviral defences. j. gen. virol. 84: 975-980, 2003. nagaraja gm, nagaraju j. genome fingerprinting of the silkworm bombyx mori using random arbitrary primers. electrophoresis 16: 16331638, 1995. nicholson aw. function, mechanism and regulation of bacterial ribonucleases. fems microbiol. rev. 23: 371-390,1999. ponnuvel km, nakazawa h, furukawa s, asaoka a, ishibashi j, tanaka h, et al. a lipase isolated from the silkworm bombyx mori shows antiviral activity against nucleopolyhedrovirus. j. virol. 77: 10725-10728, 2003 22 ponnuvel km, yamakawa m. immune resposes against bacterial infection in bombyx mori and regulation of host gene expression. curr. sci. 83: 447-454, 2002. samson mv, baig m, sharma sd, balvenkatasubbaiah to, jolly ms. survey on the relative incidence of silkworm diseases in karnataka. indian j. sericult. 29: 248-254, 1990. subbarao g, chandra ak, bhattacharya j. incidence of crop loss from adopted rearers level in west bengal due to silkworm diseases. indian j. sericult. 34: 167-170, 1991. robaino j, browdy cl, prior s, metz a, parnell p, gross p, et al. induction of antiviral immunity by double-stranded rnai in a marine invertebrate. j. gen. virol. 78: 10442-10448, 2004. uhlirova m, foy bd, beaty bj, olson ke, riddiford lm, jindra m. use of sindbis virus-mediated rna interference to demonstrate a conserved role of broad-complex in insect metamorphosis. proc. natl. acad. sci, usa 100: 15607-15612, 2003. vance v, vaucheret h. rna silencing in plants defense and counter defense. science 292: 2277-2280, 2001. wang xh, aliyari r, li wx, li hw, kim k, carthew r, et al. rna interference directs innate immunity against viruses in adult drosophila. science 312: 452-454, 2006. zamore pd, tuschl t, sharp pa, bartel dp. rnai: double-stranded rna directs the atpdependent cleavage of mrna at 21 to 23 nucleotide intervals. cell 101: 25-33, 2000. 23 abstract materials and methods isj 3: 89-96, 2006 isj 3: 103-110, 2006 issn 1824-307x minireview the multiple functions of the pgrp family in drosophila immunity a goto, s kurata graduate school of pharmaceutical sciences, tohoku university, japan accepted november 13, 2006 abstract the innate immune system discriminates between infectious non-self and self using germ-lineencoded pattern recognition receptors (prrs) that are highly conserved from insects to mammals. peptidoglycan recognition protein (pgrp) is one of the hallmark pattern recognition receptors responsible for detecting unique bacteria-derived peptidoglycans. the pgrp family comprises several members (13 in drosophila, 7 in anopheles, and 4 in mammals) and are differentially expressed on immune-responsive organs. some pgrps have amidase or bactericidal activities and function as immune modulators, whereas others have lost their enzymatic activity, but still have crucial roles in the activation of innate immune signaling. evidence from recent drosophila studies suggests that pgrps have a role in a variety of immune reactions, such as in the activation of the prophenoloxidase cascade, the production of antimicrobial peptides through the activation of the toll and imd pathways, intracellular bacteria recognition, and phagocytosis. key words: innate immunity; pgrp (peptidoglycan recognition protein); prr (pattern recognition receptor); pamp (pathogen-associated molecular pattern); amidase; antimicrobial peptide; phagocytosis __________________________________________________________________________________________ introduction insects are diverse group of organisms and comprise more than 80 % of the animal kingdom. to prevail as such a vast majority of species among other organisms, insects have developed excellent defense mechanisms against bacterial infection. in contrast to adaptive immunity, which requires highly specific receptors created by somatic gene rearrangement, the innate immune system is a defense mechanism that exploits germ-line encoded gene products. the principal mechanisms of innate immunity are difficult to study in vertebrates largely due to the effects of the adaptive immune response. thus, insects have become a favorite model system because, as invertebrates, they depend solely on the innate immune system to fight off infection. the powerful genetic and molecular techniques with the complete genome sequencing make drosophila an attractive model for deciphering the precise mechanisms of the innate immune response (hoffmann, 2003; hultmark, 2003). ___________________________________________________________________________ corresponding author: shoichiro kurata graduate school of pharmaceutical sciences, tohoku university, sendai 980-8578, japan e-mail: kurata@mail.pharm.tohoku.ac.jp although the innate immune system is composed of multiple complex processes including cellular and humoral responses, much of the attention in drosophila has focused on humoral reactions. the primary response is the most immediate response (within minutes) against microorganism invasion and depends on constitutively present endogenous molecules in the hemolymph, such as coagulation factors, cell surface recognition receptors or prophenoloxidase (propo) for wound healing, phagocytosis and melanization which confine microorganisms in small spaces, respectively (theopold et al., 2003; goto et al., 2003; ashida, 2004; kurata et al., 2006). shortly after the primary response, the secondary response begins and ultimately leads to the production of antimicrobial peptides (amps) from the fat body, the functional equivalent of the mammalian liver. to date, there are seven known distinct antimicrobial peptides in drosophila and they are basically small cationic molecules with specific membraneattackable bactericidal activity against various types of microorganisms. the transcriptional regulation of amp expression is under the control of two distinct pathways, the toll and immune deficiency (imd) pathways (kurata, 2004; kurata et al., 2006). the toll pathway was originally identified as a 103 mailto:kurata@mail.pharm.tohoku.ac.jp signaling cascade involved in dorsoventral patterning in embryos (morisato and anderson, 1995; roth, 2003). subsequent genetic analysis serendipitously led to the discovery that it also controls the expression of the antifungal peptide drosomycin, which is predominantly triggered by gram-positive or fungal infection (lemaitre et al., 1996). activation of the toll pathway is initiated through the binding of the proteolytically cleaved cytokine spaetzle to the toll receptor, which then transduces its signal to the cells. in the cell, a toll receptor-adaptor complex composed of dmyd88, tube, pelle, and other unidentified factors induces the phosphorylation and degradation of the ankyrinrepeat inhibitor protein cactus. the nuclear factor nf-κb protein dif, which is normally retained in the cytoplasm by binding to cactus, is then translocated into the nucleus and triggers the expression of the antifungal peptide drosomycin together with numerous effector molecules (hoffman and reichhart, 2002; hoffmann, 2003). this discovery unambiguously led to the identification of the tolllike receptor (tlr) family in mammals (takeda et al., 2003; royet et al., 2005). in contrast, the imd pathway is predominantly activated by gram-negative bacterial infection. while analyzing the expression of antibacterial genes in a phenoloxidase cascade mutant, imd was serendipitously discovered as the first recessive mutation that impairs the inducibility of all antibacterial peptides in the drosophila immune response (lemaitre et al., 1995). six years later, the imd gene product was identified. it is a 25-kda protein containing a death domain with significant sequence similarity to that of the mammalian tumor necrosis factor-receptor interacting protein rip (georgel et al., 2001). activation of the imd pathway triggers the kinase cascade of dtak (a map 3 kinase homologue) and the ikk complex, and ultimately leads to phosphorylation of the rel protein relish (leulier et al., 2002; naitza et al., 2002; vidal et al., 2001). phosphorylated relish is then cleaved by dredd (a mammalian homolog of caspase-8), and its dna binding domain leaving the i-κb domain in the cytoplasm is translocated into the nucleus and triggers amp expression (leulier et al., 2000; stoven et al., 2003). thus, over the past decade, our understanding of drosophila innate immunity has dramatically centered on the field of intracellular signaling mechanisms. there remain crucial questions, however, about how the innate immune system recognizes microorganisms, and discriminates between self and non-self infections, and how the signals from outside the cells subsequently reach the cognate innate receptors to activate the toll or imd pathway. the discovery of pattern recognition receptors (prr) such as peptidoglycan recognition proteins (pgrps) that recognize unique bacterial cell component peptidoglycans (pgns) led to our deeper understanding of the innate immune recognition concept (medzhitov and janeway, 2002; royet, 2004). here, we review recent advances in the understanding of the multifunctional pgrp family, a representative pattern recognition molecule, in drosophila. discovery of the pgrp family the name ”peptidoglycan recognition protein” was first introduced by ashida’s group (yoshida et al., 1996). they purified a 19-kda protein from the hemolymph and cuticles of silkworm (bombyx mori) and demonstrated that it binds to gram-positive bacteria and pgn, and activates the prophenoloxidase cascade (yoshida et al., 1996). later, its corresponding cdna was cloned (ochiai et al., 1999). pgrp gene homologs from a moth (trichoplusia ni) and subsequently from mouse and human were in turn cloned, indicating that the pgrp family is highly conserved from insects to mammals (kang et al., 1998). completion of various genome projects (e.g., drosophila melanogaster, anopheles gambiae, human, mouse, etc.) also boosted the discovery of diverse pgrp family. the drosophila genome encodes at least 13 pgrp family members, which are subdivided into seven short transcripts (pgrp-s; ~200 amino acids long) and six long transcripts (pgrp-l; from 200 to 600 amino acids long). the short transcripts are predicted to be secreted molecules due to the presence of signal peptides and are induced in response to bacterial infection, whereas the long transcripts are constitutively present on the cell surface or integrated into the plasma membrane, except for pgrp-lb (werner et al., 2000). extracellular recognition by the pgrp family pgrp roles in the immune response a significant breakthrough with regard to the role of pgrp in the immune response came through a large-scale ethylmethane sulfonate mutagenesis screen from drosophila. michel et al isolated a mutation called semmelweis (seml), named after the hungarian physician ignaz phillipp semmelweis who is a pioneer of antiseptic treatments (celine, 1999; michel et al., 2001). the pgrp-saseml mutants lead to defects in amp expression and render flies more susceptible to several gram-positive bacterial infections. the mutated region was found to be on a highly conserved cysteine residue that is changed to a tyrosine in pgrp-sa. hemolymph transfer experiments revealed that pgrp-sa is secreted into the hemolymph. surprisingly, activation of the toll pathway by fungi and of the imd pathway by gram-negative bacteria is normal compared to wildtype, raising the possibility that there are other prrs specific for fungi and gram-negative bacteria (michel et al., 2001). consistent with this hypothesis, three independent groups identified pgrp-lc mutations (ird7 or totem) (choe et al., 2002; gottar et al., 2002; ramet et al., 2002). pgrp-lc mutant flies are highly susceptible to some gram-negative bacterial infections and do not express imd-mediated amp genes (such as diptericin and attacin). epistatic analysis indicates that pgrp-lc encoding a single transmembrane protein acts upstream of imd, suggesting that pgrp-lc functions in the activation of the imd pathway in response to gram-negative bacterial infection. pgrp-sa binds to lysine (lys)-type pgn 104 from gram-positive bacteria, whereas pgrp-lc binds to meso-diaminopimelic (dap)-type pgn from gram-negative bacteria, reinforcing the structural basis for the recognition mechanisms (leulier et al., 2003; kaneko et al., 2004). cooperative roles of pgrps for detecting pathogens curiously, the pgrp-lc mutant phenotypes are not as severe as that of ikk� or relish null mutants in terms of both amp expression and survival rates upon some gram-negative bacterial infections (gottar et al., 2002). accordingly, some gram-positive bacteria such as staphylococcus saprophyticus, staphylococcus aureus, or enterococcus faecalis induce normal activation of the toll pathway in a pgrp-sa mutant background, but this activation is completely blocked in dif mutants (bischoff et al., 2004). those findings led to the observation that the one to one prr/ pathogen associated molecular pattern (pamp) concept is inappropriate for innate immune sensing mechanisms. this interpretation was soon validated by the discovery of multi-functional pgrp-le, which contains a unique acidic domain, functions in the activation of the imd pathway and also triggers the propo cascade when it is ectopically expressed (takehana et al., 2002). more importantly, pgrplc/pgrp-le double mutants are more susceptible against gram-negative bacteria (e. coli) infection than either single mutant alone, suggesting that these two proteins cooperate to recognize gramnegative bacteria and lead to the activation of the imd pathway (takehana et al., 2004). similarly, in the case of gram-positive bacteria sensing, other cooperative receptor complexes are required upstream of toll. gram-negative bacteria binding protein (gnbp), which binds gram-negative bacteria, was first identified from silkworm (lee et al., 1996). gobert et al (2003) subsequently reported that gnbp1 mutants are not resistant against some gram-positive bacterial infections, showing a similar immune defective phenotype as the pgrp-sa mutant. the finding that gnbp1 physically interacts with pgrp-sa in the circulation to activate the toll pathway supports this observation (gobert et al., 2003; pili-floury et al., 2004; wang et al., 2006). moreover, the generation of another pgrp family member mutant, the pgrpsd loss-of-function mutant, has been reported (bischoff et al., 2004). pgrp-sd mutants are highly susceptible to some types of gram-positive bacterial infection, and this mutation exacerbates the pgrpsa and gnbp1 mutant phenotypes, suggesting that pgrp-sd is another gram-positive bacteria recognition molecule involved in the activation of the toll pathway. taken together, there are four known pgrp family members, pgrp-sa, -sd, -lc, and –le, that are crucial for microbe recognition and for activating the innate immune signaling pathways (toll, imd , and propo cascades) (fig. 1). fig. 1 extracellular recognition by pgrp family. in hemolymph, meso-diaminopimelic (dap)-type peptidoglycans (pgns) from gram-negative and some gram-positive bacteria (bacillus species) are recognized by pgrp-lc or pgrp-le and activate the imd pathway to trigger diptericin or attacin expression. in the case of lysine (lys)-type gram-positive bacterial infection, either pgrp-sc1a, -sd, -sa, gnbp1 alone or in combination is responsible for the activation of the toll pathway through binding of the endogenous ligand spaetzle (spz). 105 intracellular recognition by pgrp-le large-scale gain-of-function genetic screening using amp reporters led to the identification of pgrp-le. pgrp-le overexpression leads to constitutive activation of the imd pathway as well as the propo cascade, which ultimately leads to the formation of large melanotic tumors in the hemolymph (takehana et al., 2002). pgrp-le lossof-function mutants were isolated in subsequent studies 2 years after the identification of pgrp-le (takehana et al., 2004). the phenotypes of a single pgrp-le gene mutation are fairly weak in terms of their survival rates against e. coli infection and amp expression. however, when combined with the pgrp-lc mutation, the pgrp-le/pgrp-lc double mutants exhibit high susceptibility to e. coli and to other dap-type pgn-containing bacteria (takehana et al., 2004). this finding clearly suggests that pgrp-le synergistically or redundantly acts with pgrp-lc to activate the imd pathway. to gain a better understanding of the cooperative recognition mechanisms between pgrp-lc and pgrp-le, two independent groups recently conducted very exciting experiments using monomeric dap-type pgn tracheal cytotoxin (tct) (kaneko et al., 2006; lim et al., 2006). tct is a disaccharide-tetrapeptide fragment of pgn that contains a 1,6-anhydro-arranged muramic acid and a dap residue at the third position of the stem peptides (cookson et al., 1989), and is continuously released from gram-negative bacteria as a turnover product of the pgn in the cell wall (park, 2001). the presence of such a small diffusible monomeric pgn is an ideal signature for the innate immune recognition system by prrs, as the pgn layer of gram-negative bacteria is basically hidden under the outer lipopolysaccharide (lps) layers. in vitro cell culture studies demonstrated that tct potently activates the imd pathway via two different pgrplc isoforms: pgrp-lca and pgrp-lcx (kaneko et al., 2004). deletion mutant studies demonstrated that the rhim-like motif of pgrp-lc is responsible for the signaling, but is dispensable for the interaction with imd (kaneko et al., 2006; choe et al., 2005). whereas a single pgrp-lc or pgrp-le mutant responds normally to tct, e. coli pgn, or live e. coli, the response of pgrp-lc/pgrp-le double mutants is completely abolished. clonal expression studies demonstrated that pgrp-le acts in a cell-autonomous manner on some immune responsive tissues, such as malpighian tubes. direct delivery of tct into the cytosol by calcium phosphate transfection triggers an enhanced amp expression that is dependent on pgrp-le, suggesting that pgrp-le is a second receptor for tct (kaneko et al., 2006). this idea is supported by analysis of the crystal structure of pgrp-lepg (pgrp domain of pgrp-le)/tct complex, which indicates that tct strongly binds to pgrp-lepg with an apparent kd of 27 nm, and induces pgrp-le multimerization through head-to-tail dimer formation (lim et al., 2006). collectively, these results indicate that pgrple potentially has dual functions depending on its localization; on the outside of the cells, it cooperates with pgrp-lc through its pgrp domain and triggers the propo cascade, and inside the cells, it acts as an intracellular recognition receptor for activation of the imd pathway (fig. 2). fig. 2 recognition mechanisms of pgrp-le. monomeric dap-type pgns (tct) binds to the pgrp domain of pgrp-le. in the hemolymph, this complex triggers the propo cascade. tct can also bind to pgrp-lcx to form three potential types of receptor complexes (lepg/tct/lc, lcx/tct/lca or lcx/pgn/lcx) on the cell surface. the rhim-like motif of pgrp-lcx and lca is responsible for the activation of the imd pathway. the pgrp-le/tct complex also functions in intracellular recognition. immune modulator activities of the pgrp family a representative feature of the pgrp family is that all pgrps in insects and mammals have in common an approximately 160 amino acid-long pgrp domain with similarity to the bacteriophaget7 lysozyme, a zinc-dependent n-acetyl-muramyl-lalanine amidase (steiner, 2004; royet et al., 2005). among the 13 pgrp family members in drosophila, the first group is categorized as non-catalytic pgrps, which lack the zinc binding residues required for amidase activity, but retain the binding capacities to pgn (pgrp-sa, -sd, -la, -lc, ld, le, -lf). the second group is the catalytic pgrps, which have zinc-dependent amidase activity that either reduces or eliminates the biological activities of the pgns (pgrp-sc1a, -sc1b, -lb, -sb1, sc2). the first striking discovery of the potential role of catalytic pgrps in the drosophila immune response was reported for pgrp-sc1b (mellroth et al., 2003). in vitro studies performed by mellroth et al. (2003) demonstrated that recombinant sc1b hydrolyzes the lactylamide bond between the glycan strand and the stem peptides of pgn, and the degraded pgn has less immune stimulatory activity compared with undigested pgn, indicating that 106 pgrp-sc1b has scavenger activity. unexpectedly, chang et al. (2004), in the course of crystal structure analysis, demonstrated that recombinant pgrp-sa has l,d-carboxypeptidase activity that cleaves the dap-type muropeptide, but not the lystype compound. moreover, three independent groups reported the roles of catalytic pgrps: pgrp-sc1/2 (bischoff et al., 2006), pgrp-lb (zaidman-rémy et al., 2006), and pgrp-sb1 (mellroth and steiner, 2006). first, bischoff et al. (2006) used rna interference techniques to generate pgrp-sc1/2 loss of function mutants and reported that the mutants overactivate the imd pathway after bacterial infection, and feeding-induced infection by erwinia carotovora carotovora is enhanced compared to controls. thus, pgrp-sc1/sc2 might modulate the activation of the imd pathway in the gut, which is constantly threatened by bacterial infection (bischoff et al., 2006). the effects of phenotypes of recently isolated pgrp-sc1a mutants (picky) on the toll pathway are confusing with respect to the complex immune modulator mechanisms (garver et al., 2006). second, biochemical analysis indicates that pgrp-lb has specific amidase activity for daptype pgn, including tct. similar to the rnainterference generated pgrp-sc1/2 mutants, the analysis of the time course of amp expression induced by gram-negative bacteria or tct infection revealed a strong immune response by the rnainterference generated pgrp-lb mutants compared to wild-type controls (zaidman-rémy et al., 2006). therefore, they concluded that pgrp-lb negatively regulates the imd pathway and functions as a scavenger receptor for gram-negative bacterial pgn. finally, recent biochemical analysis demonstrated that pgrp-sb1 is an nacetylmuramoyl l-alanine amidase that preferentially hydrolyzes dap-type pgn. in contrast to pgrp-lb, pgrp-sb1 lacks enzymatic activity against tct. in addition, this report first demonstrated that pgrp-sb1 possesses bactericidal activity against b. megaterium (mellroth and steiner, 2006). taken together, these findings suggest that non-catalytic pgrps preferentially function in the detection of pgn, and catalytic pgrps serve as scavenger receptors to detoxify harmful pgn and modulate the signaling pathways, although catalytic pgrps that specifically cleave lys-type pgn have yet to be identified. roles of pgrps in phagocytosis phagocytosis is a phenomenon whereby invading microbes or altered self-like apoptotic or infected cells are engulfed and, eventually, digested by host cells called phagocytes (aderem and underhill, 1999). in drosophila, most humoral immune responses leading to toll and imd pathway activation are mainly mediated by the fat body, whereas cellular responses such as phagocytosis, encapsulation, or melanization are mediated by blood cells called hemocytes (hoffmann and reichhart, 2002). there are three known hemocyte types in drosophila. plasmatocytes account for approximately 90 % of hemocytes and are the main executors of phagocytosis. crystal cells, approximately 5% of the hemocyte population, contain a set of substrates and enzymes responsible for propo-mediated humoral melanization. lamellocytes, the third hemocyte type, show a large flattened morphology. under non-infected conditions, lamellocytes are absent, but once large invaders, such as wasps, are infected, lamellocytes are differentiated from the lymph glands, the main hemocyte-producing organs in larvae (meister and lagueux, 2003). in addition to several phagocytic receptors, such as croquemort (“catcher of death” ) (franc et al., 1996, 1999; stuart et al., 2005), scavenger receptor (sr) -ci (ramet et al., 2001; ulvila et al., 2006), peste (philips et al., 2005), eater (kocks et al., 2005), and dscam (watson et al., 2005), pgrp family members are also involved in phagocytosis. pgrp-lc was the first pgrp family member discovered to be involved in phagocytosis. a double-stranded (ds)rna interference-based screen of the drosophila macrophage cell line s2 identified 34 genes, including pgrp-lc, that are essential for phagocytosis. fluorescence activated cell sorting analysis indicated that s2 cells with pgrp-lc knock-down had substantially decreased phagocytic activity towards gram-negative bacteria e. coli, but not gram-positive bacteria s. aureus. they also demonstrated that long transcripts of the same types of pgrp family, pgrp-la or pgrp– ld, are not involved in phagocytosis (ramet et al., 2002). conversely, another group recently reported that pgrp-sc1a mutation (picky) impairs phagocytic ability s. aureus, but not e. coli or s. cerevisiae (yeast), based on the results of a forward genetic ethylmethane sulfonate screen. moreover, activation of the toll pathway is significantly impaired in pgrp-sc1a mutants. transgenic rescue experiments indicated that the amidase activity of pgrp-sc1a does contribute to toll pathway activation, but is essential for the uptake of s. aureus (garver et al., 2006). conclusion over the past 5 years, the concept of the prr/pamp theory originally introduced by charles janeway has been verified experimentally using versatile molecular and genetic strategies (janeway and medzhitov, 2002). in mammals, functional characterization of toll-like receptor family has considerably deepened our understanding of the recognition mechanisms of innate immunity (takeda and akira, 2005). in contrast, it seems that insect tolls are not involved in immune functions as pattern recognition receptors, but are more likely to have other functions. drosophila toll is a kind of cytokine receptor that is activated by the endogenous proteolytically cleaved ligand spaetzle (imler and hoffmann, 2002). now, the pgrp family members are coming to the forefront as molecular sensors against microbial infection in insects. recent functional characterization of the pgrp family members in drosophila has enabled us broaden the partial picture of pattern recognition mechanisms: 1) specific types of pgn (lys-type or dap-type) can be detected by different pgrps; 2) 107 catalytic amidase pgrps act as immune modulators; 3) microbe detection by pgrps triggers the activation of innate immune signaling pathways; and 4) pgrps possess receptor functions for phagocytosis. still, a fundamental question remains as to how the signals are transmitted into the cells to kill microbes after pgns are recognized by pgrps. further studies will address this issue to complete the picture of the prr/ pamp mechanisms. acknowledgement this work was supported by grants-in-aid for scientific research from the ministry of education, culture, sports, science, and technology of japan; the japan society for the promotion of science; the program for the promotion of basic research activities for innovative biosciences; and the naito foundation. references aderem a, underhill dm. mechanisms of phagocytosis in macrophages. annu. rev. immunol. 17: 593-623, 1999. ashida m. activation of prophenoloxidase cascade with peptidoglycan. tanpakushitsu kakusan koso 49: 1168-1173, 2004. bischoff v, vignal c, boneca ig, michel t, hoffmann ja, royet j. function of the drosophila pattern-recognition receptor pgrpsd in the detection of gram-positive bacteria. nat. immunol. 5: 1175-1180, 2004. bischoff v, vignal c, duvic b, boneca ig, hoffmann ja, royet j. downregulation of the drosophila innate immune response by peptidoglycan recognition proteins sc1 and sc2. plos pathog. 2: e14, 2006. celine lf. semmelweis. l'imaginaire. gallimard, paris, 1999. chang ci, pili-floury s, herve m, parquet c, chelliah y, lemaitre b, et al. a drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual l,dcarboxypeptidase activity. plos biol.2: e277, 2004. choe km, lee h, anderson kv. drosophila peptidoglycan recognition protein lc (pgrp-lc) acts as a single-transducing innate immune receptor. proc. natl. acad. sci. usa 102: 11221126, 2005. choe km, werner t, stoven s, hultmark d, anderson kv. requirement for a peptidoglycan recognition protein (pgrp) in relish activation and antibacterial immune responses in drosophila. science 296: 359-362, 2002. cookson bt, tyler an, goldman we. primary structure of the peptidoglycan-derived tracheal cytotoxin of bordetella pertussis. biochemistry 28: 1744-1749, 1989. franc nc, dimarcq jl, lagueux m, hoffmann j, ezekowitz ra. croquemort, a novel drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. immunity 4: 431-443, 1996. franc nc, heitzler p, ezekowitz ra, white k. requirement for croquemort in phagocytosis of apoptotic cells in drosophila. science 284: 19911994, 1999. garver ls, wu j, wu lp. the peptidoglycan recognition protein pgrp-sc1a is essential for toll signaling and phagocytosis of staphylococcus aureus in drosophila. proc. natl. acad. sci. usa 103: 660-665, 2006. georgel p, naitza s, kappler c, ferrandon d, zachary d, swimmer c, et al. drosophila immune deficiency (imd) is a death domain protein that activates antibacterial defense and can promote apoptosis. dev. cell 1: 503-514, 2001. gobert v, gottar m, matskevich aa, rutschmann s, royet j, belvin m, et al. dual activation of the drosophila toll pathway by two pattern recognition receptors. science 302: 2126-2130, 2003. goto a, kadowaki t, kitagawa y. drosophila hemolectin gene is expressed in embryonic and larval hemocytes and its knock down causes bleeding defects. dev. biol. 264: 582-591, 2003. gottar m, gobert v, michel t, belvin m, duyk g, hoffmann ja, et al. the drosophila immune response against gram-negative bacteria is mediated by a peptidoglycan recognition protein. nature 416: 640-644, 2002. hoffmann ja, reichhart jm. drosophila innate immunity: an evolutionary perspective. nat. immunol. 3: 121-126, 2002. hoffmann ja. the immune response of drosophila. nature 426: 33-38, 2003. hultmark d. drosophila immunity: paths and patterns. curr. opin. immunol. 15: 12-19, 2003. imler jl, hoffmann ja. toll receptors in drosophila: a family of molecules regulating development and immunity. curr. top. microbiol. immunol. 270: 63-79, 2002. janeway ca jr, medzhitov r. innate immune recognition. annu. rev. immunol. 20: 197-216, 2002. kaneko t, goldman we, mellroth p, steiner h, fukase k, kusumoto s, et al. monomeric and polymeric gram-negative peptidoglycan but not purified lps stimulate the drosophila imd pathway. immunity 20: 637-649, 2004. kaneko t, yano t, aggarwal k, lim jh, ueda k, oshima y, et al. pgrp-lc and pgrp-le have essential yet distinct functions in the drosophila immune response to monomeric dap-type peptidoglycan. nat. immunol. 7: 715-723, 2006. kang d, liu g, lundstrom a, gelius e, steiner h. a peptidoglycan recognition protein in innate immunity conserved from insects to humans. proc. natl. acad. sci. usa 95: 10078-10082, 1998. kocks c, cho jh, nehme n, ulvila j, pearson am, meister m, et al. eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in drosophila. cell 123: 335-346, 2005. kurata s, ariki s, kawabata s. recognition of pathogens and activation of immune responses in drosophila and horse crab innate immunity. immunobiology 211: 237-249, 2006. kurata s. recognition of infectious non-self and activation of immune responses by peptidoglycan recognition protein (pgrp)-family in drosophila. dev. comp. immunol. 28: 89-95, 2004. 108 lee wj, lee jd, kravchenko vv, ulevitch rj, brey pt. purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, bombyx mori. proc. natl. acad. sci. usa 93: 7888-7893, 1996. lemaitre b, kromer-metzger e, michaut l, nicolas e, meister m, georgel p, et al. a recessive mutation, immune deficiency (imd), defines two distinct control pathways in the drosophila host defense. proc. natl. acad. sci. usa 92: 94659469, 1995. lemaitre b, nicolas e, michaut l, reichhart jm, hoffmann ja. the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults. cell 86: 973-983, 1996. leulier f, parquet c, pili-floury s, ryu jh, caroff m, lee wj, et al. the drosophila immune system detects bacteria through specific peptidoglycan recognition. nat. immunol. 4: 478-484, 2003. leulier f, rodriguez a, khush rs, abrams jm, lemaitre b. the drosophila caspase dredd is required to resist gram-negative bacterial infection. embo rep. 1: 353-358, 2000. leulier f, vidal s, saigo k, ueda r, lemaitre b. inducible expression of double-stranded rna reveals a role for dfadd in the regulation of the antibacterial response in drosophila adults. curr. biol. 12: 996-100, 2002. lim jh, kim ms, kim he, yano t, oshima y, aggarwal k, et al. structural basis for preferential recognition of diaminopimelic acid-type peptidoglycan by a subset of peptidoglycan recognition proteins. j. biol. chem. 281: 82868295, 2006. medzhitov r, janeway ca jr. decoding the patterns of self and nonself by the innate immune system. science 296: 298-300, 2002. meister m, lagueux m. drosophila blood cells. cell microbiol. 5: 573-580, 2003. mellroth p, karlsson j, steiner h. a scavenger function for a drosophila peptidoglycan recognition protein. j. biol. chem. 278: 70597064, 2003. mellroth p, steiner h. pgrp-sb1: an nacetylmuramoyl l-alanine amidase with antibacterial activity. biochem. biophys. res. commun. 350: 994-999, 2006. michel t, reichhart jm, hoffmann ja, royet j. drosophila toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. nature 414: 756-759, 2001. morisato d, anderson kv. signaling pathways that establish the dorsal-ventral pattern of the drosophila embryo. annu. rev. genet. 29: 371399, 1995. naitza s, rosse c, kappler c, georgel p, belvin m, gubb d, et al. the drosophila immune defense against gram-negative infection requires the death protein dfadd. immunity 17: 575-581, 2002. ochiai m, ashida m. a pattern recognition for peptidoglycan. cloning the cdna and the gene of the silkworm, bombyx mori. j. biol. chem. 274: 11854-11858, 1999. park jt. identification of a dedicated recycling pathway for anhydro-n-acetylmuramic acid and n-acetylglucosamine derived from escherichia coli cell wall murein. j. bacteriol. 183: 38423847, 2001. philips ja, rubin ej, perrimon n. drosophila rnai screen reveals cd36 family member required for mycobacterial infection. science 309: 1251-1253, 2005. pili-floury s, leulier f, takahashi k, saigo k, samain e, ueda r, et al. in vivo rna interference analysis reveals an unexpected role for gnbp1 in the defense against gram-positive bacterial infection in drosophila adults. j. biol. chem. 279: 12848-12853, 2004. ramet m, manfruelli p, pearson a, mathey-prevot b, ezekowitz ra. functional genomic analysis of phagocytosis and identification of a drosophila receptor for e. coli. nature 416: 644-648, 2002. ramet m, pearson a, manfruelli p, li x, kozeil h, gobel v, et al. drosophila scavenger receptor ci is a pattern recognition receptor for bacteria. immunity 15: 1027-1038, 2001. roth s. the origin of dorsoventral polarity in drosophila. philos. trans. r. soc. lond. b. biol. sci. 358: 1317-1329, 2003. royet j, reichhart jm, hoffmann ja. sensing and signaling during infections in drosophila. curr. opin. immunol. 17: 11-17, 2005. royet j. infectious non-self recognition in invertebrates: lessons from drosophila and other insect models. mol. immunol. 41: 1063-1075, 2004. steiner h. peptidoglycan recognition proteins: on and off switches for innate immunity. immunol. rev. 198: 83-96, 2004. stoven s, silverman n, junell a, hedengren-olcott m, erturk d, engstrom y, et al. caspasemediated processing of the drosophila nf-κb factor relish. proc. natl. acad. sci. usa 100: 5991-5996, 2003. stuart lm, deng j, silver jm, takahashi k, tseng aa, hennessy ej, et al. response to staphylococcus aureus requires cd36-mediated phagocytosis triggered by the cooh-terminal cytoplasmic domain. j. cell biol. 170: 477-485, 2005. takeda k, akira s. toll-like receptors in innate immunity. int. immunol. 17: 1-14, 2005. takeda k, kaisho t, akira s. toll-like receptors. annu. rev. immunol. 21: 335-376, 2003. takehana a, katsuyama t, yano t, oshima y, takada h, aigaki t, et al. overexpression of a patternrecognition receptor, peptidoglycan-recognition protein-le, activates imd/relish-mediated antibacterial defense and the prophenoloxidase cascade in drosophila larvae. proc. natl. acad. sci. usa 99: 13705-13710, 2002. takehana a, yano t, mita s, kotani a, oshima y, kurata s. peptidoglycan recognition protein (pgrp)-le and pgrp-lc act synergistically in drosophila immunity. embo j. 23: 4690-4700, 2004. theopold u, schmidt o, soderhall k, dushay ms. coagulation in arthropods: wound closure and healing. trends immunol. 25: 289-294, 2003. 109 ulvila j, parikka m, kleino a, sormunen r, ezekowitz ra, kocks c, et al. double-stranded rna is internalized by scavenger receptormediated endocytosis in drosophila s2 cells. j. biol. chem. 281: 14370-14375, 2006. vidal s, khush rs, lulier f, tzou p, nakamura m, lemaitre b. mutations in the drosophila dtak1 reveal a conserved function for mapkkks in the control of rel/nf-�b-dependent innate immune responses. genes dev. 15: 1900-1912, 2001. wang l, weber an, atilano ml, filipe sr, gay nj, ligoxygakis p. sensing of gram-positive bacteria in drosophila: gnbp1 is needed to process and present peptidoglycan to pgrpsa. embo j. 25: 5005-5014, 2006. watson fl, puttmann-holgado r, thomas f, lamar dl, hughes m, kondo m, et al. extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 18741878, 2005. werner t, liu g, kang d, ekengren s, steiner h, hultmark d. a family of peptidoglycan recognition proteins in the fruit fly drosophila melanogaster. proc. natl. acad. sci. usa 97: 13772-13777, 2000. yoshida h, kinoshita, ashida m. purification of a peptidoglycan recognition protein from hemolymph of the silkworm, bombyx mori. j. biol. chem. 271: 13854-13860, 1996. zaidman-rémy a, herve m, poidevin m, pili-floury s, kim ms, blanot d, et al. the drosophila amidase pgrp-lb modulates the immune response to bacterial infection. immunity 24: 463-473, 2006. 110 the multiple functions of the pgrp family in drosophila immunity a goto, s kurata discovery of the pgrp family references isj 3: 84-88, 2006 isj 3: 125-136, 2006 issn 1824-307x research report colony specificity in botrylloides leachi. i. morphological aspects g zaniolo, l manni, l ballarin department of biology, university of padova, padova, italy accepted december 20, 2006 abstract we studied colony specificity in the colonial ascidian botrylloides leachi which, as in other botryllid ascidians, leads to either fusion or non-fusion between contacting colonies. fusion requires the prior disappearance of contacting tunic cuticles and contact between facing ampullar epithelia. the epithelial cells of the ampullar tip show “pad regions” rich in ribosomes, which contribute to the synthesis of new tunic and cuticle. blood cells, mainly phagocytes and pigment cells, increase their concentrations inside the ampullar lumen and phagocytes can cross the ampullar epithelium and enter the tunic, where they can contribute to the digestion of tunic cuticles and cells of the ampullar epithelium in order to establish a common circulation. non-fusion reaction, as studied in the colony allorecognition assay, resembles the subcuticular rejection described in japanese botrylloides, characterised by limited tunic fusion, hemocyte leakage, and necrotic spots. conversely, in the cut surface assay, a more intense cytotoxic reaction is observed along the contact border. in this case, morula cells crowd massively inside the facing ampullae, enter the tunic, and release their vacuolar contents which are probably required for the formation of necrotic spots. key words: botrylloides; ascidians; colony specificity; allorecognition _________________________________________________________________________________________________________________ introduction the term allorecognition defines the capability of intraspecific non-self recognition; in clonal organisms, it is known as colony specificity and has been described in many species of compound ascidians, in which it leads to either fusion of facing, genetically compatible colonies into a larger chimerical colony, or non-fusion, when contacting colonies are genetically incompatible (taneda et al., 1985). botryllid ascidians are a group of compound tunicates, characterised by palleal budding, with colonies formed of many zooids embedded in a common tunic containing a vascular network, which connects and synchronises all the zooids. a marginal vessel runs along the border of the colony, from which many sausage-like blind endings, known as ampullae, sprout. ampullae sited at the growing edge allow the adhesion of the colony to the substratum ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padova via ugo bassi 58/b, 35100 padova, italy e-mail: loriano.ballarin@unipd.it and have a columnar epithelium on their tips, provided with pad cells (katow and watanabe, 1980; rinkevich et al., 1998). the outcome and intensity of the non-fusion reaction in botryllid ascidians depends on the tissues involved in the recognition. according to rinkevich (1992) and saito et al. (1994), allorecognition can occur: i) after the disappearance of most of the contacting cuticular layers, and consequent fusion of the tunics. in this case, the ampullae penetrate the opposite tunic and face the basal side of alien ampullae (tip-to-side interaction); massive crowding of hemocytes inside the ampullar tips then occurs, followed by leakage of cells into the tunic, contributing to the formation of a clear necrotic area. the ampullar tips then shrink, are amputated, and new tunic cuticles are formed by the two colonies to isolate the necrotic region along the contact border. this is typical of botryllus primigenus (taneda and watanabe, 1982); ii) after partial fusion of the tunic, restricted to regions in front of the facing (tip-to-tip) ampullae, which then can (woods hole botryllus schlosseri) or 125 fig. 1 a) living colony of botrylloides leachi from the dorsal side. zooids and buds are embedded in the common tunic, crossed by vessels with terminal ampullae. b) interacting ampullae of fusible colonies (a, b). arrows indicate the contact region. c-d) contacting ampullar tips of fusible colonies (a, b). the ampullar tips can contact either the tip or the apical side of the facing counterparts. bar = 150 µm. e) megaloampullae (m) in one of the contacting colony. bars = a, 1 mm; b-e, 150 µm. 126 fig. 2 interacting ampullae of contacting fusible colonies (a, b). a, c) contacting tips, semithin sections. bar = 50 and 15 µm, respectively. b, d) contact between tip and ampullar apical side. bars = 50 and 15 µm, respectively. e) contact between fusible colonies, ultrathin section. the two cuticles show numerous papillae (inset) and are separated by a narrow space; cells of the ampullar epithelium are cylindrical, with large nuclei (n) and welldeveloped rough endoplasmic reticulum (rer). bar = 2 µm (inset: 0.6 µm). f, highly folded plasma membrane in baso-lateral region of epithelial cells of the ampullar tip. ph, phagocytes; pc, pigment cells. bar = 2 µm. 127 cannot (monterey and mediterranean b. schlosseri) enter the facing tunic. cells crowd inside the ampullar tips, leak into the tunic, and contribute to the formation of a series of necrotic spots along the contact border. the ampullae then shrink and withdraw; in the woods hole population, their tips are amputated near the colonial boundary (boyd et al., 1990; sabbadin et al., 1992); iii) at the outer part of the tunic, just underneath the cuticle (subcuticular), which dissolves in very narrow regions in front of the facing ampullae. a few hemocytes leak from the ampullar lumen into the tunic, and contribute to the formation of small necrotic regions, which are not recognisable under the binocular microscope, along the contact border. this is reported in botrylloides simodensis, botrylloides fuscus and botrylloides violaceus (hirose et al., 1988, 1997); iv) after the fusion of the facing ampullae and an initial blood exchange. this characterises the non-fusion reaction of botryllus scalaris (saito and watanabe, 1982; shirae et al., 1999). non-fusion reaction has been widely investigated in b. schlosseri, in which morula cells (mc), representing the majority of the circulating hemocytes, massively crowd inside the tips of facing ampullae before crossing the ampullar epithelium and entering the tunic, where they degenerate and contribute to the formation of the necrotic masses observed along the contact border (ballarin et al., 1995, 1998). these cells degranulate and release the enzyme phenoloxidase, which is responsible for the cytotoxicity observed, due to the induction of oxidative stress (ballarin et al., 2002). botrylloides leachi is a compound ascidian, common in the mediterranean, and it can easily grow and reproduce in laboratory conditions. unlike from the sympatric species b. schlosseri, in which zooids are grouped in star-shaped systems and colonies are characterised by high chromatic polymorphism, b. leachi is easily distinguishable, thanks to the organisation of zooids in laddershaped systems and their orange to brown colonies (fig. 1a) . in spite of its abundance, colony specificity has been poorly investigated in this species (rinkevich et al., 1994; rinkevich, 1995) to fill this gap, we carried out a detailed investigation, at morphological and cellular level, of fusion and non-fusion in b. leachi, using both colony allorecognition assay (caa) and cut surface assay (csa) as defined by rinkevich (1992). results are discussed in the context of colony specificity in botryllid ascidians, and cellular events compared with those known in b. schlosseri. materials and methods animals b. leachi colonies, collected in the lagoon of venice, were allowed to adhere to glass slides (5 x 5 cm) and reared in aerated aquaria, filled with filtered seawater (fsw), at a temperature of 19 °c. they were fed with liquifry marine (liquifry co., dorking, england) and water was changed every other day. colony specificity assays in colony allorecognition assay (caa), colonies were juxtaposed on a supporting glass slide at a distance of 1-2 mm, their growing edges facing each other, in a moist chamber for 30 min, before being returned to the aquarium. within 24-48 h, the colonies extended towards each other and their facing ampullae contacted. cut surface assay (csa) was carried out to better investigate the non-fusion reaction: in this case, colonies were cut with razor blades and pieces of the same size from different colonies were brought into contact at their cut surface on a supporting glass slide and allowed to adhere for 30 min in a moist chamber. they were then returned to the aquarium. in each case, colonies were observed under a binocular microscope for up to 48 h, until either fusion or nonfusion occurred. light microscopy contacting colonies, at various stages of both fusion and non-fusion, were fixed for 2 h in 4 % paraformaldehyde plus 1 % glutaraldehyde in saline buffer (0.2 m na-cacodylate buffer, ph 7.4, plus 1.7 % nacl and 1 % sucrose), rinsed in saline buffer, dehydrated and embedded in paraplast x-tra (oxford labware). sections (7 µm thick) were obtained with a leitz 1212 microtome and stained with either haematoxylin/eosin or 2 % eosin g (shirae et al., 2002). electron microscopy fusing and non-fusing colonies were fixed for 2 h in 1.5 % glutaraldehyde in saline buffer, post-fixed for 1 h in oso4 in saline buffer, dehydrated and embedded in epon. thick sections (1 µm) were obtained with a lkb 2128 ultratome, stained with a hot solution of 1 % toluidine blue and 1 % borax in distilled water, and observed under the light microscope. thin (60 nm) sections were collected on copper grids, stained with uranyl acetate and lead citrate, and observed under a hitachi h 600 transmission electron microscope. results fusion interacting colonies extend the ampullae of their growing edges towards those of the alien colony to a close contact, in which ampullar tips are separated only by a thin layer of tunic (figs 1b-d). the ampullar tips can contact either the tips (figs 1c, d, 2a, c) or the apical sides (figs 1c, d, 2b, d)) of the alien counterpart. in both cases, facing tunic cuticles of early contacting colonies appear separated by a narrow space (figs 1c, d, 2c-e); they present numerous protruding papillae, 90 nm in height (fig. 2e (inset)). the tunic matrix appears rich in fibres, and tunic cells can be observed in the contacting region (figs 2c, e). the cells of the epithelium of the ampullar tips have a central nucleus, many mitochondria and a well-developed rough endoplasmic reticulum (rer), organised in a series of overlapping cisternae (fig. 2e); the basolateral plasma membranes are tightly folded (fig. 2e). the epithelium appears cylindrical in shape, 128 fig. 3 interacting ampullae of contacting fusible colonies (a, b), ultrathin sections. a) tight contact between cuticles. the epithelium appears fenestrated (asterisks) and cells show tight junctions in their apical region. b) magnification of a tight junction of the ampullar epithelium. c) pad regions (arrows) in epithelial cells of the ampullar tips. rer, rough endoplasmic reticulum. bars = 2 µm in a, c, d; 0.2 µm in b. 129 fig. 4 interacting fusible colonies. a-c) electron micrographs of the contact region between fusible colonies showing phagocytes (ph) in the lumen of facing ampulla (a), a phagocyte crossing the ampullar epithelium (b) and the complete fusion of contacting tunics of contacting colonies a and b in the framed region (c). bars = 3 µm for a and b; 1 µm for c. d-f) light micrographs of the interacting region between fusible colonies showing close contact between the tips of two facing ampullae (d) and formation of a new vessel allowing blood exchange between the two colonies a and b (e, f; arrows). bar = 250 µm. 130 fig. 5 a) light micrograph of the contact region between non-fusible colonies in caa. arrows indicate the region of close adhesion between the cuticles. bar = 0.5 mm. b) electron micrograph of the tip epithelium of interacting ampullae, cylindrical, rich in rer, and fenestrated (asterisks). scale bar: 1.5 µm. c-d) semithin sections of interacting ampullae showing crossing hemocytes (c; arrow) and fenestrated epithelium (d). bar = 20 µm. e) degenerated cells in the common tunic in front of interacting ampullae. electron-dense granules leaked from morula cells are indicated by arrowheads. bar = 2 µm. 131 lying on a thick basal lamina and provided with wide tight junctions in the apex region of the cells (figs 2e, 3a, b). once contact is established, the ampullae can grow to form megaloampullae (fig. 1e), as defined by rinkevich et al. (1994). no “wavy” epithelium, as described by rinkevich et al. (1994) in botrylloides from the mediterranean coast of israel, was observed in interacting ampullae. within 24-48 h from the first touch, a tight contact between the cuticles is observed (figs 3a, c). the epithelium of the ampullar tips is always continuous, although it now appears fenestrated (figs 3a, c); cells form irregular expansions at their apex (pad regions, according to katow and watanabe (1980)), containing ribosomes, finely granular material, and some small vesicles (fig. 3d). an increase in the concentration of various hemocytes is observed inside the facing ampullar tips (figs 2a-d), mainly represented by phagocytes and pigment cells (figs 2c, d, 4a). blood cells, particularly phagocytes, can be seen crossing the epithelial cells towards the tunic (fig. 4b). within 24-48 h of contact, the cuticles disappear, and tunic fusion is attained along the whole contact border (fig. 4c). tunic fusion is followed by fusion of the ampullar tips, so that blood can now flow from one colony to the other (figs 4d-f). non-fusion, caa at the beginning of the reaction, the tunic cuticles are well developed and separate the two contacting colonies (fig. 5a). the morphology of the ampullar tip epithelium is comparable to that observed in fusing colonies. megaloampullae may form (fig. 6a). in later stages, the contact between tunic become tighter, and the cuticles disappear in a narrow region in front of the facing ampullae where fusion of the tunic occurs; crowding of hemocytes is observed inside the facing ampullae. at about 24 h from contact, hemocytes begin to cross the ampullar tip epithelium (fig. 5c), which appears fenestrated (figs 5b, d) and, on entering the tunic, they degenerate and contribute to the formation of small, dark cytotoxic spots (fig. 6b). some of these cells are easily recognisable (e.g., phagocytes and mc; figs 5c, d); others appear degenerated, and granules with electron-dense contents are visible in the tunic (fig. 5e). leakage of cells is followed by the withdrawal of ampullae. non-fusion, csa in early stages of csa (fig. 6c), tunics are still separated, and several ampullae are observable in the region pushing towards the contact border. their lumen are crowded with hemocytes, particularly mc (figs 6d; 7a), which are easily recognisable due to their morphology and eosinophily, and are characterised by the presence of many small vacuoles, 2 µm in size, which give them a typical mulberry shape. the epithelium of the ampullar tips is flattened (figs 6d; 7a-c), with large nuclei and a well-developed rer (fig. 7b). within 24 h, tunic fusion occurs in limited regions and, over the next 24 h, many blood cells, mainly mc, leak from the ampullae (figs 7b, c) and crowd in the tunic of the contact region (fig. 6e), together with some granular cells (fig. 6f), as defined by cima et al. (2001). in this phase, mc alter their morphology and show empty vacuoles of larger size (fig. 6g). filamentous eosinophilic material is visible around the mc (fig. 6h). in advanced stages of the nonfusion reaction, mc aggregate along the contact border and contribute to the formation of a series of clearly visible, pigmented necrotic spots (fig. 6i). discussion as compound organisms, botryllid ascidians share the ability for intraspecific recognition, observable when colonies come into contact, leading to either fusion or non-fusion of genetically compatible or incompatible colonies, respectively (saito et al., 1994). the genetic bases of allorecognition have been deeply studied in b. schlosseri and b. primigenus, in which colonies can fuse when they share at least one allele at a highly polymorphic fu/hc locus; the absence of common alleles results in non-fusion (oka and watanabe, 1957, 1960; sabbadin, 1962; oka, 1970;). other botryllid species seem to follow the same kind of genetic control (saito et al., 1994). we studied fusion and non-fusion reactions between contacting colonies of the compound ascidian b. leachi. both tip-to-tip and tip-to-side interactions occur between the ampullae of the growing edges, although they never enter the opposite tunic in advanced stages of the reactions. a typical feature of this species is the frequent enlargement of contacting ampullae to form megaloampullae, already described in the case of non-fusion (rinkevich et al., 1994), and now described also for fusion. in the case of fusion, vascular anastomosis is preceded by the disappearance of the contacting cuticles and fusion of the thin layer of tunic covering the apex of growing-edge ampullae, which begins in front of the facing ampullae and extends rapidly to the whole contact border. blood cells, especially phagocytes, crowd inside the ampullar lumen and, after crossing the epithelium, enter the tunic. they can contribute to the digestion of tunic cuticles and ampullar tips, in order to allow the establishment of a common circulation. the epithelium of the ampullar tips changes its morphology, as reported by katow and watanabe (1980) in b. primigenus: numerous fenestrae appear between cells which remain closely adherent through well-developed tight junctions, and their apexes form pad regions which may contribute to the synthesis of new tunic and cuticle (katow and watanabe, 1980). the non-fusion reaction has been widely investigated in b. schlosseri: in this species incompatible colonies lead to limited fusion of the tunic in front of the ampullae facing tip-to-tip. soluble factors diffusing from the alien tunic and activated hemocytes attract blood cells, mainly mc, which crowd inside the apex of the facing ampullae, before crossing the epithelium of the ampullar tips and entering the tunic. during this process, mc degranulate, release their vacuolar contents, and contribute to the formation of a series of necrotic spots scattered along the contact border (sabbadin et al., 1992; ballarin et al., 1995, cima et al., 2006). according to our results, the non-fusion reaction in 132 fig. 6 a-b) caa. megaloampullae (m) in contact region between non-fusible colonies (a) and dark, cytotoxic spots in the contact area (b). bars = 250 and 150 µm, respectively. c-i) csa between two colonies (a, b) showing several ampullae in the contact region (c). the lumen of the ampullae in the contact area is filled with hemocytes, mainly morula cells (d), which leak in the tunic and crowd in the contact area (e), together with some granular cell (f, arrowhead), where morula cells degranulate, changing their morphology and showing large, empty vacuoles (g). eosinophilic material (arrowheads) is visible around morula cells (h). in advanced stages of the non-fusion reaction, diffuse necrotic regions are visible along the contact area (i). bars = 1 mm for c and i, 25 µm for d, f, g; 50 µm for e. 133 fig. 7 interacting ampullae in csa. a) semithin section of ampullae with flattened epithelium and blood cells, many morula cells (arrowheads) are visible inside their lumen. bar = 50 µm. b-c) electron micrographs of a morula cell (mc) interacting with the epithelium of the ampullar tip (b) and crossing it (c). bar = 1 µm b. leachi, as observed in caa, is characterised by limited tunic fusion and blood cell leakage from facing ampullae which do not penetrate the opposite tunic. giant ampullae sometimes, appear, but the outcome of the reaction seems to be similar in both the presence and absence of megaloampullae: hemocytes crowd inside the facing ampullae, and later move into the tunic through the fenestrated epithelium of the ampullar tips. the ampullae then, withdraw, and the colonies change their preferential direction of growth. in any case, the extent of the leakage of hemocytes and the size of the necrotic spots are very small when compared with the case in b. schlosseri (sabbadin, 1982; scofield and nagashima, 1983; sabbadin et al., 1992), and the reaction resembles the subcuticular rejection described in japanese species of the genus botrylloides (hirose et al., 1988, 1997), in which a few blood cells leak through the ampullar epithelium in front of the ampullar tips, and cytotoxic foci are poorly visible. in order to study better the role of blood cells in the non-fusion reaction, we used csa, which gives a more intense cytotoxic reaction in those species characterised by subcuticular rejection in caa (hirose et al., 1990, 1997, 1998). with this assay, we were able to demonstrate that mc are involved in the non-fusion reaction in b. leachi. similarly to what occurs in b. schlosseri (ballarin et al., 1995, 1998; rinkevich et al., 1998; cima et al., 2006), mc selectively accumulate inside 134 facing ampullae. this suggests chemotactic activity by soluble factors from the alien colony, which may be reinforced by the release of chemotactic chemokines by activated hemocytes. similar behaviour by activated mc has recently been demonstrated in b. schlosseri (cima et al., 2006). once inside the ampullae, mc leak into the tunic, together with some granular cells, and change their morphology: the latter event is probably related to the release of vacuolar contents which, as in b. schlosseri (ballarin et al., 1995, 1998), may be responsible for the induction of cytotoxicity. the presence of filamentous material, sharing staining affinity with the contents of morula cell vacuoles in the tunic surrounding morula cell aggregates, fits the above hypothesis. the intense dark pigmentation along the contact region suggests that phenoloxidase is involved in the induction of cytotoxicity in csa, as reported for b. schlosseri and other ascidian species (ballarin et al., 1995, 1998; shirae and saito, 2000; shirae et al., 2002). future investigations will be directed towards both better comprehension of the role of phenoloxidase in the non-fusion reaction of b. leachi and the search for the immunomodulatory molecules, secreted by activated hamocytes, involved in the process. acknowledgements this work was supported by the italian miur. the authors wish to thank m del favero for technical help. references ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri. dev. comp. immunol. 22: 479-492, 1998. ballarin l, cima f, floreani m, sabbadin a. oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian botryllus schlosseri. comp. biochem physiol. 133c: 411-418,2002. boyd hc, weissman il, saito y. morphologic and genetic verification that monterey botryllus and woods hole botryllus are the same species. biol. bull. 178: 239-250, 1990. cima f, perin a, burighel p, ballarin l. morphofunctional characterisation of haemocytes of the compound ascidian botrylloides leachi (tunicata, ascidiacea). acta zool. 82: 261-274, 2001. cima f, sabbadin a, zaniolo g, ballarin l. colony specificity and chemotaxis in the compound ascidian botryllus schlosseri. comp. biochem. physiol. 145: 376-382, 2006. hirose e, saito y, watanabe h. a new type of the manifestation of colony specificity in the compound ascidian, botrylloides violaceus oka. biol. bull. 175: 240-245, 1988. hirose e, saito y, watanabe h. allogeneic rejection induced by cut surface contact in the compound ascidian, botrylloides simodensis. invertebr. reprod. dev. 17: 159-164, 1990. hirose e, saito y, watanabe h. subcuticular rejection: an advanced mode of the allogeneic rejection in the compound ascidian botrylloides simodensis and b. fuscus. biol. bull. 192: 5361, 1997. katow h, watanabe h. fine structure of fusion reaction in the compound ascidian botryllus primigenus oka. dev. biol. 76: 1-14, 1980. oka h. colony specificity in compound ascidians. the genetic control of fusibility. in: h yukawa (ed), profiles of japanese science and scientists, kodansha, tokyo, pp 196-206, 1970. oka h, watanabe h. colony specificity in compound ascidians as tested by fusion experiments (a preliminary report). proc. japan. acad. 33: 657-659, 1957. oka h, watanabe h. problems of colony specificity in compound ascidians. bull. mar. biol. stat. ashamiushi 10: 153-155, 1960. rinkevich b. aspects of the incompatibility nature in botryllid ascidians. anim. biol. 1: 17-28, 1992. rinkevich b. characteristics of allogeneic resorption in botrylloides from the mediterranean coast of israel. dev. comp. immunol. 19: 21-29, 1995. rinkevich b, lilker-levav t, goren m. allorecognition/xenorecognition responses in botrylloides (ascidiacea) subpopulations from the mediterranean coast of israel. j. exp. zool. 270: 302-313, 1994. rinkevich b, tartakover s, gershon h. contribution of morula cells to allogeneic responses in the colonial ascidian botryllus schlosseri. mar. biol. 131: 227–236, 1998. sabbadin a. le basi genetiche della capacità di fusione fra colonie in botryllus schlosseri (ascidiacea). red. accad. naz. lincei 32: 1021-1035, 1962. sabbadin a. formal genetics of ascidians. amer. zool. 22: 765-773, 1982. sabbadin a, zaniolo g, ballarin l. genetic and cytological aspects of histocompatibility in ascidians. boll. zool. 59: 167-173, 1992. saito y, watanabe h. colony specificity in the compound ascidian botryllus scalaris. proc. jpn. acad. 58b: 105-108, 1982. saito y, hirose e, watanabe h. allorecognition in compound ascidians. intl. j. dev. biol. 38: 237247, 1994. scofield vl, nagashima ls. morphology and genetics of rejection reaction between oozoids from the tunicate botryllus schlosseri. biol. bull. 733-744, 1983. shirae m., saito y. a comparison of hemocytes and their phenoloxidase activity among botryllid ascidians. zool. sci. 17: 881-891, 2000. shirae m, hirose e, saito y. behavior of hemocytes in the allorejection reaction in two compound ascidians, botryllus scalaris and symplegma reptans. biol. bull. 197: 188-197, 1999. shirae m, ballarin l, frizzo a, saito y, hirose e. involvement of quinones and phenoloxidase in the allorejection reaction in a colonial ascidian, botrylloides simodensis: histochemical and 135 immunohistochemical study. mar. biol. 141: 659-665, 2002. taneda y, watanabe h. studies on colony specificity in the compound ascidian, botryllus primigenus oka. initiation of “nonfusion” reaction with special reference to blood cells infiltration. dev. comp. immunol. 6: 43-52, 1982. taneda y, saito y, watanabe h. self or non-self discrimination in ascidians. zool. sci. 2: 433442, 1985. 136 department of biology, university of padova, padova, italy 9 isj 14: 9-17, 2017 issn 1824-307x short communication immune response in the larvae of the black soldier fly hermetia illucens a zdybicka-barabas1, p bulak2, c polakowski2, a bieganowski2, a waśko3, m cytryńska1 1department of immunobiology, institute of biology and biochemistry, faculty of biology and biotechnology, maria curie-skłodowska university, akademicka 19 st., 20-033 lublin, poland 2institute of agrophysics, polish academy of sciences, doświadczalna 4 st., 20-290 lublin, poland 3department of biotechnology, human nutrition and science of food commodities, university of life sciences in lublin, skromna 8 st., 20-704 lublin, poland accepted december 16, 2016 abstract the black soldier fly hermetia illucens is an ecological decomposer used for biodegradation of organic waste. its larvae can develop on a wide range of decaying plant and animal matter, including manure and food scraps, i.e., habitats that are extremely rich in various microorganisms. living in such conditions requires very well-functioning immune mechanisms. however, the immune response processes have not been examined so far in h. illucens larvae. in order to shed light on the immune system in the black soldier fly, in the present study we have examined h. illucens hemocytes and analyzed the effects of immune challenge of h. illucens larvae on the activity of the key components of insect humoral immune response, i.e., phenoloxidase, lysozyme, and antimicrobial peptides. key words: hermetia illucens; innate immunity; hemocytes; antimicrobial peptides; lysozyme; phenoloxidase introduction the black soldier fly hermetia illucens is an ecological decomposer used for biodegradation of organic waste (čičkova et al., 2015). its larvae develop through six larval instars on a wide range of decaying plant and animal matter, including manure, food scrapes, municipal garbage, and rotting plant material (sheppard et al., 1994, 2002; diener et al., 2011). living in an environment that is extremely rich in various microorganisms, including many pathogenic ones, requires a very well-functioning immune system. insect immunity relies on cellular and humoral innate mechanisms, which have been well characterized in e.g., drosophila melanogaster, a dipteran model organism (lemaitre and hoffmann, 2007; buchon et al., 2014; kleino and silverman, 2014; lindsay and wasserman, 2014; cytryńska et al., 2016). the hemolymph cells, hemocytes, are involved in processes of the cellular immune response, i.e., phagocytosis, nodulation, and encapsulation (lavine and strand, 2002; dubovskiy et al., 2016). in addition to prohemocytes, three types of hemocytes, i.e., plasmatocytes, crystal cells, ___________________________________________________________________________ corresponding author: agnieszka zdybicka-barabas department of immunobiology faculty of biology and biotechnology maria curie-skłodowska university akademicka 19 st. 20-033 lublin, poland e-mail: barabas@poczta.umcs.lublin.pl and lamellocytes were characterized in d. melanogaster (ribeiro and brehélin, 2006), whereas those in anopheles gambiae and aedes aegypti were classified as granulocytes and oenocytoids (strand, 2008; hillyer and strand, 2014). an important role in humoral immune response against pathogens is played by phenoloxidase (po) as well as antimicrobial peptides and proteins. po activity is a result of fast activation of the po system by pathogen-associated molecular patterns (pamps), including components of microbial cell walls, i.e., bacterial lipopolysaccharide (lps), peptidoglycan (pgn), and fungal β-1,3-glucan (cerenius et al., 2008; bidla et al., 2009; lu et al., 2014). po activity leads to formation of quinones and other cytotoxic intermediates of melanin, and finally to melanin deposition at the site of injury and around invading pathogens. melanin as well as cytotoxic intermediate products exhibit strong antimicrobial activity and prevent spreading of the pathogens in an insect organism (suguraman, 2002; lee and miura, 2014). in addition to activation of the po system, recognition of pathogens results in induction of synthesis of defense peptides able to kill the invaders. the inducible antimicrobial peptides are mainly synthesized in the insect fat body and released into hemolymph, where they are essential components of systemic immune response. seven families of defense peptides with different biochemical and antimicrobial properties have been 10 described in d. melanogaster, i.e., attacins, cecropins, insect defensin, diptericins, drosocin, drosomycins, and metchnikowin (uvel and engström, 2007). in other insect species, a number of various defense peptides have been characterized to date, including anionic antimicrobial peptides (cytryńska et al., 2007a; scocchi et al., 2011; mylonakis et al., 2016). beside the defense peptides, hemolymph lysozymes play an important role in antimicrobial immune response in insects largely due to enzymatic muramidase activity (hultmark, 1996). lysozymes are usually constitutive components of insect hemolymph; however, they can act synergistically with defense peptides, and pathogen recognition may also result in a dramatic increase in their level and activity in hemolymph, which contributes considerably to effective antimicrobial defense (yu et al., 2002; chapelle et al., 2009; zhang et al., 2009; zdybicka-barabas et al., 2012, 2013; sowa-jasiłek et al., 2014; beckert et al., 2015). the habitat of h. illucens larvae, which is extremely rich in various microorganisms, implies that the immune system of this insect species functions very efficiently and effectively. recently, park et al. (2015) have reported on purification and characterization of a h. illucens defensin-like peptide with activity against gram-positive bacteria. however, although h. illucens larvae are used in composting piles, the immune response processes in these insects have not been examined so far and there are no data currently available in this area. in order to shed light on the immune system in the black soldier fly, in the present study we have examined h. illucens hemocytes and analyzed the effects of immune challenge of h. illucens larvae on activity of the key components of insect humoral immune response, i.e., phenoloxidase, lysozyme, and antimicrobial peptides. materials and methods insect culture conditions the larvae of hermetia illucens (diptera: striatomyidae) were reared in laboratory conditions at 29 oc and substrate humidity of 50 80 % in the institute of agrophysics of the polish academy of sciences in lublin, poland. the larvae were fed with feed consisting of protein 25 %, fat and oil 5 %, crude fiber 5.8 %, ash 5.7 %, lysine 1.25 %, calcium 1 %, phosphorus 0.97 %, methionine 0.4 %, and sodium 0.05 %. last instar larvae were used in the experiments. microorganisms gram-negative bacterium escherichia coli d31 and gram-positive bacterium micrococcus luteus atcc 10240 were grown in lb (lysogeny broth) at 37 c and 28 c, respectively, until the logarithmic growth phase. filamentous fungus aspergillus niger was grown on solid pda medium (5 % potato extract, 0.5 % dextrose, 1.7 % agar) at 28 oc until conidial spores were obtained and then stored at 4 oc. a conidial suspension for the antifungal activity assay (see below) was prepared as described in our previous paper (mak et al., 2010). insect immune challenge, hemolymph collection, and preparation of hemolymph methanolic extracts an immune challenge with live gram-negative bacterium e. coli or gram-positive bacterium m. luteus was performed with a piercing method, essentially as described previously for galleria mellonella larvae (mak et al., 2010). before immunization, the larvae were washed with sterile water and the puncture sites were disinfected with 70 % ethanol. the larvae were punctured with a sterile needle (control) or with a needle dipped in a pellet of live bacteria (40 larvae per group). next, the larvae were stored in petri dishes provided with food or in sterile conditions, and the hemolymph was collected 6 h, 24 h and 48 h after the treatment as well as from the unchallenged (naive) larvae. the hemolymph (5 µl per larva) was combined to obtain pooled samples. hemocyte-free hemolymph was obtained by centrifugation at 200xg for 5 min and subsequently at 20,000g for 15 min at 4 c (mak et al., 2010). an acidic/methanolic extraction method was used for partial purification of antimicrobial peptides. the hemolymph was diluted ten times with the extraction solution (methanol/acetic acid, glacial/water; 90:1:9), mixed thoroughly, and centrifuged (20,000×g, 30 min 4 oc) in order to pellet precipitated proteins. the supernatant containing mainly proteins of mr below 30kda and peptides was collected and vacuum dried, and the pellet was stored at -20 oc until needed (cytryńska et al., 2007a; mak et al., 2010). antimicrobial activity assays well diffusion assay the hemolymph antibacterial activity against e. coli d31 and m. luteus was detected by a growth inhibition zone assay on lb agar plates, essentially as reported (mak et al., 2010). to improve the sensitivity of the method against gram-negative bacteria, egg white lysozyme (ewl) at the final concentration of 2.5 mg/ml was added (cytryńska et al., 2001). the hemolymph antifungal activity was detected using pda agar plates containing a. niger conidia, as described previously (mak et al., 2010). lysozyme activity in the hemolymph was estimated using agarose plates containing freeze-dried m. luteus as reported (jarosz, 1995). the activity of lysozyme was calculated from a standard curve made with egg white lysozyme (ewl, ec 3.2.1.17; sigma-aldrich). each well on the petri dish was filled with 4l of five times diluted hemolymph and the plates were incubated at 37 oc (e. coli) or 28 oc (m. luteus, a. niger). the diameters of bacterial and fungal growth inhibition zones were measured after 24-h and 48-h incubation, respectively. the level of anti-e. coli activity was calculated using the algorithm described previously (hultmark et al., 1982) with cecropin b of hyalophora cecropia (sigma-aldrich) as a standard. bioautography (sds gel overlay method) detection of antibacterial activity in the hemolymph after sds/page and subsequent renaturation of polypeptides was performed as described previously (cytryńska et al., 2001). 11 briefly, after separation of proteins (300 µg of total protein per sample), the gels were washed in 2.5 % triton x-100 (bio rad) for removal of sds. next, the polypeptides were renatured in 50mm tris-hcl ph 7.5 and subsequently in lb. finally, the gels were overlaid with nutrient agar containing e. coli d31 cells and 2.5 mg/ml ewl, and the zones of bacterial growth inhibition were observed after incubation for 6 12 h at 37 oc. phenoloxidase activity assay the phenoloxidase activity in the hemolymph was determined on the basis of melanin formation according to the method described previously (park et al., 2005; zdybicka-barabas et al., 2014). briefly, 2 μl of non-diluted hemolymph was added to 18 μl of tbs (50 mm tris-hcl ph 7.4, 150 mm nacl) containing 5mm cacl2 in the wells of a 96-well plate and the mixture was incubated for 20 min at room temperature. next, 180 μl of 2 mm dopamine in 50 mm sodium phosphate ph 6.5 was added and absorbance of the mixture was measured at 490 nm over 90 min at a 5-min interval using a microtiter plate reader (biorad). the experiment was performed in triplicate on three independent occasions. other methods polyacrylamide gel electrophoresis of proteins was performed by 13.8 % glycine sds/page according to laemmli (1970). separation of proteins below 30 kda and peptides was carried out by tricine sds/page (16.5 % t, 3 % c) (schägger and von jagow, 1987). the proteins and peptides were stained using coomassie blilliant blue r-250 (0.25 %) or g-250 (0.025 %), respectively, after glycine sds/page and tricine sds/page. the protein concentration was estimated by the bradford method using bovine serum albumin (bsa) as a standard (bradford, 1976). for microscopic observations of hemocytes, the samples of freshly collected hemolymph (5µl) were placed onto microscopic slides, covered with coverslips, and the hemocytes were observed immediately using a contrast-phase microscope olympus bh-2 (lens magnification 40×). results and discussion h. illucens hemocytes the microscopic examination of the hemolymph revealed the presence of at least three types of morphologically different hemocytes, i.e. crystal celllike, plasmatocyte-like, and granule-containing hemocytes (fig. 1a). the oval shaped crystal celllike hemocytes (approx. dimensions 18 µm × 15 µm) were non-adherent cells and contained evident crystal-like inclusions (approx. 12 µm in length and 2.5 µm in width), similar to those reported in d. melanogaster crystal cells (ribeiro and brehélin, 2006). based on these characteristics, these cells may be involved in the melanization process. the two other types were adherent cells that formed filopodia-like projections, a feature that implicates their engagement in cellular immune response processes. the plasmatocyte-like cells were morphologically similar to some lepidopteran plasmatocytes (cytryńska et al., 2007b; hori et al., 2013; wu et al., 2016) and exhibited a tendency to aggregate (fig. 1b). the granule-containing hemocytes resembled morphologically culex pipiens quinquefasciatus granulocytes. interestingly, in c. pipiens quinquefasciatus, a dipteran species, three hemocyte types, i.e., oenocytoids, plasmatocytes, and granulocytes, were identified beside prohemocytes (wang et al., 2011). although the h. illucens granule-like cells resembled c. pipiens quinquefasciatus granulocytes, the plasmatocyte-like cells were morphologically distinct from those in c. pipiens quinquefasciatus. in addition, hemocytes with undefined features were observed (fig. 1b). notably, nodule-like structures with a diameter approx. 25 35 µm were observed in the hemolymph of the bacteria-challenged h. illucens larvae (fig. 1c), suggesting that nodulation may be one of the cellular immune response processes involved in fast elimination of invaders in h. illucens larvae. phenoloxidase activity in h. illucens hemolymph the immune challenge of h. illucens larvae with the gram-negative and gram-positive bacteria led to a considerable increase in hemolymph po activity. in comparison with the level of po activity in the hemolymph of naive larvae (control), the enzyme activity increased 1.27-fold and 1.6-fold after the challenge with e. coli and m. luteus, respectively (fig. 2a). interestingly, the po activity after sterile puncturing was by approx. 22 % lower than in the control hemolymph. if one takes this into consideration, the po activity level after the challenge with e. coli and m. luteus was 1.6-fold and 2-fold higher, respectively (fig. 2a). in addition to the changes in the hemolymph po activity, effects of local po activation that led to melanin deposition at the site of injury were observed on the surface of larval body (fig. 2b). the results clearly indicate an important role of po activity in h. illucens immune response against invading gramnegative and gram-positive bacteria. antimicrobial activity in h. illucens hemolymph in addition to the evident changes in the po activity level, the immune challenge of the h. illucens larvae induced antimicrobial activity in the hemolymph. the lysozyme and anti-gram-positive bacterium m. luteus activity, both detected in the hemolymph of the naive larvae, increased considerably after the challenge and reached the highest level in the e. coli-challenged larvae (tables 1, 2). in contrast, antibacterial activity measured against gram-negative bacterium e. coli d31 was detected only in the hemolymph of the challenged larvae. it was induced by the bacterial challenge and by the sterile puncturing of the larvae. the level of anti-e. coli activity in the hemolymph of the sterile punctured as well as m. luteusand e. colichallenged larvae corresponded to the activity of 0.34 µm, 0.55 µm, and 1.2 µm of a cecropin b solution, respectively (table 2). no antifungal activity against a. niger was detected in h. illucens hemolymph in our experimental conditions. on the other hand, the lysozyme activity in the hemolymph of naive larvae suggests constitutive synthesis of 12 fig. 1 hermetia illucens hemocytes. the hemocytes (a, b) and cell aggregates or nodules (c) were observed in a contrast-phase microscope olympus bh-2. the white arrows and arrowheads indicate, respectively, crystal-like intrusions and filopodia-like projections. bar = 10 µm. 13 fig. 2 phenoloxidase activity in h. illucens larvae. (a) po activity in the hemolymph. the larvae were immunized with sterile injury or bacteria-challenged, and the phenoloxidase activity was determined in the hemolymph collected 24 h after the treatment. the results are presented as ±sd from three independent experiments. the inset demonstrates melanin formed in the wells after 90 min incubation with dopa as a substrate. c naive larvae; in sterile-injured larvae; ml and ec m. luteusand e. coli-challenged larvae, respectively. (b) melanization localized at a site of injury is indicated by the red arrowheads. this antibacterial factor in h. illucens larvae and its role in elimination of gram-positive bacteria (e.g., m. luteus). it should be noted that a considerable increase in the lysozyme activity and induction of anti-e. coli activity was detected in the hemolymph of the larvae stored in the non-sterile conditions (i.e., provided with food) after the sterile puncturing (table 2). such conditions reflect a possibility of invasion of pathogens present in food through mechanical injury in the natural habitat of h. illucens larvae. the results also indicate great adaptation of the h. illucens immune system to fight against pathogens. interestingly, when the immune-challenged larvae were incubated in the sterile conditions, the challenge with e. coli induced the lysozyme, anti-m. luteus, and anti-e. coli activity, whereas the challenge with sterile puncturing and m. luteus caused only an increase in the lysozyme and antim. luteus activity. in the hemolymph of the sterile punctured and m. luteus-challenged larvae, no antie. coli activity was detected (table 1). this suggests discrimination between gram-positive and gram-negative bacteria by the h. illucens immune system, similarly to d. melanogaster, in which such a phenomenon is well documented (goto 14 table 1 antimicrobial activity in the hemolymph of h. illucens larvae kept in sterile conditions antimicrobial activity time after challenge (h) experimental group control larvae sterileinjured larvae m. luteuschallenged larvae e. colichallenged larvae lysozyme activity [µg/ml] 6 24 48 2.24±0.2 2.63±0.27 3.98±0.95 2.82±0.18 3.98±0.7 6.03±1.1 3.08±0.18 12.59±0.8 4.47±0.34 4.08±0.27 20.42±0.3 8.51±0.4 anti-m. luteus activity (mm) 6 24 48 12.6±0.3 10.4±0.25 12.9±0.2 16.4±0.1 17.0±0.2 16.4±0.3 16.0±0.15 18.6±0.25 17.2±0.1 16.3±0.3 20.4±0.15 18.0±0.5 anti-e. coli activity [µm] 6 24 48 nd nd nd nd nd nd nd nd nd 1.3±0.26 1.66±0.42 nd h. illucens larvae were sterile-injured or immunized with e. coli or m. luteus and kept in sterile conditions for 48 h. the hemolymph was collected 6 h, 24 h, and 48 h after the challenge. next, the lysozyme, anti-m. luteus, and anti-e. coli activity was determined by a well diffusion assay. the results are presented as ±sd from three independent experiments. nd = not detected and kurata, 2006; silverman et al., 2009; reumer et al., 2010). the hemolymph antibacterial activity estimated against e. coli with the diffusion well assay was determined by the presence of inducible antimicrobial peptides with molecular mass corresponding to cecropin b, as revealed by bioautography (fig. 3a) and tricine sds/page of the hemolymph methanolic extracts (fig. 3b). electrophoretic analysis of hemolymph proteins indicated that, despite induction of defense peptides, the bacterial challenge of h. illucens larvae led to appearance of an additional protein with molecular mass approx. 43 kda. interestingly, the e. coli challenge resulted in appearance of two other proteins with molecular masses approx. 58 kda and 27 kda (fig. 3c). determination of the identity of these proteins and their role in h. illucens immune response requires further study. in conclusion, it is important to note that the immune challenge of h. illucens larvae with grampositive m. luteus induced only anti-gram-positive bacterial activity, possibly resulting from increased lysozyme activity. in contrast, in the hemolymph of larvae immunized with gram-negative e. coli, besides anti-gram-positive bacterial activity also anti-gram-negative bacterial activity was detected. this is consistent with the data reported recently (park et al., 2015). these authors demonstrated that a defensin-like peptide purified from the hemolymph of h. illucens larvae challenged with gram-positive staphylococcus aureus was active against gram-positive bacteria and not against gram-negative ones (park et al., 2015). our results suggest that, depending on the bacteria used for the immune challenge (containing diaminopimelic-type pgn or lysine-type pgn in the cell wall), synthesis of different sets of antimicrobial peptides is induced in h. illucens, as in the case of d. melanogaster (silverman et al., 2009; lindsey and wasserman, 2014; kleino and silverman, 2014) and g. mellonella (mak et al., 2010) reported previously. table 2 antimicrobial activity in the hemolymph of h. illucens larvae kept in non-sterile conditions experimental group antimicrobial activity lysozyme activity [µg/ml] anti-e. coli activity [µm] control larvae 7.2±3.6 nd sterile-injured larvae 11.47±0.73 0.34±0.06 m. luteus-challenged larvae 26.12±5.84 0.55±0.09 e. coli-challenged larvae 35.48±10.83 1.2±0.48 h. illucens larvae were sterile-injured or immunized with e. coli or m. luteus and kept in non-sterile conditions for 24 h. next, the hemolymph was collected, and the lysozyme and anti-e. coli activity was determined by a well diffusion assay. the results are presented as ±sd from three independent experiments. nd = not detected. 15 fig. 3 antimicrobial activity (a) and changes in peptide (b) and protein (c) patterns in the hemolymph of immunized h. illucens larvae. the hemolymph was collected from naive larvae (control c) and from sterileinjured (in), m. luteus (ml)-, or e. coli (ec)-challenged larvae 24 h after the treatment. (a) antimicrobial activity detected by bioautography after separation of the hemolymph polypeptides (300 µg of total protein) in 13.8 % polyacrylamide gel and subsequent renaturation. cecropin b (1µg) was used as a control peptide. a gel fragment presenting clear zones (darker areas) of e. coli growth inhibition is shown. (b) electrophoretic analysis of h. illucens hemolymph peptides. the peptides extracted from 5 µl of the hemolymph with the acidic/methanolic extraction method were diluted in 20 µl of sample buffer and separated by tricine sds/page. additional peptide bands appearing in the larval hemolymph after the bacterial challenge are encircled by a red frame. (c) electrophoretic analysis of h. illucens hemolymph proteins. the hemolymph samples (40 µg of total protein) were prepared in 20 µl of laemmli sample buffer and separated by sds/page in 13.8 % polyacrylamide gels. additional protein bands appearing in the larvae hemolymph after bacterial challenge are indicated by red asterisks. references beckert a, wiesner j, baumann a, pöppel ak, vogel h, vilcinskas a. two c-type lysozymes boost the innate immune system of the invasive ladybird harmonia axyridis. dev. comp. immunol. 49: 303-312, 2015. bidla g, hauling t, dushay ms, theopold u. activation of insect phenoloxidase after injury: endogenous versus foreign elicitors. j. innate immun. 1: 301-308, 2009. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254, 1976. buchon n, silverman n, cherry s. immunity in drosophila melanogaster from microbial recognition to whole-organism physiology. nat. rev. immunol. 14: 796-810, 2014. cerenius l, lee bl, söderhäll k. the propo system: pros and cons for its role in invertebrate immunity. trends immunol. 29: 263-271, 2008. chapelle m, girard pa, cousserans f, volkoff na, duvic b. lysozymes and lysozyme-like proteins from the fall armyworm, spodoptera frugiperda. mol. immunol. 47: 261-269, 2009. čičková h, newton gl, lacy rc, kozánek m. the use of fly larvae for organic waste treatment. waste manag. 35: 68-80, 2015. cytryńska m, mak p, zdybicka-barabas a, suder p, jakubowicz t. purification and characterization of eight peptides from galleria mellonella immune hemolymph. peptides 28: 533-546, 2007a. cytryńska m, wojda i, jakubowicz t. how insects combat infections. in: ballarin l, cammarata m (eds), lessons in immunity: from single-cell organisms to mammals, elsevier academic press, pp 117-128, 2016. cytryńska m, zdybicka-barabas a, jablonski p, jakubowicz t. detection of antibacterial polypeptide activity in situ after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. anal. biochem. 299: 274-276, 2001. cytryńska m, zdybicka-barabas a, jakubowicz t. protein kinase a activity and protein phosphorylation in the haemocytes of immunechallenged galleria mellonella larvae. comp. biochem. physiol. 148b: 74-83, 2007b. diener s, studt solano nm, gutierrez fr, zurbrugg c, tockner k. biological treatment of municipal organic waste using black soldier fly larvae. waste biomass valor. 2: 357-363, 2011. 16 dubovskiy im, kryukova na, glupov vv, ratcliffe na. encapsulation and nodulation in insects. inv. surv. j. 13: 229-246, 2016. goto a, kurata s. the multiple functions of the pgrp family in drosophila immunity. inv. surv. j. 3: 103-110, 2006. hillyer jf, strand mr. mosquito hemocyte-mediated immune responses. curr. opin. insect sci. 3: 14-21, 2014. hori t, kiuchi t, shimada t, nagata m, katsuma s. silkworm plasmatocytes are more resistant than other hemocyte morphotypes to bombyx mori nucleopolyhedrovirus infection. j. invertebr. pathol. 112: 102-104, 2013. hultmark d, engström a, bennich h, kapur r, boman hg. insect immunity: isolation and structure of cecropin d and four minor antibacterial components from cecropia pupae. eur. j. biochem. 127: 207-217, 1982. hultmark d. insect lysozymes. in: jollès p (ed.), lysozymes: model enzymes in biochemistry and biology, birkhäuser verlag, basel, switzerland, pp 87-102, 1996. jarosz j. haemolymph immune proteins protect the insect body cavity from invading bacteria. comp. biochem. physiol. 111c: 213-220, 1995. kleino a, silverman n. the drosophila imd pathway in the activation of the humoral immune response. dev. comp. immunol. 42: 25-35, 2014. laemmli uk. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227: 680-685, 1970. lavine md, strand mr. insect hemocytes and their role in immunity. insect biochem. mol. biol. 32: 1295-1309, 2002. lee wj, miura m. mechanisms of systemic wound response in drosophila. curr. top. dev. biol. 108: 153-83, 2014. lemaitre b, hoffmann j. the host defense in drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. lindsay sa, wasserman sa. conventional and nonconventional drosophila toll signaling. dev. comp. immunol. 42: 16-24, 2014. lu a, zhang q, zhang j, yang b, wu k, xie w, et al. insect prophenoloxidase: the view beyond immunity. front. physiol. 11: 252, 2014. mak p, zdybicka-barabas a, cytryńska m. a different repertoire of galleria mellonella antimicrobial peptides in larvae challenged with bacteria and fungi. dev. comp. immunol. 34: 1129-1136, 2010. mylonakis e, podsiadlowski l, muhammed m, vilcinskas a. diversity, evolution and medical applications of insect antimicrobial peptides. philos. trans. r soc. lond. b biol. sci. 371(1695), doi: 10.1098/rstb.2015.0290, 2016. park si, kim jw, yoe sm. purification and characterization of a novel antibacterial peptide from black soldier fly (hermetia illucens) larvae. dev. comp. immunol. 52: 98-106, 2015. park sy, kim ch, jeong wh, lee jh, seo sj, han ys, et al. effects of two hemolymph proteins on humoral defense reactions in the wax moth, galleria mellonella. dev. comp. immunol. 29: 43-51, 2005. reumer a, van loy t, schoofs l. the complexity of drosophila innate immunity. inv. surv. j. 7: 3244, 2010. ribeiro c, brehélin m. insect haemocytes: what type of cell is that? j. insect physiol. 52: 417429, 2006. schägger h, von jagow g. tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kda. anal. biochem. 166: 368-379, 1987. scocchi m, tossi a, gennaro r. proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action. cell. mol. life sci. 68: 2317-2330, 2011. sheppard dc, newton gl, thompson sa, savage s. a value added manure management system using the black soldier fly. bioresour. technol. 50: 275-279, 1994. sheppard dc, tomberlin jk, joyce ja, kiser bc, sumner sm. rearing methods for the black soldier fly (diptera: stratiomyidae). j. med. entomol. 39: 695-698, 2002. silverman n, paquette n, aggarwal k. specificity and signaling in the drosophila immune response. inv. surv. j. 6: 163-174, 2009. sowa-jasiłek a, zdybicka-barabas a, stączek s, wydrych j, mak p, jakubowicz t, et al. studies on the role of insect hemolymph polypeptides: galleria mellonella anionic peptide 2 and lysozyme. peptides 53: 194-201, 2014. strand mr. the insect cellular immune response. insect sci. 15: 1-14, 2008. suguraman m. comparative biochemistry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects. pigment cell res. 15: 2-9, 2002. uvell h, engström y. a multilayered defense against infection: combinatorial control of insect immune genes. trends genet. 23: 342-349, 2007. wang z, lu a, li x, shao q, beerntsen bt, liu c, et al. a systematic study on hemocyte identification and plasma prophenoloxidase from culex pipiens quinquefasciatus at different developmental stages. exp. parasitol. 127: 135141, 2011. wu g, liu y, ding y, yi y. ultrastructural and functional characterization of circulating hemocytes from galleria mellonella larva: cell types and their role in the innate immunity. tissue cell 48: 297-304, 2016. yu kh, kim k, lee j, lee h, kim s, cho k, et al. comparative study on characteristics of lysozymes from the hemolymph of three lepidopteran larvae, galleria mellonella, bombyx mori, agrius convolvuli. dev. comp. immunol. 26: 707-713, 2002. zdybicka-barabas a, mak p, jakubowicz t, cytryńska m. lysozyme and defense peptides as suppressors of phenoloxidase activity in galleria mellonella. arch. insect biochem. physiol. 87: 1-12, 2014. zdybicka-barabas a, mak p, klys a, skrzypiec k, mendyk e, fiołka mj, et al. synergistic action of galleria mellonella anionic peptide 2 and lysozyme against gram-negative bacteria. biochim. biophys. acta 1818: 2623-2635, 2012. 17 zdybicka-barabas a, stączek s, mak p, skrzypiec k, mendyk e, cytryńska m. synergistic action of galleria mellonella apolipophorin iii and lysozyme against gram-negative bacteria. biochim. biophys. acta 1828: 1449-1456, 2013. zhang y, huang j, zhou b, zhang c, liu w, miao x, et al. up-regulation of lysozyme gene expression during metamorphosis and immune challenge of the cotton bollworm, helicoverpa armigera. arch. insect biochem. physiol. 70: 18-29, 2009. isj 5: 41-42, 2008 isj 5: 41-42, 2008 issn 1824-307x visions and perspectives developed to cull: how a master control gene of development turned into a regulator of innate immune homeostasis v zappavigna department of animal biology, university of modena and reggio emilia, modena, italy accepted march 13, 2008 abstract a striking novel role for the caudal "master control" gene of development in the regulation of innate immune functions in insects has emerged. a recent study now adds further insight into the function of this homeobox gene in the maintenance of the immune homeostasis that is required to preserve the normal commensal community within the drosophila gut. these results point to a possible more widespread co-option of developmental regulatory genes during evolution to add tissue and/or organ-specific regulatory plasticity to innate immune systems. key words: innate immunity; homeostasis; insects; drosophila melanogaster; homeobox genes; caudal in metazoa innate immunity represents the first barrier of defense against infections caused by various types of microorganisms. insects, for instance, rely primarily on innate immunity to fight infectious microbes. an important branch of the immune defence system in these organisms is based on the production and secretion of antimicrobial peptides (amps). the inducible production of amps is one of the best-studied mechanisms of immune defence in insects. in drosophila melanogaster the synthesis of amps can be triggered by the activation of three main signalling pathways: the toll, immune deficiency (imd), and jnk pathways. both the toll and imd pathways control the transcription of amp genes via the activation of two drosophila homologs of the nfkb transcription factor, dorsal and relish. amps, however, are synthesized not only in response to infection, but are also produced constitutively in healthy individuals in a tissue-, sex-, and genespecific manner (reviewed in uvell and engstrom, 2007). understanding the molecular mechanisms underlying the constitutive and inducible expressions of amps in various tissues represents thus an intriguing novel field of study. one of the innovative concepts that have already begun to emerge in this field is that the regulation of amp expression may rely on the same gene regulatory ___________________________________________________________________________ corresponding author: vincenzo zappavigna department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: vincenzo.zappavigna@unimore.it networks that control cell fate during developmental processes. a beautiful example of this was published in a recent issue of science by ryu et al.(2008). in their work ryu and co-wokers analyzed at the molecular level the immune interactions between commensal microbiota and the drosophila gut. they found that, despite the chronical imdmediated high-level activation of relish by gut commensal microorganisms, only a subset of its target genes was expressed, remarkably excluding amps. the reason for this selective exclusion of amp gene expression was found in the specific repressive action of the caudal (cad) homeodomain transcription factor, as ryu et al. (2008) elegantly demonstrate. caudal had been previously shown to act as a master control gene in several crucial developmental processes in drosophila, including the definition of the anteroposterior axis and gut development (mlodzik and gehring, 1987; moreno and morata, 1999; lengval and iwaki, 2002). in addition, cad has been recently found to be crucial for constitutive amp expression in salivary glands and ejaculatory duct epithelia, implicating for the first time this developmental regulatory gene in a constitutive innate immune strategy (ryu et al., 2004). ryu et al. (2008) now show that the knockdown of cad expression in gut cells via transgenic rnai restores the production of amps in the gut, indicating that cad acts as a gut-specific transcriptional repressor of commensal-induced nfkb-dependent amp expression. strikingly, cad knockdown in intestinal cells was also accompanied by a significant rise in gut epithelial cell apoptosis. as it turned out, gut cell death was induced as a secondary effect to amp hyperactivation due to 41 drastic changes in the gut commensal community structure. in particular two bacterial strains were shown to vary considerably in their abundance upon cad knock-down. a911 bacteria, which represents the dominant strain in the gut microbial community, were significantly reduced in number in cad knockdown flies, whereas the g707 strain, which is a normally a minor member of the commensal community in the drosophila gut, emerged as a dominant commensal. g707 bacteria revealed to be responsible for the observed induction of apoptosis in gut cells and the consequent rise in mortality of host flies. interestingly, g707 bacteria fed to animals with a normal gut commensal community did not induce apoptosis, and germ-free animals first colonised with a911 bacteria prevented the growth of g707 bacteria, indicating that the normal gut microbial community is sufficient to suppress the growth of pathogenic bacteria. a911 bacteria were furthermore found to be sensitive to amps, whereas g707 bacteria were much less so, thus explaining their rise in number in cad knock-down ampexpressing drosophila guts. overall, the results by ryu et al. (2008) show that the maintenance of the immune homeostasis required for the preservation of the normal commensal community of the drosophila gut ultimately rests on the gut-specific repressive action of the cad homeodomain transcription factor on amp genes. drosophila intestinal epithelia have thus evolved a remarkable immune strategy, which entails the recruitment of a developmental regulatory gene whose repressive action allows the selective survival of non-pathogenic commensal bacteria capable of maintaining homeostasis via colonization resistance. on a broader perspective, it is tempting to speculate that the co-option of developmental regulatory genes expressed in a tissueand/or organ-specific manner may offer the unique evolutionary advantage of adding regulatory plasticity to the innate immune system. indeed, the transcriptional control of amps by tissue-specific transcription factors would meet the needs for an infection-independent costitutive expression (or repression, as in the case of the drosophila gut) that is tailored to obtain a spatially-differentiated immune defense. it is therefore not unlikely that cad will represent the first example of a whole series of developmental "master control" genes that will reveal in the near future to play fundamental roles in the tissueand/or organ-specific regulation of the innate immune system function. references lengyel ja, iwaki dd. it takes guts: the drosophila hindgut as a model system for organogenesis. dev. biol. 243: 1-19, 2002. mlodzik m, gehring wj. expression of the caudal gene in the germ line of drosophila: formation of an rna and protein gradient during early embryogenesis. cell 48: 465-478, 1987. moreno e, morata g. caudal is the hox gene that specifies the most posterior drosophile segment. nature 400: 873-877, 1999. ryu jh, kim sh, lee hy, bai jy, nam yd, bae jw, et al. innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in drosophila. science 319: 777782, 2008. ryu jh, nam kb, oh ct, nam hj, kim sh, yoon jh, et al. the homeobox gene caudal regulates constitutive local expression of antimicrobial peptide genes in drosophila epithelia. mol. cell. biol. 24: 172-185, 2004. uvell h, engstrom y. a multilayered defense against infection: combinatorial control of insect immune genes. trends genet. 23: 342-349, 2007. 42 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj101.pdf 69 isj 2: 69-74, 2005 issn 1824-307x minireview are matrix metalloproteinases the missing link? f mannello1, g tonti1, s papa2 1istituto di istologia e analisi di laboratorio, università studi “carlo bo”, urbino, italy 2istituto di scienze morfologiche, università studi “carlo bo”, urbino, italy accepted may 25, 2005 abstract it is more and more evident that the matrix metalloproteinase (mmp) system is not a characteristic feature of vertebrate animals only, as it can also be found in many invertebrate organisms. this endopeptidase family has been widely studied since its first member was described 40 years ago during metamorphosis in tadpole tails. many researches have been carried out in mammals in order to elucidate and analyze the several and important roles these endopeptidases play, both in physiological pathways and in pathological processes. the evolving researches of these multifaceted enzymes enter the very interesting and fascinating world of the invertebrates, where these enzymes seem to be in the front line during important biological events. mmp-like enzymes and their inhibitors have been found in insects, crustaceans, mussels, sea urchins and also in organisms as simple as hydra. in these species mmps partake in several fundamental processes, such as extracellular matrix (ecm) remodelling, embryonic development, cell growth and differentiation and also in defense mechanisms thus highlightening their intriguing and unexpected functional importance in invertebrate life too. key words: development; embryogenesis; extracellular matrix; invertebrates; metalloproteinases; tissue inhibitors of metalloproteinases introduction the discovery of the first member of the matrix metalloproteinase (mmp) family set the way to a new line of research dealing with this novel class of enzymes. an extraordinary number of new discoveries are contributing to put together the many pieces of this intruiging mmp puzzle world, adding more and more informations on how these enzymes work. the first enzyme with the capacity of degrading interstitial collagenase was found in the tail of a tadpole (gross and lapiere, 1962) and from then onwards the search continued and brought to the identification of more than 25 vertebrate mmps (brinckerhoff and matrisian, 2002; mott and werb, 2004; mannello et al., 2005a). corresponding author: ferdinando mannello istituto di istologia ed analisi di laboratorio, via e. zeppi, snc, università studi “carlo bo”, 61029 urbino (pu), italy e-mail: f.mannello@uniurb.it at the beginning there were the “vertebrate mmps”... mmps are endopeptidases characterized by their zinc-dependece and by a highly conserved sequence that contains three histidines necessary for binding the zinc ion at the catalytic site, and a conserved methionine turn that lies beneath the active site zinc. (stöcker et al., 1995). mmps are classified according to their substrate specificity and are subdivided into collagenases, gelatinases, elastases, stromelysins and membrane-type mmps; moreover, they are also classified depending on their domain structure. the nterminal portion of all mmps (pre-domain) is a signal sequence due to be removed whose function is to guide the mmp synthesis to the endoplasmic reticulum and their following secretion in the extracellular environment. mmps are secreted in a zymogenic form and the latency of these enzymes is maintained by a mechanism known as the “cysteine switch”; the unpaired cysteine in the pro-domain of latent mmps forms a bridge with the catalytic zinc, thus maintaining the enzyme inactive as zymogen until this interaction is abolished by mechanical disruption or cleavage by 70 other proteinases yielding a fully active enzyme (vu and werb, 2000). the third fundamental domain of mmps is the catalytic domain where the zinc ion is coordinated with three histidine residues and its fourth ligand is represented by a water molecule (overall, 2004). most mmps are characterized by a hemopexin/vitronectin-like sequence that is linked to the catalytic domain by a hinge region that can vary in length (baragi et al., 1994). two mmps, known as gelatinases, are peculiar as they show the insertion of three fibronectin-like repeats within their catalytic domain which are necessery for binding and degrading specific substrates (shipley, 1996). mmps are not only secreted enzymes, as there are membrane-type mmps which have a single-pass transmembrane domain and a short cytoplasmic cterminal tail, or a short c-terminal hydrophobic region (itoh et al., 1999). mmp activity is specifically inhibited by endogenous tissue inhibitors of metalloproteinases (timp) which reversibly bind previously activated mmps in a 1:1 stoichiometric ratio and differ in their expression patterns and mmp affinity (gomez et al., 1997; mannello and gazzanelli, 2001). also nonspecific inhibitors (e.g., á2-macroglobulin) can control mmp activity (mannello et al., 2005b). the level of expression of mmps by unstimulated cells and in intact tissues is generally low. the mmp expression is inducible by cytokines, growth factors, physical stress, oncogenic transformation, by cell-cell and cellmatrix interactions (stamenkovic, 2003), even if their expression is regulated primarily at the level of transcription and their proteolytic activity requires zymogen activation (mannello et al., 2005b). mmps can degrade almost every component of the extracellular scaffold but their role is not limited only to the breakdown of structural components, as they have been found to be strongly involved in many physiological processes, such as cell migration, tissue morphogenesis, wound healing and in the modulation of the bioavailability of active molecules (vu and werb, 2000), and in pathological situations such as inflammation, cancer development and metastasis (stamenkovic, 2003; mannello et al., 2005b). ...but something else emerges from the depth of the sea world... one of the first invertebrates that has been analyzed in this context is the sea urchin in its different embryogenetic stages and it has been demonstrated that normal developmental processes (i.e. spiculogenesis and gastrulation) necessitate collagen deposition and ecm modifications (spiegel et al., 1989), thus evidencing the necessity of enzymes with proteolytic activity (wessel et al., 1984). during the blastula early stages the sea urchin regulates the transcription and secretion of the hatching enzyme (envelysin) which degrades the protective envelope. this collagenase-like enzyme is structurally very similar to the vertebrate counterpart as it is characterized by domains displaying the same functional roles found in mammalian collagenases; in addition, it posesses its own distinctive sequences. (lepage and gache, 1990; roe and lennarz, 1990; nomura et al., 1997). during the following embryonic developmental stages, the sea urchin expresses a 41 kda protease which shows substrate specificity towards gelatin and extraembryonic collagen components (mayne and robinson, 1996; mayne and robinson, 1998). this gelatinase has been found in both the hyaline and basal lamina, which contain components similar to those found in vertebrate ecms (wessel et al., 1984). this 41 kda enzyme is secreted on the apical surface of the embryo and prior to its secretion it can be detected in the cortical and in the yolk granules (mayne and robinson, 1998). the structural organization of this collagenase is closely related to that of vertebrate mmps as it has a signal and a pro-peptide, a zn2+-binding catalytic domain and a hemopexin-like c-terminal domain (nomura et al., 1991). in the gastrula and pluteus stages the sea urchin expresses an 87 kda protease which can specifically cleave gelatin and can control shapechanges, cell-cell and cell-ecm interactions during the gastrula and pluteus stages through the regulation of ecm composition (robinson, 1997). this enzyme is ca2+ and zn2+ dependent, but its activation mechanism may be different from that of the known “cysteine switch”, as demonstrated by inhibitory and activating studies. the two proteinases (41 and 87 kda) described up to now are both zn2+-dependent and also need low affinity ca2+ binding for activity; moreover, mg2+ seem to have an inhibitory effect on the enzyme (robinson, 2000). this contrasting effect of ca2+ and mg2+ on the gelatinase activity could cause the fine regulation and modulation of the enzymatic activity on the cell surface, as small variations in the ion concentrations, obtained through the binding capacity of extraembryonic matrix, can regulate the enzyme activity (robinson and mayne, 1998). the hyaline layer of the sea urchin was analyzed for mmp activity during the transition from early to late stage embryos and enzymes (initially secreted as proenzymes and subsequently proteolytically activated) with molecular masses of 94/117, 90 and 45 kda with gelatin specificity and ca2+-zn2+ dependence were found and suggested to be matrix metalloproteinases (flood et al., 2000). the exact relationship between the 94/117 kda and 90 kda species and those found in the sea urchin embryo is still unclear. also the process of skeleton formation and the process of spiculogenesis seems to be correlated to the action of metalloproteinases, as inhibitors of these enzymes block these morphological events (ingersoll et al., 2003). in echinoderms, mmps are not only involved during embryonic development, but play important roles also during tissue/organ regeneration due to the modifications that need to occur in the ecm. evidence of this is the expression and activity of mmps during early stages of intestinal regeneration in the sea cucumber holothuria glaberrima (quinones et al., 2002). a gelatinase has also been found in the digestive tissues of the crab scylla serrata. all the identified crustacean collagenases belong to the serine protease family, while this novel enzyme results as a metalloproteinase with gelatin specificity. this enzyme has high proteolytic activity at low temperatures and acts on a wide range of substrates (this could be due to the fact that lower animal collagenases are necessary for the digestion of collagen containing tissues of the prey that these animals feed on) but its 71 preferential substrate is represented by denatured collagen (sivakumar et al., 1999). the barnacle balanus amphitrite is a thoracican cirriped crustacean that is due to undergo many complex and substantial morphological changes before it metamorphoses to the final stage of larval development: the lecithotrophic cypris larva. these developmental processes depend on the activity of specific extracellular matrix-degrading enzymes as b. amphitrite naupliar stages contain several proteinases specific towards different gelatin substrates evidencing their involvement in all phases of larval growth and development. substrate and activity analyses collocate these proteolytic enzymes in the mmp family as they are specific towards gelatin substrates and dependend on zn2+ and ca2+ ions (mannello et al., 2003). these enzymes could also be dependent on mg2+ suggesting that the enzyme activity could be regulated by these cations, as can be seen also in the sea urchin embryo (robinson, 2000) and in the mussel mytilus galloprovincialis (mannello et al., 2001). even though the activation mechanism of the barnacle mmps is quite similar to the one observed for mammals it is probably not regulated by the same cysteine switch trigger but has its own unique setup (robinson, 1997; mannello et al., 2001). the serum and hemocytes of the eastern oyster crassostrea virginica have been analyzed for ecmdegrading activity underlining the role of ecm ptoteolytic enzymes in both normal and diseased molluscs (ziegler et al., 2002). a mmp-like enzyme has been fonud in the hemocytes, but not in the serum of c. virginica. this enzyme has been proven to be an mmp due to its gelatin and collagen degrading activity and to its inhibition profile. this mmp is not involved in the degradation of interiorized phagocytosed materials, but it is probably active in the external environment. these findings support the hypothesis that hemocyte derived mmp-like activity may control the remodelling and development of ecm and may be also involved in the response towards pathogen invasion (ziegler et al., 2002). the new exciting role mmp may have during deseased or damaged states has been further investigated in the pacific oyster crassostrea gigas and a mmp inhibitor named cg-timp with functional characteristics extremely similar to vertebrate was identified (montagnani et al., 2001). this inhibitor binds and blocks mmps but posesses other functions probably regulated by an additional pair of cysteine residues in the carboxy-terminal domain (this could be a characteristic of invertebrate timps as it is present also in drosophila timp). cg-timp could be strongly involved in pathogen protection or in wound healing processes as it was only expressed in hemocytes and showed increased activity after shell damage or bacterial infection, suggesting new insights for the anti-microbial defense of marine invertebrates (bachere et al., 2004). gelatinolytic activity similar to that found in c. virginica was discovered in the hemocyte and serum homogenates of the mussel m. galloprovincialis (mannello et al., 2001). this proteolytic enzyme was similar to known mmps due to its gelatinase and collagenase activity and to its ionic requirements, but exhibited different activating and inhibiting processes suggesting that in molluscs the activation mechanism differs from the vertebrate “cysteine switch”. in healthy mussels this gelatinase may regulate normal physiological functions such as cell migration and tissue infiltration, but mmp activity goes beyond these roles being of great importance during cell-mediated and humoral immune responses (chen and bayne, 1995) and moreover in wound repair, inflammation, internal defense and also in pathological conditions as hematopoietic neoplasia (riginos and cunningham, 2005). it is clearly emerging that these mmp-like enzymes touch several aspects of oyster and mussel biology as they take part to tissue homeostasis and are also strongly involved in defence mechanisms, or because they get produced directly by the pathogenic agent (norqvist et al., 1990; lepore et al., 1996) or because they are stimulated to be secreted by the pathogen itself (okamoto et al., 1997). mmp-like enzymes have been found in a member of the cnidaria family as hydra; the entire body wall of this organism is structurally reduced to an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix that contains basement membrane components such as laminin and interstitial matrix components such as a unique type i fibrillar collagen. the simple structure of this metazoan and the relation between developmental processes and cell-ecm interaction led to the search for mmp-like enzymes (zhang and sarras, 1994). a single hydra matrix metalloproteinase, hmmp, with a strong sequence similarity to human mmps was identified, (even though it contains some unique amino acid stretches) (leontovich et al., 2000). hmmp is not only structurally similar to vertebrate mmps, but it also shares functional characteristics as inhibition by specific mmp-inhibitors and substrate specificity towards hydra ecm molecules and gelatin. activation studies on hmmp demonstrate that it can be activated intracellularly by a furin-like enzyme and that there can be an intermediate step before reaching the fully active enzymatic form. hmmp has been studied during foot and head regeneration processes (leontovich et al., 2000). these results clearly evidence a direct implication of this enzyme during morphogenetic events as hmmp is involved in cell transdifferentiation (werb and chin, 1998) and regenerative processes being implicated in biogenesis (shimizu et al., 2002). hmmp is secreted by the cells belonging to the endoderm evidencing that although hydra ecm has a symmetrical structure, its components are synthesized in a non-symmetrical manner (shimizu et al., 2002). besides having a important role during regenerative events and also in the maintainance of the differentiated state of certain cells (leontovich et al., 2000), hmmp may participate in the regulation of the bioavailability of signalling molecules which are sequestered by the ecm, and can be released by a hmmp dependent proteolytic cleavage (muller, 1996). hmmp demonstrates to be a fundamental element in several processes of the hydra underlining the importance of ecm-related mechanisms. molecules similar to mmp inhibitors have been found in low metazoans such as sponges; callyspongia truncata produces callysponginol sulphate a (fujita et al., 2003a) and agelas nakamurai expresses a novel mmp inhibitor, ageladine a which also possesses antiangiogenic activity (fujita et al., 2003b). 72 ... and spreads into the sky... drosophila melanogaster expresses two matrix metalloproteinases: dm1-mmp and dm-2-mmp. dm1mmp, as vertebrate mmps, contains a signal sequence necessary for secretion, a pro-peptide with a cys residue that maintains the enzyme in an inactive zymogenic form, a catalytic domain with the zinc-binding site and finally a hinge region and a nterminal hemppexin domain. one more aspect of this newly discovered proteinase is that its activation could also be regulated by furin-like proteases, due to the presence of a furin-like cleavage site located at the end of the pro-peptide (roebroek et al., 1993; llano et al., 2000). studies on substrate specificity of recombinant dm1-mmp evidenced proteolytic activity towards extracellular matrix and basement membrane proteins, such as fibronectin and type iv collagen which are present in drosophila (fessler and fessler, 1989). on the bases of several reports it can be to hypothesized that this mmp could be directly involved in the guidance and extention of axons during the nervous system development, as it was found to be secreted by the glial cells of larval tissues (menne et al., 1997; llano et al., 2000); this could depent or on the brakdown of extracellular barriers or on the release of hidden, not yet available signal molecules. dm2-mmp, is similar to dm1-mmp by posessing the mmp distinctive structural domains, but it differs from dm1-mmp because of the presence of a 200 amino acid-long insertion in the hinge region (llano et al., 2002) and also because this enzyme is expressed in all of the developmental stages of the fly, while dm1mmp is present in the developing embryo at stages 12 and 13 (llano et al., 2000). one more difference is that dm1-mmp is a secreted enzyme, while dm2mmp is a membrane bound proteinase. due to the differential pattern of expression of the two mmps it could be possible that they have different functional roles. it has been hypotesized that dm2-mmp may be involved in photoreceptor growth cone guidance and cell rearrangement in the retina and nervous system (llano et al., 2002). dm1-mmp seems to be determinant for larval tracheal growth and pupal head reversion, while dm2-mmp participates to larval tissue histolysis and epithelial fusion during metamorphosis and both enzymes seem to be required for tissue remodeling (page-mccaw et al., 2003). for a regulated mmp activity the presence of mmp inhibitors is fundamental, and infact one inhibitor of matrix metalloproteinases has been found in the fly (pohar et al., 1999) and it is structurely closely related to mammalian timps (wei et al., 2003). it is clear that drosophila in its relative semplicity unravels a highly regulated functional proteolytic system involved in several biological and physiological processes which could account for a common origin with the fully evolved vertebrate mmp system (wei et al., 2003). the larvae of the greater wax moth, galleria mellonella, is peculiar in that it containes the first insect inhibitor of metalloproteinases (impi) with no similarity at all with other known vertebrate or invertebrate counterparts. it is possible that this impi is implicated during the response of g. mellonella to invading pathogens as it is released during the humoral immune response (probably stimulated by particular peptidic fragments) and is able to protect the insect from exogenous metalloproteinases of pathogen origin such as bacterial thermolysin (wedde et al., 1998; vilcinskas and wedde, 2002). ... and into every nook and craggy ... three gene products (mmp-c31, h19 and y19) encoding matrix metalloproteinases have been found in the nematode caenorhabditis elegans. these enzymes have a domain organization very similar to human mmps, in particular the catalytic sites of these newly discovered mmps show high sequence homology with human interstitial collagenase. interestingly, two of these nematode mmps were inhibited by specific human mmp inhibitors evidencing a similar mmp system between mammalian and c. elegans mmps (wada et al., 1998). a glycoprotein similar to c. elegans mmps was analyzed in the larvae of gnathostoma spinigerum and this protein showed to possess an n-terminal signal peptide necessary for its secretion and the catalytic domain which are two elements distinctive of other known mmps (uparanukraw et al., 2001). considerations it is evident that in all organisms the role of the ecm goes beyond that of a mere scaffold having only structural functions, but controls many more complex and important processes such as cell shape, growth, migration and differentiation as a consequence of its capacity of sequestering signal molecules. the involvement of the ecm components in such biological pathways led to a great amount of rersearch in order to better understand the mechanisms that regulate the organization of extracellular environment. key elements in the regulation of ecm components are the proteinases belonging to the mmp family; these enzymes seem to hide many surprises as their functional roles touch many aspects of physiological and pathological processes and have been widely studied not only in vertebrate models but also in invertebrates (massova et al., 1998). mmp-like enzymes with their relative inhibitors have been discovered in animals belonging to distantly related taxa, evidencing the existence and importance of such a proteolytic system also in relatively simple organisms. even though mmps and relative inhibitors are present in such a varied range of organisms, they share many features characteristic of the vertebrate mmp family, suggesting a possible common ancient origin. these vertebrate and invertebrate enzymes may share structural similarities, but it is amazing to note that invertebrate mmps take part in many processes such as embryogenesis, differentiation, cell migration, wound healing and immune defense, evidencing that also in these organisms mmps are far more than simple structure destroyers. it is evident that in all these organisms mmps and their inhibitors are closely linked to ecm remodelling displaying multiple roles that touch many aspects of animal physiology and demonstrating that in such evolutionary distant organisms several biological pathways follow similar laws. 73 finally, this overview add force to the emerging concept that mmps and timps are universal and ubiquitous in animals, and that invertebrates may provide further novel information for the understanding of the multifaceted physiological roles of this ancient proteolytic system (mannello et al., 2005a); numerous evidences suggest that both mmp and timp expression and their remodelling functions appear well conserved in invertebrates, laying the hypothetical basis on their possible biological link between proterostomata and deuterostomata. references bachere e, gueguen y, gonzalez m, de lorgeril j, garnier j, romestand b. insights into the antimicrobial defense of marine invertebrates: the penaeid shrimps and the oyster crassostrea. immunol. rev. 198: 149-168, 2004. baragi vm, fliszar cj, conroy mc, ye qz, shipley jm, welgus hg. contribution of the c-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases. c-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-lenght stromelysin. j. biol. chem. 269: 12692-12697, 1994. brinckerhoff ce, matrisian lm. matrix metalloproteinase: a tail of a frog that became a prince. nat. rev. mol. cell biol. 3: 207-214, 2002. chen jh, bayne cj. bivalve mollusc hemocyte behaviours: characterization of hemocyte aggregation and adhesion and their inhibition in the california mussel (mytilus californianus). biol. bull. 188: 255-256, 1995. fessler jh, fessler li. drosophila extracellular matrix. annu. rev. cell biol. 5: 309-359, 1989. flood j, mayne j, robinson jj. identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo. biochem. cell. biol. 78: 455-562, 2000. fujita m, nakao y, matsunaga s, van soest rw, itoh y, seiki m, fusetani n. callysponginol sulfate a, an mt1mmp inhibitor isolated from the marine sponge callyspongia truncata. j. nat. prod. 66: 569-571, 2003a. fujita m, nakao y, matsunaga s, seiki m, itoh y, yamashita j, van soest rw, fusetani n. ageladine a: an antiangiogenic matrix metalloproteinase inhibitor from the marine sponge agelas nakamurai. j. chem. soc. 125: 15700-15701, 2003b. gomez de, alonso df, yoshiji h, thorgeirsson up. tissue inhibitors of matrix metalloproteinases: structure, regulation and biological functions. eur. j. cell biol. 74: 111-122, 1997. gross j, lapiere cm. collagenolytic activity in amphibian tissues: a tissue culture assay. proc. natl. acad. sci. usa 48: 1014-1022, 1962. ingersoll ep, mcdanald kl, wilt fh. ultrastructural localization of spicule matrix proteins in normal and metalloproteinase inhibitor-treated sea urchin primary mesenchyme cells. j. exp. zool. a comp. exp. biol. 300: 101-112, 2003. itoh y, kajita m, kinoh h, mori h, okada a, seiki m. membrane type 4 matrix metalloproteinase (mt4-mmp, mmp-17) is a glycosylphosphatidylinositol-anchored proteinase. j. biol. chem. 274: 34260-34266,1999. leontovich aa, zhang j, shimokawa k, nagase h, sarras mp jr. a novel hydra matrix metalloproteinase (hmmp) finctions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot process. development 127: 907-920, 2000. lepage t, gache c. early expression of a collagenase-like hatching enzyme gene in the sea urchin embryo. embo j. 9: 3003-3012,1990. lepore ls, roelvink pr, granados rr. enhancin, the granulosis virus protein that facilitates nucleopolyhedrovirus (npv) infections, is a metalloproteinase. j. invert. pathol. 68: 131-140, 1996. llano e, pendas am, aza-blanc p, kornberg tb, lopez-otin. dm1-mmp, a matrix, metalloproteinase from drosophila with a potential role in extracellular matrix remodelling during neural development. j. biol. chem. 275: 3597835985, 2000. llano e, adam g, pendas am, quesada v, sànchez m, santamarìa i, stèphane n, lòpez -otin c. structural and enzymatic characterization of drosophila dm2-mmp, a membrane bound matrix metalloproteinase with tissuespecific expression. j. biol. chem. 277: 23321-23329, 2002. mannello f, gazzanelli g. tissue inhibitors of metalloproteinases and programmed cell death: conundrums, controversies, and potential implications. apoptosis 6: 479-482, 2001. mannello f, canesi l, gazzanelli g, gallo g. biochemical properties of metalloproteinases from the hemolymph of the mussel mytilus galloprovincialis lam. comp. biochem. physiol. 128b: 507-515, 2001. mannello f, canesi l, faimali m, piazza v, gallo g, geraci s. characterization of metalloproteinase-like activities in barnacle (balanus amphitrite) nauplii. comp. biochem. physiol. 135b: 17-24, 2003. mannello f, luchetti f, falcieri e, papa s. multiple roles of metalloproteinases during apoptosis. apoptosis 10: 1924, 2005a. mannello f, tonti g, papa s. matrix metalloproteinase inhibitors as anticancer therapeutics. curr. cancer drug targets 5: 285-298, 2005b. massova i, kotra lp, fridman r, mobashery s. matrix metalloproteinases: structure, evolution, and diversification. faseb j. 12: 1075-1095, 1998. mayne j, robinson jj. purification and metal ion requirement of a candidate matrix metalloproteinase: a 41 kda gelatinase activity in the sea urchin. biochem. cell biol. 74: 211-218, 1996. mayne j, robinson jj. calcium-protein interactions in the extracellular environment: calcium binding, activation, and immunolocalization of a collagenase (gelatinase activity expressed in the sea urchin embryo. j. cell. biochem. 71: 546-558, 1998. menne tv, luer k, technau gm, klambt c. cns midline cells in drosophila induce the differentiation of lateral neural cells. development 124: 4949-4958, 1997. montagnani c, le roux f, berthe f, escoubas jm. cg-timp, an inducible tissue inhibitor of metalliproteinase fron the pacific oyster crassostrea gigas with a potential role in wound healing and defense mechanisms. febs lett. 500, 64-70, 2001. mott jd, werb z. regulation of matrix biology by matrix metalloproteinases. curr. opin. cell biol. 16: 558-564, 2004. muller wa. head formation at the basal end and mirror-image pattern duplication in hydra vulgaris. int. j. dev. biol. 40:1119-1131, 1996. nomura k, tanaka h, kikkawa y, yamagachi m, suzuki n. the specificity of the sea urchin hatching enzyme (envelysin) places it in the mammalian matrix metalloproteinase family. biochem. 30: 6115-6123, 1991. nomura k, shimizu t, kinoh h, sendai y, inomata m, suzuki n. sea urchin hatching enzyme (envelysin): cdna cloning and deprivation of protein substrate specificity by autolytic cleavage. biochem. 36: 7225-7238, 1997. norqvist a, norman b, wolf-watz h. identification and characterization of a zinc metalloproteinase associated with invasion by the fish pathogen vibrio anguillarum. infect immun. 58: 3731-3736, 1990. okamoto t, akaike t, suga m, tanase s, horie h, miyaima s, ando m, ichinose y, maeda h. activation of human 74 matrix metalloproteinases by various bacterial proteinases. j. biol. chem. 272: 6059-6066, 1997. overall cm. dilating the degradome: matrix metalloproteinase 2 cuts to the heart of the matter. biochem. j. 383: 5-7, 2004. page-mccaw a, serano j, sante jm, rubin gm. drosophila matrix metalloproteinases are required for tissue remodeling, but not embryonic development. dev. cell. 41: 95-106, 2003. pohar n, godenschwegw ta, buchner e. invertebrate tissue inhibitor of metalloproteinase: structural and nested gene organization with the synapsin locus is conserved from drosophila to humans. genomics 57: 293-296, 1999. quinones jl, rosa r, ruiz dl, garcia-arraras je. extracellular matrix remodeling and metalloproteinase involvement during intestine regeneration in the sea cucumber holothuria glaberrima. dev. biol. 250: 181197, 2002. riginos c, cunningham cw. local adaptation and species segregation in two mussel (mytilus edulis x mytilus trossulus) hybrid zones. mol. ecol. 14: 381-400, 2005. robinson jj. characterization of a metalloproteinase: a late stage specific gelatinase activity in the sea urchin embryo. j. cell biochem. 66: 337-345, 1997. robinson jj, mayne j. the effects of ca2+ and mg2+ on the major gelatinase activities present in the sea urchin embryo. biochem. biophys. res. commun. 243: 326330, 1998. robinson jj. effects of calcium and magnesium on a 41 kda serine-dependent protease possessing collagencleavage activity. cell. biochem. 80: 139-145, 2000. roe jl, lennarz wj. biosynthesis and secretion of the hatching enzyme during sea urchin embryogenesis. biol. chem. 265: 8704-8711, 1990. roebroek aj, creemers jw, pauli ig, bogaert t, van de ven wj. generation of structural and functional diversity in furin-like proteins in drosophila melanogaster by alternative splicing of the dfur 1 gene. embo j. 12: 1853-1870, 1993. shipley jm, doyle ga, fliszar cj, ye qz, johnson ll, shapiro sd, welgus hg, senior rm.the structural basis for the elastolytic activity of the 92-kda and 72kda gelatinases. role of the fibronectin type ii-like repeats. j. biol. chem. 271: 4335-4341,1996. shimizu h, zhang x, zhang j, leontovich a, fei k, yan l, sarrs mp jr. epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases. development 129: 15211532, 2002. sivakumar p, sampath p, chandrakasan g. collagenolytic metalloprotease (gelatinase) from the hepatopancreas of the marine crab, scilla serrata. comp. biochem. physiol. 123b: 273-279, 1999. spiegel e, howard l, spiegel m. extracellular matrix of sea urchin and other marine invertebrates. j. morphol. 199: 71-92, 1989. stamenkovic i. extracellular martix remodelling: the role of matrix metalloproteinases. j. pathol. 200: 448-464, 2003. stöcker w, grams f, baumann u, reinemer p, gomis-rüth fx, mckay db, bode w.the metzincins-topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. protein sci. 4: 823-840, 1995. uparanukraw p, morakote n, harnnoi t, dantrakool a. molecular cloning of a gene encoding matrix metalloproteinase-like protein from gnathostoma spinigerum. parasitol. res. 87: 751-757, 2001. vilcinskas a, wedde m. insect inhibitors of metalloproteinases. iubmb life 54: 339-343, 2002. vu th, werb z. matrix metalloproteinases: effectors of development and normal physiology. genes dev. 14: 2123-2133, 2000. wada k, sato h, kinoh h, kajita m, yamamoto h, seiki m. cloning of three caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases. gene 211: 57-62,1998. wedde m, weise c, kopacek p, franke p, vilcinskas a. purification and characterization of an inducible metalloproteinase inhibitor from the hemolymph of greater wax moth galleria melonella. eur. j. biochem. 255: 535-543, 1998. wei s, xie z, filenova e, brew k. drosophila timp is a potent inhibitor of mmps and tace: similarities in structure and function to timp-3. biochem. 42: 1220012207, 2003. werb z, chin jr. extracellular matrix remodelling during morphogenesis. ann. ny acad. sci. 857: 110-118, 1998. wessel gm, marchase rb, mcclay dr. ontogeny of the basal lamina in the sea urchin embryo. dev. biol. 103: 235-245, 1984. zhang x, sarras mp jr. cell-extracted matrix interactions under in vivo conditions during interstitial migration in hydra vulgaris. development 120: 425-432, 1994. ziegler g, paynter k, fisher d. matrix metalloproteinase-like activity fron hemocytes of the eastern oyster, crassostrea virginica. comp. biochem. physiol. 131b: 361-370, 2002. minireview isj 7: 157-164, 2010 issn 1824-307x minireview defensins and cystein rich peptides: two types of antimicrobial peptides in marine molluscs g arenas díaz laboratorio de genética e inmunología molecular, instituto de biología, campus curauma, pontificia universidad católica de valparaíso, chile accepted june 3, 2010 abstract this review focuses on defensins and cystein rich peptides, which are the most abundant natural antimicrobial peptides (amps) described in molluscs. these are compact peptides, 3-5 kda in molecular mass, cationic and amphipatic; the presence of at least six cysteine residues forming three or four disulfide bridges is their prime structural characteristic. a 3-d structural characterization of these molecules has been included in recent investigations, using currently-available techniques. amps have been purified from hemocytes, epithelial tissue and plasma as well as cloned and chemically synthesized. their antibacterial activity against gram-positive and gram-negative bacteria and fungi has been shown; only a synthetic mytilin fragment has displayed activity against viruses. key words: defensins; cystein rich peptides; antimicrobial peptides; innate immunity; marine molluscs introduction marine molluscs are exposed to microbial pathogens in their environment, which can number up to 106 bacteria/ml and 109 virus/ml of seawater (ammerman et al., 1984). in order to defend themselves against such condition, molluscs have developed very effective mechanisms that are part of their innate immunity (tincu and taylor, 2004). antimicrobial peptides (amps) are the major component of the innate immune system in marine invertebrates (destoumieux et al., 1997, mercado et al., 2005; arenas et al., 2009; de zoysa et al., 2009). the first research on amps in bivalve molluscs through reverse genomics was done at the end of the 90s (hubert et al., 1996). amps are distinguished by their net positive charge and amino acidic residue amphipathic distribution; these key features explain their mode of action with the membrane of target microorganisms (marshall and arenas, 2003). in order for the synthetic peptides to maintain the antimicrobial activity, they must be able to form an amphipathic structure, i.e., they must be organized in hydrophobic and hydrophilic amino acid zones (zasloff, 2002; arenas et al., 2009). a general mechanism of action has been proposed describing the sequence of associated ___________________________________________________________________________ corresponding author: gloria arenas díaz laboratorio de genética e inmunología molecular, instituto de biología, campus curauma, pontificia universidad católica de valparaíso, chile e-mail: garenas@ucv.cl events occurring once the peptides are initially attracted to the target membrane of microorganisms by electrostatic attraction. then, hydrophobic interactions with the membrane ensue, followed by accumulation of the peptide until a threshold concentration is achieved. the peptide then adopts a new dynamic conformation that causes a deformation of the membrane, followed by a transient peptide conformation which enables it to insert into the membrane. in the next step the peptides multimerize forming complexes such as barrel-staves or toroid pores. in the final stages the peptide is translocated to the cytoplasmic face of the membrane to exert its action on membranous cytosolic components. different types of amps follow some or all of the steps described above. (cudic and otvos, 2002; zasloff, 2002; yeaman and yount, 2003). the purification procedure is summarized as follows: the homogenized samples are suspended in cold acetic acid 11 % (1:10) in order to solubilize cationic molecules and sonicated for 3x30 sec at 11 rms in ice. the crude extract is centrifuged at 11,000xg, 35 min at 4 °c and the pellet is discarded. the supernatant is called acid extract (ae) and is further shaked at 37 °c for 1 h to favor sugar hydrolysis. the acid extract is loaded on a sulfoethyl (se) sephadex c-50 cation-exchange chromatography column (biorad), eluted with 1 m nacl 1 % acetic acid ( ph 3.0), in order to enrich cationic peptides. the eluate is applied onto a seppak c18 vac cartridge (waters associates) 157 equilibrated in acidified water (0.05 % trifluoroacetic acid in upw (ultra pure water). after a wash with acidified water, the peptides are eluted with 5 %, 20 %, 40 %, 60 % and 80 % acetonitrile (acn), to obtain several hydrophobic fractions. the samples obtained are lyophilized and reconstituted in milliq water, total protein content determined by the bicinchoninic acid (bca) microplate assay (pierce) and tested for antibacterial activity. only those with antimicrobial fraction activity are subjected to reversed phase hplc. all purification steps are performed on a rp-hplc model lachrom d-7000 with a lachrom model l-7455 photodiode array detector. column effluents are monitored by uv absorption at 225 nm. eluates are selected for further purification and loaded on a sephasil c-18 (250x4.1 mm) column (lichrocart). elution is performed with a linear gradient of 5-60 % acn in acidified water over 90 min at a flow rate of 0.6 ml min-1. the resulting fractions are collected, lyophilized, reconstituted in ultra-pure water (upw ) and frozen at -20 °c until antimicrobial activity testing (bulet et al., 1991; charlet et al., 1996; mercado et al., 2005). among the different natural amps, those containing pairs of cysteine residues forming intramolecular disulfide bridges are particularly common (dimarcq et al., 1998; bulet et al., 2004; reddy et al., 2004; yount et al., 2006). this highly complex 3 5 kda group has been extensively studied in mussels, mytilus edulis and mytilus galloprovincialis, where they were classified into four groups: defensins, mytilins, myticins and mytimycin (charlet et al., 1996; mitta et al., 1999a; pallavicini et al., 2008; costa et al., 2009; parisi et al., 2009). defensins have been also recently described in oysters crassostrea virginica and crassostrea gigas (seo et al., 2005; gueguen et al., 2006; gonzález et al., 2007) and abalone haliotis discus discus (de zoyza et al., 2010); mytilins and myticins, on the other hand, have been also described in clams ruditapes decussates (gestal et al., 2007). defensins and cystein rich peptides from marine molluscs express a stronger activity against gram-positive and gram-negative bacteria and fungi (charlet et al., 1996; mitta et al., 1999a, b; seo et al., 2005; gueguen et al., 2006; gestal et al., 2007) and one synthetic mytilin fragment displayed activity against the white spot syndrome virus (dupuy et al., 2004; roch et al., 2008). defensins and cystein rich peptides from mussels for the summary of the amps described in mussels and the relative alignments see table 1 and fig. 1. defensins a (4314.3 da) and b (4392.4 da) were purified from the hemolymph of m. edulis using chromatographic methods. both exhibited six cysteines, forming three intramolecular disulfide bridges positioned in a highly conserved array, thus allowing a complex three-dimensional structure the cysteine consensus motif is identical to that found in the large family of arthropod defensins, phormia defensin, described in detail for the phormia terranovae (charlet et al., 1996). the latter corresponds to a central amphipathic α-helix with an extended nh2-terminal loop and a cooh-terminal antiparallel β-sheet. the helix is stabilized through two disulfide bridges to the β-sheet and the nh2terminal loop is linked to one of the strands of this sheet via the third disulfide bridge (cornet et al., 1995). using the liquid growth inhibition method (bulet et al., 1993) it was determined the mytilus defensins a and b were consistently more active against the gram-positive strain m. luteus (mic: 0.6 1.2 μm) than to the gram-negative strain e. coli (mic: 2.5 10 μm). the minimal inhibitory concentration (mic) values are expressed as an interval (a b), where (a) represents the highest peptide concentration tested at which bacteria are still growing and (b) the lowest concentration that causes 100 % growth inhibition (charlet et al., 1996). the defensin isoforms mgd-1 and mgd-2 (4 kda), containing eight cysteines, were purified from the plasma and hemocytes of mussels, m. galloprovincialis, using conventional chromatographic methods. two extra cysteines and one modified amino acid suggested that these molecules are new members of the arthropod defensins family (mitta et al., 1999b). the 3-d structure of mgd1 was established using nmr analysis, which mainly consists on the classical csαβ structural motif (cys4 cys25, cys10 cys33 and cys14 cys35 disulfide bonds). the two extra cysteines (cys21 cys38) form an original fourth disulfide bond. synthetic mgd1, correctly folded to form the four disulfide bonds, retains the antibacterial activity of the native molecule and presents a similar effect than the insect defensin a, thus proving the fourth disulfide bond of mgd1 was not significant for the biological activity (yang et al., 2000). a series of synthetic peptides, conforming the main known secondary structures of mgd1, allowed the location of the nonapeptide cggwhrlrc corresponding to the residues between cys25 and cys33. the bacteriostatic activity of such sequence was strictly dependent on the bridging of cys25 and cys33. the antibacterial activity of this synthetic nonapeptide clearly evidenced an effect on grampositive when tested against the gram-positive bacteria micrococcus lysodeikticus, staphylococcus aureus, staphylococcus epidermidis, bacillus megaterium (mic 0.6 0.8 μm) and the gramnegative bacteria vibrio alginolyticus, vibrio metschnikowii, escherichia coli 363, salmonella newport (mic>75 μm) and fusarium oxysporum (mic 5 μm) (romestand et al., 2003). modelling studies evidenced that positively charged and hydrophobic residues of mgd1 were organized in two discrete domains; this feature would support the hypothesis that positive charges allow the initial attraction of the peptide with the membrane, as well as the hydrophobic domain insertion into the bacterial lipid bilayer (romestand et al., 2003). the tissue location analysis through optical and ultrastructural levels showed mgds were mainly located in the granular structures of the hemocytes and enterocytes, where they were synthesized as an 81 amino acid precursor and processed as active 158 table 1 shows a summary of the amps described in marine bivalves peptide origin acces number mw da sequence reference defensin a m. edulis sp | p81610 4314.3 gfgcpndypchrhcksipgrxggycggxhrlrctcyr charlet et al., 1996 defensin b m. edulis sp | p81611 4392.4 gfgcpndypchrhcksipgryggycggxhrlrctc charlet et al., 1996 mgd-1 m. galloprovincialis sp | p80571 4000 gfgcpnnyqchrhcksipgrcggycggwhrlrctc mitta et al., 1999b mgd-2 m. galloprovincialis gb | aad52660 4000 gfgcpnnyachqhcksirgycggycaswfrlrctc mitta et al., 1999b aod c. virginica sp | p85008 4265.0 gfgcpwnryqchshcrsigrlggycagslrltctcyrs seo et al., 2005 cg-def c. gigas gb | aj565499 (est) emb | caj19280 n.a. gfgcpgnqlkcn nhcksiscragycdaatlwlrctc gueguen et al., 2006 cg-defh1 c. gigas gb |dq400101 n.a. gfgcprdqykcnshcqsigcragycdavtlwlrctc gonzalez et al., 2007 cg-defh2 c. gigas gb |dq400102 n.a. gfgcpgdqyecnrhcrsigcragycdavtlwlrctc gonzalez et al., 2007 abalone defensin h. discus discus gb |fj864724 4900 krvtcdllslqimgnsfgdsacaahcig lhhsgghcsggvcvcr de zoyza et al., 2010 mytilin a m. edulis sp | p81612 3773.7 gcasrckakcagrrckgwasasfrgrcyckcfrc charlet et al., 1996 mytilin b m. edulis sp | p81613 3974.3 scasrckghcrarrcgyyvsvlyrgrcyckclrc charlet et. al., 1996 mytilin b m. galloprovincialis gb |aad52661 n.a. spsdmmpqmnenentefgqdmptgeteqgetgi mitta et al., 2000 mytilin c m. galloprovincialis gb |aad45013* n.a. scasrcksrcrarrcryyvsvryggfcycrc mitta et al., 2000 mytilin d m. galloprovincialis gb |acf21701 n.a. gcasrckakcagrrckgwasasfrrrcyckcfrc mitta et al., 2000 mytilin g1 m. galloprovincialis n.a. n.a. tcgslckahctfrkcgyfmsvlyhgrcycrcllc mitta et al., 2000 myticin a m. galloprovincialis gb |aad47638 4438 hshactsywcgkfcgtakmcacvhcsrvnnpfrvnqvaksindldytpim mitta et al., 2000 myticin b m. galloprovincialis gb |aad47639 4562 hphvctsyycskfcgtaklcfclhcsrvkfpfgatqdaksmneleytpim mitta et al., 2000 myticin 1 r. decussatus n.a. n.a. qsvactsyycskfcgsakicyclhcrraesplalsgsarnvndknnemdnspvm gestal et al., 2007 myticin 2 r. decussatus n.a. n.a. vpcastycarfcgsakicyclhcrraesplalsgsarnvndknnemdnspvm gestal et al., 2007 myticin 3 r. decussatus n.a. n.a. vpcastlcsrfcgsakicyclhcrraesplalsgsarnvndqnkemdnspvm gestal et al., 2007 mytimycin m. edulis n.a. 6233.5 dccrkpfrkhcwdctagtpyygystrnifgctc charlet et al., 1996 aminoacidic sequences of the amps presented in the text with their accession number (from swissprot and genbank data bases), molecular weight and references. *the amino acids different in genbank database are highlighted in grey. n.a.: not available. peptides. the bacterial challenge caused the release of mgd-1 and mgd-2 from the stimulated hemocytes (mitta et al., 1999b, 2000). the mgd-1 and mgd-2 antibacterial test at 10ul/100ul against m. luteus, using microtitration plates that were measured 24 h post incubation at 30 °c (od 600 nm), showed inhibitory effects. antifungal activity was monitored against spores from f. oxysporum; growth inhibition was observed microscopically after 24 h incubation at 30 °c with 10ul/80ul, and quantified by measurement of optical density at 600 nm after 48 h (felhbaum et al., 1994, mitta et al., 1999b). mytilin comprise isoforms a, b, c, d and g1, containing eight cysteines, represent the second group of amps described in mussels. isoforms a and b were isolated from m. edulis plasma (charlet et al, 1996); isoforms b, c, d and g1, on the other hand, were isolated from m. galloprovincialis hemocytes (mitta et al., 2000). mytilins a (3773.7 da) and b (3974.3 da) were isolated by conventional chromatographic methods. the concentration of mytilins in the blood of mussels may be estimated at approximately 2 μm. this is a range of the mic determined for most of the tested bacteria. mytilins appeared primarily effective against gram-positive bacteria (0.6-1.2 um) and less active against gram-negative bacteria (2.5 10 um). minimal inhibitory concentration (mic) values are expressed as an interval (a b), where (a) represents the highest peptide concentration tested at which bacteria are still growing and (b) the lowest concentration that causes 100 % growth inhibition (charlet et al., 1996). 159 fig. 1 clustalw alignment of the peptides sequences from defensin, mytilin and myticin from m. edulis, m. galloprovincialis, c,virginica, c. gigas, h. discus discus and r. decussatus. the color code is basic blue, acidic red, polar without charge green and hydrophobic white, the cys are highlighted in yellow. the figures were created with jalview (clam et al., 2004) mytilins b, c, d, and g1 were isolated from m. galloprovincialis (mitta et al., 1999a). they were synthesized as precursors and processed as active peptides within the hemocytes. they were found, by confocal microscopy analysis in two subclasses of circulating granulocytes, one containing small granules and one with large clear granules (mitta et al., 2000). the 3-d solution structure of synthetic peptides derived from the structure of mytilin b (34-residue) was established by 1h nmr. this structure consists of the common cysteine-stabilized α β motif (cs αβ) closely related to the one observed in the mussel defensin mgd-1. the 8 cysteines formed four disulfide bonds (2 27, 6 29, 10 31, and 15 34) only involving the β-strand ii. the percentage of the hydrophilic and hydrophobic areas from mytilin and mgd-1 are closely related (63 % 37 % and 64 % 36 %, respectively). the c10c (accvcyrgrcycnh2) mytilin fragment showed antiviral activities again with white spot syndrome virus (wssv). this peptide was precipitated in salty water, although it was able to recover its original structure once the salt content was lowered, and appeared to be soluble in a high ionic strength environment. this fact anticipates its potential application as an antiviral agent in both aquatic and terrestrial animals and in humans. the small size and ease of synthesis of c10c enables its biotechnology development (roch et al., 2008). diversity of mrnas from mytilin b in m. galloprovincialis has been studied from circulating hemocytes, thus defining 10 individual dgge (denaturing gradient gel electrophoresis) patterns in untreated mussels (parisi et al., 2009). further analysis is required on amps polymorphism to determine the role of the environment on the polymorphism of these molecules in molluscs. mytimycin isolated from the plasma of m. edulis (6233.5 da) involves twelve cysteines engaging in the formation of six intra-molecular disulfide bridges. mytimycin demonstrated to be strictly anti-fungal when tested against the strains neurospora crassa and f. culmorum (charlet et al., 1996). myticin isolated from m. galloprovincialis is a cysteine-rich peptide produced in two isoforms, a and b. myticins a and b were isolated from the hemocytes (a 4.438 da and b of 4.562 da); myticin a was also isolated from the plasma of the mussel. the mature molecule consists of 40 residues, with four intramolecular disulfide bridges and a cysteine array in the primary structure, which is different from that of previously characterized cysteine-rich antimicrobial peptides (mitta et al., 1999a). the sequence analysis of the cloned cdnas revealed that myticin precursors comprise 96 amino acids. 160 myticins a and b displayed antibacterial activity against gram-positive bacteria, and myticin b is active against the fungus f. oxysporum and the gram-negative bacteria e. coli d31 (mitta et al., 2000). myticin c, a novel antimicrobial peptide from m. galloprovincialis (pallavicini et al., 2008), appears to be extremely polymorphic. seventy four variants with nucleotide mutations were identified using dgge (costa et al., 2009). defensin from oyster for the summary of the amps described in oysters and the relative alignments see table 1 and fig. 1. the first mollusc defensin isolated from an oyster species was named american oyster defensin (aod) (seo et al., 2005). it was purified from a gill extract of c. virginica using classical chromatography for cationic peptides. electrospray ionization mass spectroscopy (esi-ms) of aod evidenced a mass of 4265.0 da. the aod (38 amino acids) has common structural features with arthropod defensins: i.e., 1) six cysteine residues; 2) at least 4 basic amino acid residues; 3) a hydrophobic loop in the amino terminal; 4) a tetraamino acid motif gly-gly-tyr-cys; and 5) a carboxy terminal penta amino acid motif cys-thr-cys-tyrarg. aod has high sequence homology (62 73 %) with the defensins from m. edulis and m. galloprovincialis, respectively (charlet et al., 1996; hubert et al., 1996; mitta et al., 1999b). sequence homology search of the purified peptide was performed using blastp 2.2.10 and tblastn 2.2.10 on genome net (http://www.ncbi.nlm.gov/blast). the theoretical isoelectric point (pi) and molecular mass were estimated by expasy (http://www.expasy.ch tools/peptide-mass.html). sequence alignment was performed using the clustalx program (thompson et al., 1997). the antibacterial activity of the purified peptide was tested using a double-layer radial diffusion assay (lehrer et al., 1991). using the minimal effective concentration (mec) as a parameter, significant activity against the gram-positive bacteria lactococcus lactis subsp.lactis and s. aureus at 2.4 and 3.0 ug/ml, respectively, was detected,. on the other hand, a lower effect on the gram-negative bacteria e. coli d31 and vibrio parahemolyticus, mec at 7.6 and 15.0 ug/ml, respectively, was observed. mec was calculated as described (zhao et al., 2001). another defensin from an oyster was identified in the mantle of c. gigas (cg-def) (gueguen et al., 2006). the cg-def gene is continuously expressed in the mantle. the structure of the recombinant peptide in e. coli is cs-αβ like arthropod defensins, but it includes an additional disulfide bond as the mussel defensin mgd-1. nonetheless, the difference with mgd-1 is the size of their loops and the presence of two aspartic residues. the oyster cg-def cdna contained 323 bp. the 195 bp coding region encoded a 65 amino acid propeptide (genbanktm caj19280) (gueguen et al., 2003). cg-def is not synthesized as a precursor. the antimicrobial activity of the recombinant cg-def was determined against gram-positive and gram-negative bacteria and filamentous fungi, resulting mainly effective against the gram-positive strains micrococcus lysodeikticus, s. aureus brevibacterium stationi, and microbacterium maritypicum at 0.005 1.25 um (mic values). the activity of cg-def was retained in vitro at a salt concentration similar to that of seawater (gueguen et al., 2009). it has been additionally established that the two isoforms of the defensin from the hemocytes of the oyster c. gigas, cg-defh1 and cg-defh2 (43 amino acids), have four disulfide bridges. this feature is also present in the defensins mgd-1 and mgd-2 from m. galloprovincialis hemocytes and cg-def from the c. gigas mantle (gonzalez et al., 2007). a quantitative rt-pcr (qrt-pcr) analysis indicated that cg-defh2 was continuously expressed in the hemocytes of c. gigas. in addition, the level of cg-defh2 transcripts decreased drastically in the circulating hemocyte population after a bacterial challenge, thus suggesting a possible migration of the hemocytes towards the gill and mantle tissue. this fact may be correlated with an increase of cgdefh2 transcripts in such tissues (gonzález et al., 2007). rich cystein antimicrobial peptides from clams for the summary of the amps described in clams and the relative alignments see table 1 and fig. 1. myticin isoforms 1, 2 and 3 were identified and characterized for the first time in clams, ruditapes decussatus (gestal et al., 2007), using the suppression subtractive hybridization technique (ssh). suppression subtractive hybridization libraries may facilitate the identification of genes involved on bivalve immune response (tanguy et al., 2004). clam myticins (40 aa) and mytilin (34 aa) are similar to the myticins previously reported from m. galloprovincialis, as both conserved the cystein array with four intramolecular disulfide bridges, which are characteristic of the myticin family (gestal et al., 2007). clams challenged with bacteria showed that clam myticin and clam mytilin increased the expression levels 48 h post-infection using qpcrs performed on hemocytes. during the challenge, specimens were injected 100 μl (containing 107 cells/ml) of a dead bacteria mixture into the adductor muscle, which included micrococcus lysodeikticus, vibrio splendidus and vibrio anguillarum (gestal et al., 2007). aminoacidic diversity of the clam myticin was found with the previously described mussel myticins a and b and with myticin c (pallavicini et al., 2008). the variation in aminoacidic residues of the different amps may occur as a response to the recognition of different pathogens in their environment (gestal et al., 2007). 161 defensins from gastropods for the summary of the amps described in abalone and the relative alignments see table 1 and fig. 1. an abalone defensin, with 66 aa, lower molecular mass of 4.9 kda, positive charge +5, hydrophobic residue ratio 46 %, α-helical structure and with an arrangement of six cysteine residues forming three disulfide linkages in c1-c4, c2-c5 and c3-c6, was characterized from the h. discus discus. the complete coding sequence of the defensin was obtained from the abalone cdna library est database (de zoyza et al., 2010). hemocytes were collected for rna extraction. a domain sequence was identified, 24 66 aa, exhibiting the same basic characteristics of the arthropod defensin family members. it is expressed constitutively in hemocytes, gills, muscle and digestive tract; however, the transcripts in tissues were significantly induced 48 h post-infection in abalones injected into the adductor muscle with 100 μl of a bacterial mixture containing v. alginolyticus, v. parahemolyticus and lysteria monocytogenes (5x107 cells/ml). conclusions different research groups have focused on amps since they are molecules of the innate immune system of a wide range of organisms, displaying an efficient activity against pathogenic microorganisms without cytotoxic effects on eukaryotic cells. they were discovered in marine molluscs almost 20 years ago and they have been primarily identified in mussels mainly as defensins and cys-rich peptides. since then, they have been purified from tissues and the genes associated to their expression have been identified; furthermore, the phylogenetic relationships with existing molecules from other invertebrates have also been established. all these aspects have contributed to the knowledge of the immune responses of this phylum. the study of the structures and antimicrobial effects of the different amps, on the other hand, has allowed achieving significant progress in the elucidation of the different action mechanisms, thus suggesting specific models that have been integrated as part of a sequence of events of a more general mechanism. various trends have also emerged where amps are regarded as alternative molecules to classical antibiotics due to their structural and physiological characteristics, stressing the fact that in vitro assays and clinical tests until phase iii have confirmed that resistance evolution against antimicrobial peptides is less probable than that observed for conventional antibiotics, although the broad therapeutic use of amps is still unconsolidated and a long path must be covered to overcome the technical problems limiting such aspirations. although the diversity of marine molluscs is quite large, amps have been explored only in a few species; therefore, there is still a great potential to unveil new molecules in this phylum. the development of molecular biology, bioinformatics and genetic engineering techniques have allowed to produce amps in such quantities as to be used as drugs to curtail human, animal and plant diseases (zasloff, 2002; gordon et al., 2005; keymanesh et al., 2009). genetic manipulation using antimicrobial peptide coding sequences to produce transgenic plants resistant to certain pathogens is presently an emerging research field. however, genetic engineering should also take into consideration obtaining stable recombinant genomes using genes from the same species and not different species. nevertheless, such possibility still remains controversial. although the diversity of amps is astounding they still are not a solid alternative to conventional antibiotics. therefore, there are ample opportunities for research in basic and applied aspects of antimicrobial peptide biology to fulfil expectations of their use in the control of infectious diseases. references ammerman jw, fuhrman ja, hagstrom a, azam f. bacterioplankton growth in seawater. 1. growth kinetics and cellular characteristics in seawater cultures. mar. ecol. prog. ser. 18: 31-39, 1984. arenas g, guzmán f, cárdenas c, mercado l marshall sh. a novel antifungal peptide designed from the primary structure of a natural antimicrobial peptide purified from argopecten purpuratus. peptides 30: 1405-1411, 2009. bulet p, cociancich s, dimarcq j-l, lambert j, reichhart j-m, hoffmann d, et al. isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family. j. biol. chem. 266: 24520-24525, 1991 bulet p, dimarcq j-l, hetru c, lagueux m, charlet m, hegy g, et al. antimicrobial peptides: from invertebrate to vertebrate. inmmunol. rev. 198: 169-84, 2004. charlet m, chernysh s, philippe h, hetru c, hoffmann ja, bulet p. innate immunity: isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc mytilus edulis. j. biol. chem. 271: 21808-21813, 1996. clamp m, cuff j, searle sm, barton gj. the jalview java alignment editor. bioinformatics 20: 426-427, 2004. cornet b, bonmatin jm, hetru c, hoffmann ja, ptak m, vovelle f. refined three-dimensional solution structure of insect defensin a. structure 3: 435-448, 1995. costa mm, dios s, alonso-gutierrez j, romero a, novoa b, figueras a. myticin c variability in mussel: ancient defense mechanism or self/non self discrimination? dev. comp. immunol. 33: 162-170, 2009. destoumieux d, bulet p, loew d, vandorsselaer a, rodriguez j, bachere e. penaeidins, a new family of antimicrobial peptides isolated from the shrimp penaeus vannamei. j. biol. chem. 272: 28398-28406, 1997. de zoysa m, nikapitiya ch, whang i, lee js, lee j. abhisin: a potential antimicrobial peptide 162 derived from histone h2a of disk abalone (haliotis discus discus). fish shellfish immunol. 27: 639-646, 2009. de zoysa m, whang i, youngdeuk l, sukkyoung l, lee j-s, lee j. defensin from disk abalone haliotis discus discus: molecular cloning, sequence characterization and immune response against bacterial infection. fish shellfish immunol. 28: 261-266, 2010. dimarcq jl, bulet p, hetru c, hoffmann j. cysteine-rich antimicrobial peptides in invertebrates. biopolymers 47: 465-477, 1998. dupuy jw, bonami jr, roch p. a synthetic antibacterial peptide from mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp. j. fish dis. 27: 57-64, 2004 fehlbaum p, bulet ph, michaut l, lagueux m, broekaert wf, hoffman ja. insect immunity injury of drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. j. biol. chem. 269: 33159-33163, 1994. gestal c, costa m, figueras a, novoa b. analysis of differentially expressed genes in response to bacterial stimulation in hemocytes of the carpetshell clam ruditapes decussatus: identification of new antimicrobial peptides. gene 406: 134143, 2007. gonzález m, gueguen y, desserre g, de lorgeril j, romestand b, bachère e. molecular characterization of two isoforms of defensin from hemocytes of the oyster crassostrea gigas. dev. comp. immunol. 31: 332-339, 2007. gordon yj, romanowski eg, mcdermott am. a review of antimicrobial peptides and their therapeutic potential as anti-infective drugs. curr. eye res. 30: 505–515, 2005. gueguen y, herpin a, aumelas a, garnier j, fievet j, escoubas jm, et al. characterization of a defensin from the oyster crassostrea gigas. recombinant production, folding, solution structure, antimicrobial activities, and gene expression. j. biol. chem. 281: 313-23, 2006. gueguen y, romestand b, fievet j, schmitt p destoumieux-garzón d, vandenbulcke f, et al. oyster hemocytes express a proline-rich peptide displaying synergistic antimicrobial activity with a defensin. mol. immunol. 46: 516522, 2009. gueguen y, cadoret jp, flament d, barreauroumiguiere c, girardot al, garnier j, et al. immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster crassostrea gigas. gene 16: 139-145, 2003. hubert f, noel t, roch p. a member of the arthopod defensin family from edible mediterranean mussels (mytilus galloprovincialis). eur. j. biochem. 240: 302306, 1996. keymanesh k , soltani s , sardari s. application of antimicrobial peptides in agriculture and food industry. world j. microbiol. biotechnol. 25: 933-944, 2009. lehrer ri, rosenman m, harwig ssl, jackson r, eisenhaur p. ultrasensitive assay for endogenous antimicrobial polypeptides. j. immunol. methods 137: 167-173, 1991. marshall sh, arenas g. antimicrobial peptides: a natural alternative to chemical antibiotics and a potential for applied biotechnology. electron. j. biotechnol. 6: 271-284, 2003. mercado l, schmitt p, marshall s, arenas g. gill tissues of the mussel mytilus edulis chilensis: a new source for antimicrobial peptides. electron. j. biotechnol. 8: 284-290, 2005. mitta g, hubert f, noel t, roch ph. myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel mytilus galloprovincialis. eur. j. biochem. 265: 71-78, 1999a. mitta g, vandenbulcke f, hubert f, roch p. mussel defensins are synthesized and processed in granulocytes then released into the plasma after bacterial challenge. j. cell sci. 112: 42334242, 1999b. mitta g, vandenbulcke f, hubert f, roch p. original involvement of antimicrobial peptides in mussel innate immunity. febs lett. 486: 185190, 2000. pallavicini a, del mar costa m, gestal c, dreos r, figueras a, venier p, et al. high sequence variability of myticin transcripts in hemocytes of immunestimulated mussels suggests ancient host-pathogen interactions. dev. comp. immunol. 32: 213-226, 2008. parisi mg, li h, toubiana m, parrinello n, cammarata m, roch p. polymorphism of mytilin b mrna is not translated into mature peptide. mol. immunol. 46: 384-392, 2009. reddy kr, yedery rd, aranha c. antimicrobial peptides: premises and promises. int. j. antimicrob. agents 24: 536-47, 2004. roch p, yang y, toubiana m, aumelas a. nmr structure of mussel mytilin, and antiviralantibacterial activities of derived synthetic peptides. dev. comp. immunol. 32: 227-238, 2008. romestand b, molina f, richard v, roch p. key role of the loop connecting the beta strands of mussel defensin in its antimicrobial activity. eur. j. biochem. 270: 2805-2813, 2003. seo jk, crawford jm, stone kl, noga ej. purification of a novel arthropod defensin from the american oyster, crassostrea virginica. biochem. biophys. res. commun. 338: 19982004, 2005. tanguy a, guo x, ford se. discovery of genes expressed in response to perkinsus marinus challenge in eastern (crassostrea virginica) and pacific oysters (c. gigas). gene 338: 121131, 2004. tincu ja, taylor sw. antimicrobial peptides from marine invertebrates. antimicrob. agents chemother. 48: 3645-3654, 2004. thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. the clustal x windows interface: flexible strategies for multiple alignments aided by quality analysis tools, nucleic acids res. 25: 4876-4882, 1997. 163 yang y, mitta, g, chavanieu a, calas b, sanchez j, roch p. solution structure and activity of the synthetic four disulfide bond mediterranean mussel defensin (mgd-1). biochemistry 39: 14436-14447, 2000. zhao c, nguyen t, boo lm, hong t, espiritu c, orlov d, et al. rl-37, an alpha-helical antimicrobial peptide of the rhesus monkey. antimicrob. agents chemother. 45: 2695-2702, 2001. yount ny, bayer as, xiong yq, yeaman mr. advances in antimicrobial peptide immunobiology. biopolymers 84: 435-458, 2006. zasloff m. antimicrobial peptides of multicellular organisms. nature 415: 389-395, 2002. 164 ecotoxicology of nanomaterials: the role of invertebrate testing isj 6: 78-97, 2009 issn 1824-307x review ecotoxicology of nanomaterials: the role of invertebrate testing ag cattaneo1, r gornati1, m chiriva-internati2, g bernardini1,3 1 department of biotechnology and molecular sciences (dbms), university of insubria, varese, italy 2 texas tech university health sciences center, lubbock, tx 79430, usa 3 centro interuniversitario politecnico di milano e università dell'insubria "the protein factory" accepted june 15, 2009 abstract engineered nanomaterials represent a new and expanding class of chemicals whose environmental hazard is actually poorly determined. the peculiar behavior of nanomaterials makes them much more similar to new chemicals than to the corresponding bulk materials; this feature imposes reliable and standardized evaluation protocols for toxicity and ecotoxicity assessments. general rules for assessing nanotoxicity and the state of the art are periodically published in reports by control agencies. this review highlights the role of invertebrates as valuable and validated test organisms for assessing ecotoxicity of new and/or untested chemicals. the general scarcity of experimental data, their unequal distribution among the different nanomaterials and environmental conditions, the difficulties in manipulating nanomaterials and obtaining stable and homogeneous suspensions, the confusion arising from a not well defined metrics are discussed. key words: engineered nanomaterials; nanotoxicology; invertebrates introduction assessing the ecotoxicity of previously untested substances, as in the case of nanomaterials, is a challenging task. therefore, inexpensive, rapid and reproducible methods are preferred, and a coordinated standardization could help in avoiding the waste of resources. nanomaterial is a material having at least one dimension 100 nm or less. nanomaterials can be nanoscale in one dimension (e.g., films), two dimensions (e.g., fibers and tubes), or three dimensions (e.g., particles). nanoparticles constitute a sub-fraction of what is defined as “colloids” (christian et al., 2008). chemicals fitting these requirements share protean physical chemistry, which confers very unusual properties to them. the colloid nature, the ability to form aggregates and an appealing potential for practical applications remain nevertheless common features, explaining the growing interest in engineered nanomaterials. scientists studying this field were awarded twice with the nobel prize for chemistry. this exclusive acknowledgment was firstly given in 1996 ___________________________________________________________________________ corresponding author: anna giulia cattaneo department of biotechnology and molecular sciences (dbms) university of insubria via j-h dunant 3, 21100 varese, italy e-mail: annagiulia.cattaneo@uninsubria.it for the fullerene synthesis discover (curl, 1996; kroto, 1996; smalley, 1996), and then in 2000 for research on conductive and semi-conductive nanopolymers (heeger, 2000; macdiarmid, 2000; shirakawa, 2000). since then, the production of newer engineered nanomaterials and their applications exponentially increased, spanning from cosmetics, drug delivery systems and food additives, to products for waste remediation and fuel and energy production, the so called environmentally friendly nanotechnologies (tungittiplakorn, 2005; hollins, 2007). space and military applications of nanomaterials range at the present from protective clothing, sensors and signals, to propellants and explosives, and more (for reviews see ruffin, 2004; glenn, 2005). at the same time, basic research in the fields of nanoscience and nanotechnology improved rapidly: manufactured and natural nanomaterials branched out as distinctive fields, theories and models developed about their ability to actively interact with environmental and biological systems and the issue of their potential hazard for health and environment has been posed (oberdörster, 2004; oberdörster et al., 2005; moore, 2006; chun ke and qiao, 2007; oberdörster et al., 2007; christian et al., 2008; handy et al., 2008a, 2008b; di gioacchino et al., in press). nevertheless, nanomaterials remain very poorly tested potential pollutants, in contrast with their 78 mailto:annagiulia.cattaneo@uninsubria.it large diffusion: main difficulties in assessing toxicity are a consequence of their colloidal nature and dynamics, as systems in which smaller or larger aggregates can form in poorly predictable ways, making it difficult to measure shape, size and concentration in the final sample (service, 2004; nowack and bucheli, 2007; blaser et al., 2008; diegoli et al., 2008; hassellöv et al., 2008; tiede et al., 2008, 2009). the ability of nanomaterials to interact with natural soils and porous or colloid substrates allows a long, often unexpected, passive transport to the groundwater (nowack and bucheli, 2007; loux and savage, 2008; farré et al., 2009). important tools lacking in assessing nanotoxicity are sample-related certified standards, reliable measure units and analytical chemical procedures to measure nanomaterials in the environment. despite a great concern about waste and pollution, the presence of nanoparticles in effluents, sediments or surface waters near urban areas supporting a nanotechnology industry is not yet documented. the use of approved or certified standards, a basic requirement for good practice in toxicology laboratories, seems far from fulfillment in the case of nanomaterials. in all naturally occurring systems (water, soil, air and their combinations), the organization of the dispersed nanophase depends equally from the physical-chemistry of the manufactured nanomaterials and from that of the environment, as well as from the modalities of suspension. obtaining a gold standard for every case is far-fetched, redundant and expensive, while the use of a stable internal standard could attain a satisfactory level of laboratory practice. furthermore, interlaboratory comparisons will improve the characterization and overcome the complexity of nanometrology (hassellöv et al., 2008). theoretical prediction of equilibrium represents a pivotal characterization of nanocolloids. indeed, nanoparticles partially elude the rules of derjaguin, landau, verwey and overbeek (dvlo) theory requiring remodelling, nernst equilibrium does not apply, and the charge density at the surface cannot be calculated when the surface area is unknown (loux and savage, 2008). the specific surface area exponentially increases as a function of small size, and amplifies the energy of collision between particles due to the brownian motion, a major event affecting the colloid stability (casey and rustad, 2007; christian et al., 2008; tenne and seifert, 2009). experimental evidences, positively relating this parameter to toxicity, endorse speculations about its importance in toxicology (oberdörster et al., 2005; stoeger et al., 2006). another feature characterizing the colloid stability is the zeta potential, i.e. the diffuse surface charge, linked to the chemical nature of the dispersed nanomaterials and to the properties of the continuous phase (ph, dilution, temperature and interatomic distance between others). its measure gives good information on nanomaterials mobility, aggregation rates and interactions with surfaces: when its value approaches 0 mv massive aggregation occurs (dunphy guzman et al., 2006a; loux and savage, 2008). moreover, for magnetically charged particles, the dipole moment is a key feature in their characterization and appears to be related to the toxicity potential (kumar et al., 2006; malvindi et al., 2008). the critical coagulation concentration (ccc) of electrolytes (mol/l) is particularly interesting in evaluating the stability of colloid suspensions in hard and salt water: the stability of suspension is strongly dependent from counterion valence and electrostatic potential at the interface, at least in nearly spherical nanometals (loux and savage, 2008). the recently released notes of the organization for economic co-operation and development (oecd, 2008; table 1) include these properties in the endpoints for phase one characterization of manufactured nanomaterials. in addition to them, water solubility and stability of dispersions, crystalline phase, dustiness, crystallite size, tem picture(s), particle size distribution, surface chemistry, photocatalytic activity, pour density, porosity, octanol-water partition coefficient, redox potential, and radical formation potential are listed. the second tool, i.e., a reliable measuring unit expressing toxicity, gave matter of discussion. particle size, firstly suggested as a highly appropriate unit of measure related to toxicity, declined in popularity in recent years: the size of aggregate, not particles, seems to be more informative (pauluhn, 2009). an alternative method, expressing nanomaterials toxicity based on mass or on surface area, seemed to be satisfactory (oberdörster et al., 2005; stoeger et al., 2006). however, a revision of published data in an attempt to explain non-linear dose-response toxicity of some nanomaterials revealed that surface-to-mass area seemed to be preferred to surface-to-size, and the number of particles performed as well (wittmaack, 2006). a lively discussion followed with every scientist supporting his or her own reasons (oberdörster et al., 2007; stoeger et al., 2007; wittmaack, 2007). the problem, as claimed in many works, is probably more complex, and additional information is needed to obtain optimally informative metrics (kandlikar et al., 2007; teeguarden et al., 2007; gornati et al., in press). nevertheless, the number-based metrics proposed by wittmaack (2006) adds predictive value to the existing experimental data on nanomaterial ecotoxicity, which remain mainly, if not exclusively, expressed as concentrations (ppm, molarity or w/v). this method is simple, generally accepted and comparable with the conventional units for corresponding bulk chemicals and should be used at least until adequate standard metrics and samples are available. another regrettable lacking matter is the systematic measure of elements released by dissolution. dissolution is dependent from the chemical nature and size of the nanoparticle, as well as from environmental variables, such as ph and temperature (meulenkamp, 1998; vogelsberger, 2003; hardman, 2006). consequently, the measured effect and the eventually observed toxicity could be not a feature of the nanomaterial itself, but of its degrading products. dissolution has been very rarely documented, while these kinds of events are well known and frequently postulated 79 table 1 guidelines for assessing toxicological risk of nanomaterials. national and supranational agencies agency country document id. year cst uk nanosciences and nanotechnologies: a review of government’s progress on its policy commitments (http://www2.cst.gov.uk/cst/business/files/nano_review.pdf) 2007 defra uk nanotechnologies research reports (http://www.defra.gov.uk/environment/nanotech/research/reports/index.htm) 2009 rcep uk novel materials in the environment: the case of nanotechnology (http://www.rcep.org.uk/reports/27-novel%20materials/documents/novelmaterials-report.pdf ) 2008 european commission eu european activities in the field of ethical, legal and social aspects (elsa) and governance of nanotechnology. (http://cordis.europa.eu/nanotechnology/) 2008 oecd series on the safety of manufactured nanomaterials – nr. 6 (http://www.olis.oecd.org/olis/2008doc.nsf/linkto/nt000034c6/$file/jt 03248749.pdf) 2008 oecd series on the safety of manufactured nanomaterials – nr. 8 (http://www.olis.oecd.org/olis/2009doc.nsf/linkto/nt000029e6/$file/jt 03263204.pdf) 2009a oecd nanotechnology research resources by country (http://www.oecd.org/countrylist/0,3349,en_21571361_41212117_42325 621_1_1_1_1,00.html) 2009b oecd guidelines for the testing of chemicals bioaccumulation in terrestrial oligochaetes (http://www.oecd.org/dataoecd/11/8/42551309.pdf) 2009c iso, iec, nist and oecd international workshop on documentary standards for measurement and characterization for nanotechnologies (http://www.standardsinfo.net/info/livelink/fetch/2000/148478/7746082/in dex.html) 2008 us epa usa nanoscale materials: stewardship program. interim report (http://www.oecd.org/dataoecd/33/55/42061387.pdf) 2009 scenihr eu risk assessment of products of nanotechnologies (http://nanotech.lawbc.com/tags/scenihr/) 2009 cst: the council for science and technologies; defra: the department for environment, food and rural affairs; us epa: environmental protection agency, usa; scenhir: scientific committee on emerging and newly identified health risks; ue: united europe; oecd: organisation for economic co-operation and development; iso: international organization for standardization; iec: international electrotechnical commission; nist: national institute of standards and technology; rcep: royal commission on environmental pollution (lovern and klaper, 2006; biju et al., 2008; handy et al., 2008b; heinlaan et al., 2008). the common efforts of the worldwide control agencies and governmental organisms finally achieved their goal in coordinating the resources, and periodically updated guidelines for risk assessment of nanomaterials have been published (table 1). the policy applied by control agencies to expert recruitment, research funding and targeting the point of interest have been critically reviewed over time (oberdörster et al., 2005; dunphy guzman et al., 2006b; rickerby and morrison, 2007; paradise et al., 2008; wilhelmi, 2008). the us epa in its recently released nanoscale materials stewardship program (nmsp), elaborated an assessment of knowledge about nanomaterials: data scheduled in databases (nanowerk nanomaterials database and wilson center project on emerging nanotechnologies (pen) inventory of nanomaterials in consumer products), or directly submitted to the nmsp were collected and compared. sharing a part of commercially available products from those for research use only or under development, the engineering process standardization and description was the best characterized (epa, 2009; table 1). an overall picture of the statistical elaboration of the data set evidenced that morphology, physical chemistry and production processes have been characterized by 50-90 % in all nanomaterials. this means that validation of producing processes, availability of approved standard for pristine materials and approved nomenclature are near to be reached purposes for the largest part of known engineered nanomaterials. the following best characterized point was the knowledge about professional risk factors in handling and manipulating nanomaterials. the risk was assessed for 50 % of known materials. rapid improvement in this field should be expected from the protocol planned for the period 2003-2013 and involving risk insurance companies (lauterwasser, 2003; oecd, 2009a; table 1). 80 experimental data about acute and chronic ecotoxicity and environmental fate score lower by far: tested materials do not exceed 20 % in this respect. any effort to bridge the gap with an adequate set of measures in this field should be encouraged in the near future. while natural systems represent the real target of these investigations, artificial laboratory conditions can be preferred for practical reasons. first, the steps of knowledge can speed up by using a combination of test media mimicking the natural environment, and test species adapted to the laboratory, representative of the field. acute lethality should be conveniently studied first, long-term toxicity tests following as needed, focused on growth and reproduction as well as morphology and behavioural changes. simplified or natural food webs should be tested to identify the levels and the risks for bioaccumulation and biomagnification (crane et al., 2008), and modelling of exposure has been proposed (mueller and nowack, 2008). endpoints for phase one assessment of environmental toxicity of nanomaterials comprise a list of short and long term effects on species inhabiting pelagic, sediment/benthic, soil and other terrestrial habitats (gourmelon and ahtiainen, 2007). invertebrates as test organisms for environmental pollution studies the invertebrates represent valuable organisms for environmental pollution studies. in addition to be among the most widely distributed living organisms on the earth and offer the opportunity to explore nearly every ecological niche, they have a relatively short life span, reproduce quickly at higher rates and are sensitive to pollutants. invertebrate-based tests are cost-effective, reasonably quick and easy to perform with reproducible standard protocols for multicentered trials. these organisms are indeed particularly convenient as test species to pioneering ecotoxicity studies on newer chemicals, and nanoparticles among others (gourmelon and ahtiainen, 2007). other physiological features of some invertebrates are amictic reproduction, warranting a large number of identical clones, and production of resting eggs, exploited to produce commercial test kits. wild specimens generally adapt quickly to laboratory conditions, making standardization easily obtainable. additional advantages are the highly reproducible staging for larval progression, the small size of adult individuals with transparent bodies, and a stereotyped behavior with easily recognizable disruption. these features facilitate the collection of valuable statistical data at the three levels important for species threatening: survival (quantified by lc50 or median lethal concentration), growth rate, and fertility (expressed as latency and length of reproductive life, number of offspring per life cycle, and offspring viability rates). chronic studies provide information on sub lethal effects, such as active swimming, feeding and avoiding predation capabilities: noec (non observed effect concentration) and loec (lowest observed effect concentration) are the mostly used in this type of approach. besides, an array of changes at cellular and sub cellular level can be followed in invertebrates to inform on the mode of actions of chemicals. despite their wide use in other fields, environmental and ecotoxicological genomics, proteomics and metabolomics (snape et al., 2004) in invertebrates are at the very beginning. the increasing interests in these areas raise the hope for a rapid enhancement in the near future, with useful applications to nanoparticle studies (snape et al., 2004; hines et al., 2007; iguchi et al., 2007; poynton et al., 2007; soetaert et al., 2007; ralstonhooper el al., 2008; steinberg et al., 2008). differently from prokaryotes (fang et al., 2007; heinlaan et al., 2008; holbrook et al., 2008), invertebrates are able to intake nanomaterials dispersed in the environment by different ways: direct ingestion or from contaminated preys, water filtration, inhalation, and surface contact. some degrees of biomodification occur, at least in daphnids (oberdörster et al., 2006a; roberts et al., 2007; baun et al., 2008; filella et al., 2008), and compartmentalization of nanosized contaminants in selected tissues and intracellular organelles has been documented (moore et al., 1997; leeuw et al., 2007; tortiglione et al., 2007; ingle et al., 2008; koehler et al., 2008; oughton et al., 2008; tedesco et al., 2008). invertebrates largely enter the food chain mainly at intermediate levels, and represent a powerful vehicle for recycling pollutants deposited in the sediments. as predator of bacteria, plants, algae and other invertebrates, or feeding on substrates, they become the preferential prey of larger organisms: humans, fish and birds, which, in turn, represent a great deal of the human diet. the risk for humans is possibly related to the doses, therefore, bioconcentration, bioaccumulation and biomagnification raise a matter of concern. their rates, clearance time and fate of the contaminants through the food chain should be measured whenever possible (allen et al., 2005; rocha et al., 2005; emerich and thanos, 2006). studies on ecotoxicological risk assessment for nanomaterials should follow rules and protocols accepted for previously untested chemicals; whenever possible comparison with comparable bulk materials should be considered. the weak stability of nanocolloids represents a problem to be solved: a comprehensive and recent paper in this particular issue reports the generally accepted approaching scheme (crane et al., 2008). briefly, a tired protocol evaluates first acute lethality tests of contaminated waters and sediments on simple organisms: bacteria, monocellular algae and invertebrates. long term tests should follow, if convenient, and data should be predictive of lethality, growth, reproduction and offspring viability. organisms of greater complexity (from bacteria to algae, invertebrates and eventually fish and birds) will be included as needed by the main aim of the study. the preference should be given to standardized test and to test species ecologically representative and well adapted to standard laboratory conditions: the control agencies for environmental contaminant surveillance schedule tests on daphnids, in addition to bacteria, algae and 81 fig. 1 percentage of nanotoxicological studies performed in different test media and species. fw: freshwater; sw: salt (marine or artificial sea) water. other*: liquid media (c. elegans), foods (d. melanogaster) fish, as key step for assessing aquatic toxicity. other invertebrates are recommended as standard test organisms to test benthic waters, sediments, soil, ingestion or skin contact, or for artificial food web analysis (crane et al., 2008). once the material under investigation has shown aquatic toxicity and a trend to accumulate in soils and sediments, tests on these substrates should follow. finally, a set of experiments on organisms playing a higher role in the alimentary chain should be appropriate, in the case of suspected or documented bioaccumulation or biomagnification interesting the food chain. invertebrates used in toxicity studies on nanomaterials: main characters and habitats this section will present the features which characterizes the species of invertebrates which underwent nanotoxicity assessment up until today and the rational for their selection. as shown in fig. 1, species entering nanotoxicity studies are few and unevenly distributed in different environmental samples. test on the water column are prevalent, mainly conducted on daphnids and other cladocerans, while studies on sediments are neglected, despite the emphasized trend of aqueous nanocolloids to be unstable and precipitate. again, in some cases practical reasons (kit availability, lab expertise, others) seems to prevail on recommended protocols in determining the choice of test species. the main taxonomic classification of invertebrates considered in the studies on nanotoxicity is presented in table 2, together with codes of standard tests validated by at least one of the control agencies listed in the legend of table 1. testing the water column: pelagic and littoral organisms tests on water column or water-suspended sediments should be performed in standardized media. commercial mixtures, like those used for aquaria or provided in kits, should be preferred to comparable solutions obtained at the lab bench by mixing high-grade chemicals in demineralized water. these solutions, in fact, are expensive, poorly reproducible and at higher risk of leaving unsafe concentrations of heavy metals in the test medium (ecetoc, 2003). control agencies recognize daphnids (daphnia magna, daphnia pulex and ceriodaphnia dubia) as “first choice” test organisms for validated ecotoxicological tests (joncxyk and gilron, 2005). the daphnia genomics consortium (dcg, http://daphnia.cgb.indiana.edu) promotes sequencing projects of the entire genome of daphnia sp. as model organisms for ecology. these small crustaceans, widely distributed in all aquatic habitats with the only exception for extreme environments, show all the features mentioned before as highly convenient for testing. if maintained in optimal conditions (ph = 7.2-8.5; t = 20 °c; hard water=160-180 mg/l or 80-90 mg/l caco3, related to species), daphnids adapt quickly in the laboratory. stereotype behaviors, which involve filtration feeding, jerky motion in swimming and anti-photo tactic movements, are in part genetically determined and appear to be sensitive to chemical pollution. 82 table 2 list of invertebrate species used in testing nanoparticles toxicity the tests approved by control organisms and test codes, whenever available, are reported (see even burton et al., 2003; crane et al., 2008). asterisks: not yet validated by control agencies. species class order codes of validated tests approved by astm/epa/oecd/eu/i so freshwater crustaceans: daphnia magna and d.pulex, caeriodaphnia dubia acute: epa850.1010; epa821r02.013, oecd202, astme-1209501. chydorus sphaericus* branchiopoda diplostraca sublethal: epa850.1300; oecd211, astme1193-97, astme-12095-01 all (only daphnids) crustaceans: thamnocephalus platyurus* branchiopoda anostraca none rotifera: brachionus calyciflorus monogononta ploimida acute: astme-1440-91 sublethal: astme-2317-04 astm/epa cnidaria: hydra attenuata hydrozoa hydroida astm: stp921-eb astm/epa molluscs: elliptio complanata* bivalvia unionoidea none salt (estuarine, sea water) crustaceans: harpacticoida copepods maxillopoda harpacticoida astme-2317-04, oecd 254 astm/epa/oecd molluscs: mytilus edulis bivalvia mytiloidea astme-2122-02, epa850.1050 astm/epa/oecd/eu freshwater sediments crustaceans: hyalella azteca malacostraca amphipoda astme-1706-00, oecd 251, epa850.1735, epa600/r99.064 astm/epa/oecd worms: lumbriculus variegatus oligochaeta lumbriculida astme1688-00, epa 823-f-00-002; oecd2007: new proposal astm/epa/oecd sea water sediments crustaceans: leptocheirus plumulosus malacostraca amphipoda astme1367-99, epa850.1735; epa 600/r01/020, oecd 252 astm/epa/oecd soil earthworms: eisenia sp. oligochaeta haplotaxida astme1676-04 (toxicity and bioaccumulation); epa850.6200; oecd207/211 (acute/chronic) all potworms: enchytraeus crypticus oligochaeta enchytraeidae astme1676-04 (toxicity and bioaccumulation); iso 16387:2004; oecd 207/fkz: 204 67 458 astm/iso/oecd crustaceans: porcellio scaber* isopoda oniscidea none other nematodes: caenorhabditis elegans* chromadorea rhabditida model organism arthropods: drosophila melanogaster* insecta diptera model organism 83 short life cycles and amictic reproduction are the rule: deposition of non-resting eggs in the dorsal brood chamber (from which juvenile female are released) gives rise to large, rapidly evolving clones. a great level of body transparency permits noninvasive exploration of brood chamber and of gut content. sexual cycle follows non favorable changes in natural habitats (freezing, drought and others): 12 resting eggs are deposited in the ephyppium, a detachable modification of the carapace, and male individuals are produced, whose sperm is haploid, tailless but motile. sexual cycles are more frequent in c. dubia (ebert, 2005). commercial kits, based on standardized acute and chronic toxicity tests on daphnids, contain “cysts” (ephyppia) and use hard freshwater as the test medium (persoone et al., 1994; centeno et al., 1995; ruck, 1998; lazorchak et al., 2008). testing the water column: pelagic (or littoral)/ benthic organisms chydorus sphaericus is a daphnid-like cladoceran whose small spherical body is transparent. the dorsal brood chamber is present only in adult females, and reproduction is mainly amictic. feeding and swimming patterns are similar to those described for daphnids. its ability to change habitat from pelagic to bentic confers to him the property of an useful alternative to daphnid testing, used since 1991 (havens, 1991). while not yet approved by control agencies a test for acute toxicity on c. sphaericus has been recently proposed (pieters et al., 2008; velzeboer et al., 2008). copepods are other organisms in this group, perhaps the dominant ones, at least for the very large number of species and the ability to colonize a wide range of aquatic ecosystems. studies on nanomaterials have been performed only in marine species, by far the most numerous species. different from daphnids, their body is segmented and poorly transparent. pelagic larvae (nauplia) develop slowly (within months) into sexually (male and female) differentiated adults. the life span is comparably long and variable between species. no resting eggs are produced, but in unfavorable environmental conditions adults enter diapause. benthic habits are restricted to the adult life. swimming and crawling-swimming movements on substrates are powerful (90 m/h). copepods feed on suspended particles (like algae, bacteria and organic matter), and are validated test organisms for ecotoxicology (anderson et al., 2001). thamnocephalus platyurus is a big freshwater crustacean, with a segmented and poorly transparent body. while two distinct sexes have been described (byron and ponder, 1949), resting eggs can be produced. a commercial kit supplies this material to perform acute studies. however, the test time (24 h for hatching plus 1 h for in vitro exposure to contaminants) is too short to allow a full maturation of the larvae (williams, 2007). the test is widely used, but not included in standard validated methods (centeno et al.,1995). other benthic organisms are useful for longterm toxicity studies, such as the cnidarian hydra attenuata, a freshwater, relatively small organism. lacking a mesoderm hydras belong to the group of diploblastic animals; their tubular body develops a head, terminated by an oral apparatus with a crown of tentacles, and a basal disc (adherent to the substrate) at opposite extremities. reproduction proceeds by clones; they are generated by budding from the distal third of their body, near the basal disc. a gradient of activators regulates head formation and regeneration by morphallaxis, and inhibits budding in younger individuals. differentiation into true hermaphrodite and sexual reproduction are induced by unfavorable conditions. toxicity tests have been described: clubbing movements of tentacles are early signs of exposure to toxic agents, before reproductive changes or death (davies and freeman, 1995; blaise and kusui, 1997; holdway, 2005). hydras are interesting models for aging: no tissue aging processes or age-related enhanced mortality have been observed in hydras over a period of 4 years. however, some degeneration follows the induction of sexual reproduction, at least in some species, but not in h. attenuata (martinez, 1998; austad, 2009). chronic toxicity was assessed on the bivalves, mytilus edulis (blue mussel) and elliptio complanata, two species feeding while filtering large amounts of sea and freshwater, respectively. the model was restricted to quite large, long living and mainly sessile organisms. the sea species is long 3-7 cm, and inhabits the intertidal, infra and circa-littoral zones. e. complanata, whose habitat is restricted to freshwater, can reach 25 cm in length and 10 years of age. both species reproduce sexually. their importance as test organisms for ecotoxicology is linked to the role played in the food chain. the rotifer brachionus calyciflorus was studied under exposure to nanomaterials as a component of a simplified food web: algae and bacteria were predated by a protozoan, which in turn was the rotifer prey; both fed fish (holbrook et al., 2008). testing artificial food web containing rotifers is a validated procedure (snell, 2005). the acute test has been validated, and a commercial kit for acute (24 h) and short-chronic (48 h) toxicity tests is available: the kit supplies resting eggs and the test is performed on newly hatched larvae (persoone et al., 1993). brachionidae are eutelic, small loricate organisms, with one-pieced, thin and transparent lorica, a small corona (the oral apparatus), a single gonad, and a long “foot” used for anchoring. both free swimming and sessile forms are present. amictic and sexual reproductive cycles can alternate in relation to environmental conditions. in favorable conditions, non-resting eggs externally attached to the root of the maternal foot hatch within 12 h. immediately before periods of freezing or drying, small defective males lacking a digestive system appears. insemination of females leads to deposition of resting eggs, from which a new generation of diploid females hatches when environmental conditions improve. the life span is short (ca. 2-3 weeks), but the total number of offspring is large. 84 testing sediments: fresh-water and marine benthic organisms these animals inhabit the deepest zone of water bodies and are useful to study sediments, on which they are feeding. three species have been tested in nanoecotoxicology, all representing validated test organisms. hyalella azteca (family: hyalellidae) and leptocheirus plumulosus (family: aoridae) are small shrimps inhabiting freshwater and estuarine sediments respectively, and feed on sediments or suspended particles. sexual reproduction is the rule, with two dimorphic sexes (larger males). tests for acute and chronic toxicity are performed in younger individuals and the experimental time should not exceed 4 weeks for h. azteca and 10 days for l. plumulosus (borgmann et al., 2005). the worm lumbriculus variegatus (annelida, oligochaeta, lumbriculidae) inhabits freshwater sediments in ponds, lakes and marshes, where it feeds on decaying vegetation and microrganisms. it can be occasionally found in silty sediments in deeper waters. sexual reproduction begins between true hermaphrodites, with formation of transparent cocoons containing 4-11 fertilized eggs. small worms hatch in 2 weeks without a previous larval stage. in laboratory conditions, the worms are smaller (4-6 cm), never reach sexual maturity and reproduce by spontaneous asexual fragmentation followed by rapid regeneration of a complete worm from surviving fragments. regeneration is also described in natural conditions, fragmentation is a rapid and common response to injuries, like body compression. its use as a test organism in ecotoxicology is validated (epa 823-f-00-002). testing soils: terrestrial organisms the earthworms eisenia sp. (eisenia foetida and eisenia veneta, annelida, oligochaeta, lumbricidae) and the potworm enchytraeus crypticus (oligochaeta, enchytraeidae) strictly depend on soil, as their habitat and food source, are easy available, growth rapidly and enter the food chain through fish and higher vertebrates. earthworms and potworms have a segmented body with cephalo-caudal symmetry. the cephalic portion ends in a clitellum, characterizing sexually mature individuals. toxicity tests, however, require younger individuals. both groups are true hermaphrodite, each individual developing a complete male and female set of reproductive organs, with two pairs of testes and one pair of ovaries. while able to completely regenerate from fragments if injured, sexual reproduction is the rule (dominguez et al., 2003). eisenia sp. and enchytraeidae are validated test organisms for soil ecotoxicological studies (epa850.6200; oecd207/211; iso 16387:2004; egeler et al., 2009). the isopod porcellio scaber is another terrestrial organism. it is heavily pigmented and has a ventral brood pouch which characterizes sexually mature females. it hosts fertilized eggs (by sexual reproduction with internal copulation) and hatched larvae as only young complete individuals are released. this crustacean is common in the wooded soil; its diet is mostly on vegetal debris. life span (up to 3 years) and reproduction rates are sensitive to a number of environmental changes and pollutants. the oecd recently released updated guidelines for testing the bioaccumulation of chemicals in soils, using earthand pot-worms as test organisms (oecd 2009c). p. scaber, instead, is a test species not yet validated; however, it is the only terrestrial crustacean useful for soil pollution testing. model organisms: caenorhabditis elegans and drosophila melanogaster studies on the toxicity of nanomaterials have been performed on the larvae of two species considered to be model organisms for developmental and cell biology and genetics: the nematode c. elegans and the fruit fly d. melanogaster. d. melanogaster is perhaps the prototype of model animal organisms and its genome has been completely sequenced (adams et al., 2000). it is a small fly, with sexual dimorphism and sexual reproduction. eggs mature outside the female body; larvae development is by stages. the soil nematode c. elegans is easily cultured in laboratory conditions on agar plates. viable worms can even be stored in frozen stocks indefinitely. the worm body is tiny (about 1 cm long) and transparent, eutelic (100 cells ca) and moving with swimming motion. two sexes are described: a self-fertilizing true hermaphrodite, with two intra-abdominal gonads, one producing sperm, the other eggs. it is able to give rise to about 300 larvae in its life course. males are occasionally and rarely produced (1/103). they are slightly smaller than hermaphrodites, from which they differ for the presence of the only male gonad in the abdomen and the shape of the tail, carrying the organ for the internal copulation, not always followed by fertilization. sex is genetically determined (hermaphrodites are xx, males x0), however environmental or dietary changes can convert hermaphrodites born from mating into males, but it never occurs with amictically produced hermaphrodites (hart, 2006; the c. elegans sequencing consortium, 1998). toxicity of engineered nanomaterials to invertebrates while natural and engineered nanomaterials can overlap and share common properties, this review has been limited to manufactured nanomaterials and to the related ecotoxicological risks. nanotoxicology studies, in which the invertebrate plays a significant while intermediate role, remain poorly and unevenly distributed, in spite of great attention reserved to nanotoxicity in humans, cultured cell lines and vertebrate organisms (chun ke and qiao, 2007; duffin et al., 2007; farrè et al., 2009; shvedova et al., 2009; di gioacchino et al., in press; gornati et al., in press). environmental nanotoxicology remains almost confined to the aquatic environments, mainly freshwater, while studies on contaminated sediments and non aquatic environments are scarce, accounting altogether for 30 % of the total, 85 fig. 2 percent of ecotoxicological studies on different nanomaterials. fullerene: c60 and carbon nanotubes (cnt); metal oxides: tio2 and metal oxides plus tio2 plus (*), qds: quantum dots; others: organic (sucrose polyester oil, polymethyl-methacrylate, nipam/bam) and inorganic (upconverting phosphors, glass wool, nanometals) nanomaterials not included in the chemicals classes mentioned before. including observations on model organisms maintained in artificial aqueous medium (c. elegans and d. melanogaster). the most studied test species by far are daphnids (d. magna, d. pulex and c. dubia) (fig. 1). the most studied materials are fullerenes and metal oxides that altogether represent 70 % of the available literature in this field (fig. 2). fullerenes carbon nanomaterials captured great attention for both historical reasons and convenience. fullerenes are organized as nearly spherical nanoparticles, the so called buckminsterfullerenes (nc60, nc70, and fullerols, their hydroxylated derivatives) or as single, double or multi-walled carbon nanotubes (swcnt, dwnct and mwcnt, respectively). these materials can be toxic for higher organisms: they are able to cross the membrane of eukaryote cells, accumulating in lysosomes and mitochondria, as well the bloodbrain barrier, reaching the olfactory bulb in fish and mammals via the olfactory nerve (oberdörster, 2004; oberdörster et al. 2005; tin-tin-win et al. 2008). moreover, in human cell cultures, swcnt determine changes in the expression of several genes, mainly involved in apoptosis (cui, et al., 2005; sarkar et al., 2007). fullerenes have a very wide application range, from cosmetics to computer engines, and tons are produced yearly. their waste in the environment is significant and safety is poorly granted by their hydrophobicity, which partially prevents miscibility with superficial water (heymann et al., 1996; deguchi et al., 2001; fortner et al., 2005). while protective effects of fullerenes against other more powerful contaminants were sporadically described (baun et al., 2008), the potential risk of heavy pollution with fullerene poses major environmental concern and need for studies on adequate models, invertebrates among others. the toxicity of fullerene in invertebrates (table 3) is variable and related not only to nanoparticle properties, but even to the methods adopted for suspension in water and to the test species. the link between lethality rates and methods for obtaining suspension was determined for nc60 in d. magna, perhaps the most sensitive species. suspensions prepared by long stirring alone seemed to be safe: the cmax ≅ 35 ppm did not reach the lc50 even when the test time was prolonged to 96 h. stirring plus sonication reached an intermediate toxicity, while the solution retaining some traces of thf, added as solvent, was the most toxic. this toxicity apparently is not linked to the residual solvent as, in the absence of fullerene, traces of thf were not toxic (lovern and kapler, 2006; oberdörster et al., 2006a; zhu et al., 2006; lovern et al., 2007; spohn et al., 2009; zhu et al., 2009). lc50 was higher for d. pulex while the test time in this case was shorter (klaper et al., 2009). other species like t. platyurus, h. azteca and harpacticoida copepods, seemed to be more resistant and did not show acute toxicity (oberdörster et al., 2006a; blaise et al., 2008). fullerenes added to soils seemed to be ineffective on survival rates of earthand pot-worms (baird 2007; scott-fordsmand et al., 2008). d. magna appeared to be the most sensitive species even to sublethal effects. exposure to nc60 86 table 3 summary of lethality data for fullerenes lc50: median lethal concentration; cnt: carbon nanotubes: sw: single walled, dw: double walled, mw: multi walled. (10 ppm, 48 h) increased the heart rate and amplified (and partially disrupted) stereotypical movements in swimming and feeding. fullerols had only fable effects, reversible within 30 min (lovern and klaper, 2006; lovern et al., 2007). long-term (21 days) exposure to stirred nc60 solutions reduced reproductive rates. moreover, the final population was significantly reduced, despite some degree of adaptation (noec = 1.0 ppm, loec = 2.5 ppm; oberdörster et al., 2006a). oxidative stress was observed in d. pulex (klaper et al., 2009). chronic exposure of h. attenuata to nc60 was associated with clubbing and retraction of tentacles (ec50 < 10 ppm, 96 h) (blaise et al., 2008), while d. melanogaster exposed during the larval stages showed only slight effect at smart (somatic mutation recombination test) on wing cells (loec = 2.24 ppm, noec = 0.45 ppm). fullerols were not effective (noec = 2.46 ppm) (zakharenko et al., 1997). nc60 (0.1-1 % in soil) did not affect reproduction in the potworm enchytraeus crypticus (baird, 2007), but was able to inhibit cocoon formation in e. veneta (scott-fordsmand et al., 2008). bioavailability and bioaccumulation of nc60 were measured in d. magna. the maximum intake of carbon was greater than 2 ppm/mg of tissue after a 48 h exposure (oberdörster et al., 2006a), mainly localized in the gut. a near to complete excretion of carbon clumps organized at micrometer level was recorded after 48 h clearance, and no bioaccumulation of nanoparticles seemed to occur (baun et al., 2008). the complete clearance was accompanied by a complete recovery from toxic effects (lovern et al., 2007). the toxicity of cnt seemed to be lower, or even absent, mw proved to be generally more aggressive than dw and swcnt, which moderately affected the mortality rate of d. magna fed on algae. however, swcnt coated with phospholipoproteins (pl-swcnt) protected d. magna from mortality due to starvation. cladocerans seemed to be able to metabolize the lp-swcnt: when starved, they ate the lipid coating, causing aggregation and precipitation of nanotubes. nanocarbon, mainly internalized in the gut within 45 min, was completely excreted in clumps of amorphous carbon after 20 h, or sooner if fullerene species lc50 time notes d. pulex 0.5-5.0 (lc30/75) 24 h nc60 in thf 0.8, 0.46 ppm nc60 stirred/sonicated 7.9, 10.51 ppm d. magna >35 ppm 48-96 h d. pulex lc40 = 100 ppm 24 h harpacticoida copepods never reached 48-96 h h. azteca never reached 96 h nc60 stirred t. platyurus never reached 24 h no mortality effects e. veneta never reached no mortality effects nc60 added to soil e. crypticus never reached no mortality effects nc60/70 d.pulex never reached 24 h no mortality effects fullerols d.pulex lc40 = 100 ppm 24 h d.magna 2.42 ppm 48 h t. platyurus 24 h h.attenuata 96 h a. tenuiremis 35 days l.variegatus 28 days swcnt d.melanogaster never reached no mortality effects dwcnt e. veneta never reached 28 days no mortality effects d .magna 22.75 ppm 48 h c. dubia 7 % survival at 40 ppm 48 h h. azteca >264 ppm l. plumulosus 68 ppm 10 days mwcnt l.variegatus never reached 28 days no mortality effects 87 table 4 summary of sublethal toxicity for fullerenes fullerene species noec dose time effect 0.18 ppm loec = 0.26 ppm 60 min swimming and feeding movements d. magna 1.0 ppm loec = 2.5 ppm 21 days reduced reproduction d. pulex 20 ppm loec = 100 ppm 24h oxidative stress h. attenuata ec50 < 10 ppm 96 h morphological changes e. veneta 1000 mg/kg d.w.f. weight gain reduction (20 %) cocoon formation, not hatchability (by 78 %) enchytraeus crypticus 2000 ppm 14 days reproduction not affected nc60 stirred 0.45 ppm loec = 2.24 ppm 2.46 ppm d.melanogaster 24 ppm smart fullerols d.pulex 7.5 ppm loec = 20 ppm 24 h oxidative stress nc60/nc60 d.pulex 5.0 ppm loec = 7.5 ppm 24 h oxidative stress h.attenuata ec50: 10-100 ppm 96 h swcnt a.tenuiremis 10 ppm 35 days ec50: 94 mg/kg d.w.f. 28 days weight gain reduction (20 %) dwcnt e. veneta ec10 = 37 and ec50 = 176 mg/kg d.w.f. 28 days cocoon formation, not hatchability (by 60 %) ec50: median effective concentration; noec: non observed effect concentration; loec: lowest observed effect concentration; cnt: carbon nanotubes: sw: single walled, dw: double walled, mw: multi walled; smart: somatic mutation recombination test; d.w.f.: dry weight food. algae was provided for feeding (roberts et al., 2007). exposure to maximal concentrations of swcnt did not affect mortality rates in t. platyurus (24 h), h. attenuata (96 h) and in copepod amphiascus tenuiremis (28-35 days) (templeton et al., 2006; leeuw et al., 2007; blaise et al., 2008). clubbing of tentacles were present in h. attenuata exposed to sublethal doses (ec50: 10-100 ppm, 96 h; blaise et al., 2008). a. tenuiremis tolerated without side effects the presence of purified swcnt (≤10 ppm) along its entire life cycle (up to 35 days). mortality and fertility rates, sex ratio and viability of the offspring were not affected, and a delay of 1 day in offspring development was the only side effect registered. however, the presence of fluorescent byproducts of fullerenes in the solution, possibly related to procedures adopted to obtain suspension, led to an 80 % mortality rate (templeton et al., 2006). d. melanogaster larvae well tolerated the ingestion of swcnt mixed with yeast feeding paste, and the mortality and fertility rates were not affected, notwithstanding the occurrence of nanocarbon accumulation in tissues and fluids (leeuw et al., 2007). dwcnt (10-30 nm diameter, 5-15 µm length) were tested on e. veneta. nanotubes were mixed with food (50-495 mg/kg dry weight food, d.w.f.) and administered to worms according to standard protocols (10 g mixed food every 7th day for 28 days, astme1676-99, epa850.6200; oecd211). no lethality was observed, but the exposure to the highest dose reduced weight gain (ec10 = 94 mg/kg d.w.f.) and cocoon formation (ec10 = 37 and ec50 = 176 mg/kg), but not hatchability. mwcnt retained the highest toxicity, among different types of cnt: lethality rate was enhanced in c. dubia, h. azteca and l. plumulosus (kennedy et al., 2008, zhu et al., 2006). the only exception was d. magna (lc50 = 22.75 vs 2.42 ppm, mw vs swcnt, 48 h). these values were obtained in a recent work, in which disagreement with previous results were commonly observed with all the materials tested (tables 3-5): the reason, as discussed by the authors, could be found in the different modalities for suspension preparation, and especially in continuous stirring and shaking of test medium during the exposure period (zhu et al., 2009). hydroxylation, carboxylation and coating with natural organic matter (nom) reduced mortality rate, at least in c. dubia. survivors showed anomalies of the carapace, to which nanoparticles can adhere (baun et al., 2008). carbon accumulated mainly in the gut; it was completely excreted after 24 h washing-out and refeeding. (kennedy et al., 2008). 88 table 5 summary of lethality data collected for metal oxides organized at nanoscale nano-compound test species and medium lethality reference tio2: <20 nm 25-30 nm d.magna d magna d.pulex c.dubia c.elegans p.scaber 48 h lc50 = 143 ppm 48 h lc50 = 5.5 ppm 48 h lc50> 10 ppm 48 h lc50> 10 ppm 24 h lc50 = 80 ppm 14 d noec >1000 ppm zhu et al., 2009 lovern and klaper, 2006 griffitt et al., 2008 wang et al., 2009 drobne et al., 2009 al2o3 (60 nm) c.elegans 24 h lc50 = 82 ppm wang et al., 2009 ag oxide (20-30 nm) d.magna d.pulex c.dubia 48 h lc50 = 0.04 ppm 48 h lc50 = 0.067 ppm griffitt et al., 2008 al oxide: 20-30 nm; or 51 nm d.magna d.pulex c.dubia 48 h lc50> 162 ppm 48 h lc50> 10 ppm 48 h lc50 = 3.99 ppm zhu et al., 2009 velzeboer et al., 2008; griffitt et al., 2008 co oxide (10-20 nm) d.pulex c.dubia 48 h lc50> 10 ppm 48 h lc50 = 1.67 ppm griffitt et al., 2008 cuo: 30 nm, or 15-45 nm d.magna t. platyurus d.pulex c.dubia 48 h lc50 = 3.2 ppm 48 h lc50 = 2.1 ppm 48 h lc50 = 0.06 ppm 48 h lc50 = 0.419 ppm heinlaan et al., 2008 griffitt et al., 2008 ni oxide (5-20 nm) d.pulex c.dubia 48 h lc50 = .89 ppm 48 h lc50 = 0.674 ppm griffitt et al., 2008 sio2 (205>4,700 nm) d.magna 48 h, lc70 = 10 ppm adams et al., 2006 zno: 20 nm 50-70 nm 480>4,000 nm d.magna d.magna t. platyurus d. magna c.elegans 48 h lc50 = 1.5 ppm 48 h lc50 = 3.2 ppm 48 h lc50 = 0.18ppm 48 h lc73 = 0.2 ppm 24 h lc50 = 2.3 ppm zhu et al., 2009 heinlaan et al., 2008 adams et al., 2006 wang et al., 2009 cuznfe2o3/indium tin oxide/ho2o3 t. platyurus h. attenuata 48h, lc50 = 0.1-1.0 ppm 96 h, ec50 = 10-100 ppm blaise et al., 2008 niznfe2o3/o3sm2 / er2o3 t. platyurus 48h, lc50 = 1-10 ppm blaise et al., 2008 srfe12o19/tio2/fe5o12y3 t. platyurus 48h, lc50 = 10-100 ppm blaise et al., 2008 kinetic of intake and depuration rates after ingestion of mwcnt (diameter: 30-70 nm) and swcnt (1-2 nm diameter) mixed with sediments were studied in l. variegatus. data were expressed as basfs (biota–sediment accumulation factors, calculated as the ratio of the concentration of a substance in an organism normalized by the organism lipid fraction to its concentration in the sediment normalized by its organic carbon fraction, mg/g dry sediment (petersen et al., 2008b). at the time (7 days) of first observation, the accumulation had reached maximal levels and remained stable for the entire 28 days period of observation. values were slightly larger for mwcnt (0.40 vs 0.28 mg/g dry sediment). mortality did not increase; sub-lethal toxicity was not tested. clean sediments added to water accelerated the clearance, nearly complete in about 2 days, and still incomplete after 60 h in clean water without sediments (petersen et al., 2008b). a similar work performed in e. foetida gave similar results (petersen et al., 2008a). metal oxides these compounds behave differently from fullerenes. particle and aggregate size are more uniformly distributed, and their hydrophobicity is generally lower, allowing easier standardization in preparing aqueous suspensions. table 5 shows lethality data for metal oxides. it is evident that tio2 is by far the most studied among this group of compounds (fig. 2). two freshwater invertebrates, t. platyurus and h. attenuata, permitted to group metal oxides into three toxicity degrees: cuznfe2o3, indium-tin oxide and ho2o3 scored highest, niznfe2o3, o3sm2, and er2o3 retained an intermediate toxicity level, while tio2, srfe12o19, and fe5o12y3 scored the lowest. moreover, compounds listed in the two last groups did not show toxic sub lethal effects on h. attenuata (blaise et al., 2008). when metal oxides (size range: 5-50 nm) were compared in different organisms, the toxicity degree was ag> cu> ni> co = al = ti in d. pulex, and ag> cu> ni> co> al> ti in c. dubia. nanoparticles toxicity in comparison with that of the corresponding bulk salts gave contrasting results in different species: in aquatic organisms (d. pulex, c. dubia) toxicity was greater for nanoparticles, in c. elegans for bulk salts (griffitt et al., 2008; wang et al., 2009). the toxicity degree of larger particles (>200 nm) was zno> sio2> tio2 in d. magna 89 (adams et al., 2006), partially confirmed in a recent study which found the toxicity degree to be zno> tio2> al2o3 (zhu et al., 2009). highly sized (>500 nm) tio2, alo2 and ceo2 did not retain toxicity against c. sphaericus: the solutions containing the higher concentrations (10 and 100 ppm) however were cloudy and unstable (velzeboer et al., 2008). reproduction was negatively affected in c. elegans (wang et al., 2009). here again, not only the chemical species, but also the modalities of suspension preparations and the size of nanoparticles were influent on toxicity. preparing tio2 suspension by filtration lead to ultra fine particulate (25-30 nm): this suspension is much more toxic than those obtained by sonication (particle size: >100 nm). particles larger than 100 nm are poorly or nontoxic for d. magna, t. platyurus and c. sphaericus (adams et al., 2006; heinlaan et al.,2008; lovern and klaper, 2006; warheit et al., 2007; blaise et al., 2008; velzeboer et al., 2008). sublethal toxicity tests were performed in two crustaceans, d. magna, inhabiting freshwater, and p. scaber, adapted to wooden soil. filtered tio2 particles (30 nm) did not alter stereotypical movements of d. magna after 60 min exposure to the loec = 2.0 ppm (lovern and klaper, 2006; lovern et al., 2007). an immobilization test of d. magna after 48 h exposure to 100 % anatase tio2 particles (30 and 100 nm) gave unclear results: smaller and photo-catalysed particles seemed to be more toxic, but statistical difference was never reached (hund-rinke et al., 2006). nanotio2 (10, 100 and 1,000 ppm) was not toxic when mixed with food for 14 days to p. scaber. enhanced feeding rate, assimilation efficiency, levels of catalase and glutathione-s-transferase where observed: these effects appeared earlier at higher dose (3,000 ppm). again, sonicated and bigger particles were ineffective (jemec et al., 2008; drobne et al., 2009). quantum dots, qds in addition to a number of possible interesting uses in optical and computer sciences, quantum dots represent a flexible and interesting dye for living cells and organisms. their toxicity, bioaccumulation and clearance efficiency in invertebrate organisms have been tested in only a few species: c. dubia, h. attenuata and elliptio complanata, in addition to a simplified food web including rotifers. the potential for lethality of qds seems to be determined by their metal core, its position inside the shell and dissolution rate. qds with a cdse crystalline core of 4 nm inside a shell of zns coated with organic polymer (total size: 15-20 nm) and registered qds (545 itk carboxyl quantum dots) were diffusely internalized in c. dubia, whose body, and especially the gut, showed intense fluorescence. the toxicity was nevertheless absent (noec = 600 or 110 ppt, respectively; bouldin et al., 2008; ingle et al., 2008). e. complanata was sensitive to toxic effects of qds with cdte (instead of cdse) crystal core. mussels, collected in the field and exposed for 24 h to qds dispersed in tanks of freshwater, showed lipid peroxidation of gills and gut, reduced viability and immune activity of hemocytes (ec50 = 2-4 ppm; gagné et al., 2008). qds formed by cdse core asymmetrically sited inside a rod-shaped shell of cds induced nonsynchronous tentacle retraction, a behaviour anticipating the beginning of sexual reproduction in h. attenuata. the test substance was not internalized, and the dose-independent effect developed only in the presence of functioning nerves in the test organisms (intact animals or segments of their bodies). the dipole moment retained by asymmetrical qds was probably a key requisite for inducing the anomaly: particles in which the core was symmetrically disposed inside the shell were lacking both qds properties and toxicity (malvindi et al., 2008). identical particles, solubilized by coating with polymers, instead were able to enter the test species: after localization in the head (within 1 h), diffusion to the entire body followed within 24 h. these nanoparticles were able to enter dissected cells of hydra sp. (tortiglione et al., 2007). finally, a simplified food web did not reveal bioaccumulation or biomagnification: e. coli was unable to internalize qds, however those biotynilated or carboxylated adhered to the bacterial surface, and caused cells aggregation. consequently, the ciliate predator (tetrahymena pyriformis) avoided eating aggregates; but internalized qds with water ingestion. neither degradation occurred nor release of toxic metals from the core. the bioaccumulation was low and the excretion rate efficient (t1/2 = 1.5-3.6 days). the biomagnification in the rotifer predating ciliate was very low (0.29-0.62), the t1/2 = 14-21 h. no toxicity was found in protozoans or in predating rotifers (holbrook et al., 2008). other nanosized materials a pioneering study on material possibly organized at nanoscale was carried out in 1997, using as test substance, a sonicated suspension of sucrose polyester oil, proposed as food additive for humans. droplets were not measured. after having been exposed in vivo and in vitro to this suspension, m. edulis showed signs of oxidative stress, persistent lipofuscin formation and reduced lysosomes membrane stability (moore et al., 1997). nanopt coated with polyvinylpyrrolidone (size: 2.4 nm) acts as an antioxidant agent in larvae of c. elegans at l4 development stage. the mean life span was prolonged in wild-type worms and in short-living mutants mev-1, and the oxidative stress induced by paraquat was partially counteracted at the loec = 5 mm. (kim et al., 2008). m. edulis reacted to 24 h exposure to nanoau (gnp, gold 0 (stable), 13 nm, and 0.75 ppm) with enhanced stress parameters in digestive glands, mantle and haematocytes. paradoxically, gnp partially protected from the oxidative stress due to menadione (tedesco et al., 2008). nanoag (lc50 = 125 ppm, 24 h) accumulated in the gut of d.magna and on antennae in individuals near to death (oberdörster et al., 2006b). nano60co was tested on young adults e. foetida maintained on standard soil. radioactive particles obtained by neutron activation emitted 90 beta and gamma radiation (0.157 kbq/μg) used as a tracer, and were administered mixed with food for 7 days (667 g horse manure containing 87 μg nanoco, or 13.7 kbq, per g). no toxicity was observed, while radioactivity was found in many tissues: spermatogenic cells, clitellum, cocoons and blood among others. a low intestinal clearance followed the depuration period (20 % within 8 weeks): the authors considered this work a pilot study on the kinetics of nanoparticles in biological systems (oughton et al., 2008). upconverted phosphors (ucp) are a promising new class of dyes with application, as an example, in biomedical researches. these particles consist of trivalent ions of lanthanides embedded in a lattice of crystalline chromophores. these last act as an antenna and permit the transfer of one-two photons to the ions, otherwise unable to absorb light. decaying from the excited state, the trivalent ions emit a luminescence characterized by its brightness and long-decay time. ucp sized 150 nm were proved non-toxic within 24 h in c. elegans immobilized with na azide (noec = 0.5 ppm). nanospheres were internalized in the gut, and completely excreted after 2 h only in re-fed worms (lim et al., 2006). glass wool, used to absorb material from floating oil spill barriers, is a form of silica oxide organized in nanowires. those used in the study (diameter: 5-25 nm, length: several microns) accumulated in lysosomes and endosomes of gills, and in mitochondria of the hepato-pancreas of m. edulis as shorter fibres, 100-200 nm and 60 nm, respectively. membrane stability of lysosomes was reduced by 70 % within 24 h, and chronic exposure for 16 days enhanced lipofuscin in the hepatopancreas (koelher et al., 2008). the commercially available polymethylmethacrylate (pmma, diameter: 60 nm-1.08 µm) retained toxicity independent from particle size when tested by the chidotox test, on chidorus sphaericus: (lc50> 100 ppm, 48 h) (velzeboer et al., 2008). two organic nano-copolymer particles used in medicine (poly n-isopropylacrylamide, pnipam and n-isopropylacrylamide-co-n-tert-butylacrylamide, nipam/bam, with three different ratios of the comonomers) were tested in freshwater organisms, d. magna and t. platyurus in addition to primary consumers. the stability of suspension in water was greatly dependent from temperature, in the range 10-25 °c. nipam/bam, 50:50 showed the higher toxicity, and d.magna was even in this case more sensitive than t.platyurus, (noec = <50 vs <200 ppm; loec = 50 vs 200 ppm; lc50 = 61 vs 353 ppm, respectively; naha et al., 2009). conclusion the importance of invertebrates to test the potential toxicity of nanomaterials can be educed from the data discussed in this paper. invertebrate tests should not be considered as opposed to tests on other systems; instead, invertebrates add valuable information, at an intermediate level between prokaryotes and vertebrates, on pollution affecting the environment. moreover, the wide degree of standardization, time and cost effectiveness, and suitability to study acute lethality as well as long-term toxicity represent additional advantages. in conclusion, nanoparticles showed their elusive nature in ecotoxicological studies, leading to partially inhomogeneous results. the chemical nature of the compound, the sensitivity of the test species, the dose and the time scheduling obviously played a significant role. to them, however, the effects exerted by the nanoparticle properties should be added, so that different manipulations of pristine material and suspension generated confusion. the aggregates shape and dimension greatly influenced the results, in strict dependence from test media composition and physical-chemistry variable, ph and temperature among others. (lovern and klaper, 2006; oberdörster et al., 2006, zhu et al., 2006; baun et al., 2008). functionalized surfaces, by coating, or by hydroxylation or carboxylation, generally reduced the toxicity of fullerenes (lovern et al., 2007; kennedy et al., 2008; klaper et al., 2009). even more impressive were the results obtained by introducing apparently innocent variations, like stirring and shaking during the entire exposure time. these simple modifications were associated with unexpected changes (zhu et al., 2009). while metal oxides seemed to retain higher ecotoxicity for invertebrates, followed by buckminsterfullerenes and mwcnt, generalization is difficult and with some degree of arbitrariness. nanomaterial interactions with living systems are quite complicated: they can act as carriers for drugs, toxic chemicals, and dissolved substances, and enhance the bioavailability of other molecules (zhang et al., 2007; sun et al., 2008). daphnid sensitivity in acute testing was high with all tested substances. a dose-dependent response to concentration or exposure time was frequent, but not always present. the concentration was indeed self-limiting, being that these compounds were poorly soluble. chronic toxicity was a rare event, but persistent, and able to affect the population size. in the case of qds, generalization is difficult. the apparent low or even absent toxicity of tested formulations on invertebrates is contrasting with data from other systems (tang et al., 2008; kingheiden et al., 2009). evidences in invertebrates, however, remain quite incomplete. only very recent papers started a systematic study on bioavailability, bioaccumulation, compartmentalization and degradation of qds in exposed invertebrates (jackson et al., 2009; peyrot et al., 2009). biodegradation and bioaccumulation were poorly studied. both fullerene and nano-metal oxide mainly accumulated in the gut, at least in daphnids: a nearly complete faecal excretion of digested bulk material within 20-24 h prevented bioaccumulation, as demonstrated at least in d. magna and c. dubia. great amounts of nanoparticles (fullerene and metal oxide ot nanoag) were instead found accumulated in all tissues of dead daphnids (oberdörster et al., 2006b; roberts et al., 2007; kennedy et al., 2008). they were able to adhere to the carapace of daphnids and copepods, while the link to toxic physiological or behavioral effects was 91 only postulated, not documented (baun et al., 2008). validation protocols for assessing bioaccumulations of soil pollutants in terrestrial organisms have been planned by the oecd (egeler et al., 2009). a need for better standardization and supervision of studies seems urgent, to avoid dispersal of efforts and accumulation of anecdoctical and poorly descriptive results. planning research under a rational of feasibility and finding easy methods is a positive trend and should be encouraged, with the limit that the comparison with different and previous studies will not be compromised, and the conventional protocols fulfilled. another regrettable point is the complete lack of molecular data: 48 % of about 30 genes found overor under-expressed in human cells exposed to carbon nanotubes (cui et al., 2005; sarkar et al., 2007) are conserved in bilateria and the correspondent genes are known at least in the model organisms d. melanogaster and c. elegans. data on gene expression changes caused by exposure to nanomaterials in invertebrates should clarify the modalities of action and the underlying pathogenesis mechanisms; a wide place for new researches seems to be available in this field. acknowledgments m chiriva-internati's stay at the dbsm was partially supported by a cariplo grant. references adams lk, lyon dy, mcintosh a, alvarez pj. comparative toxicity of nano-scale tio2, sio2 and zno water suspensions. water sci. technol. 54: 327-334, 2006. adams md, celniker se, holt ra, evans ca, gocayne jd, amanatides pg, et al. the genome sequence of drosophila melanogaster. science 287: 2185–2195, 2000. allen sn, edge m, sandoval g, verran j, stratton j, maltby j. photocatalytic coatings for environmental applications. photochem. photobiol.81: 279-290, 2005. anderson hr, wollenberger l, halling-sorensen b, kusk ko. development of copepod nauplii to copepodites: a parameter for chronic toxicity including endocrine disruption. environ. toxicol. chem. 20: 2821-2829, 2001. austad sn. is there a role for new invertebrate models for aging research? j. gerontol. a biol. sci. med. sci. 64: 192-194, 2009. baird k. transport and ecotoxicity of c60 fullerenes in the terrestrial environment honour thesis, the ohio state university, 2007. baun a, hartmann nb, grieger k, kusk ko. ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing. ecotoxicology. 17: 387-395, 2008a. baun a, sørensen sn, rasmussen rf, hartmann nb, koch cb. toxicity and bioaccumulation of xenobiotic organic compounds in the presence of aqueous suspensions of aggregates of nano-c(60). aquat. toxicol. 86: 379-387, 2008b. biju v, itoh t, anas a, sujith a, ishikawa m. semiconductor quantum dots and metal nanoparticles: syntheses, optical properties, and biological applications. anal. bioanal. chem. 391: 2469-2495, 2008. blaise c, gagné f, férard jf, eullaffroy p. ecotoxicity of selected nano-materials to aquatic organisms. inc. environ. toxicol. 23: 591-598, 2008. blaise c., kusui t. acute toxicity assessment of industrial effluents with a microplate-based hydra attenuata assay. environ. toxicol. water qual. 12: 53-60, 1997. blaser sa, scheringer m, macleod m, hungerbühler k. estimation of cumulative aquatic exposure and risk due tosilver: contribution of nano-functionalized plastics and textiles. sci. tot. envinron. 390: 396-409, 2008. borgmann u, norwood wp, nowierski m. amphipod (hyalella azteca) solid-phase toxicity test using high water-sediment ratios, (chapter 12). in: blaise c, férard j-f (eds), small-scale freshwater toxicity investigations, vol. 1: toxicity tests methods, springer verlag, dordrecht, nl, pp 413-436, 2005. bouldin jl, ingle tm, sengupta a, alexander r, hannigan re, buchanan ra. aqueous toxicity and food chain transfer of quantum dots in freshwater algae and ceriodaphnia dubia. environ. toxicol. chem. 27: 1958-1963, 2008. burton ag, denton dl, ho k, ireland ds. sediment toxicity testing: issues and methods. in: hoffman dj, rattner ba, burton ag, cairns j (eds), handbook of ecotoxicology, 2nd edition, crc press llc, boca raton, usa, pp 111150, 2003. byron al, ponder lh: crustacea in eastern kansas. trans. kansas acad. sci. 52: 168-204, 1949. casey wc, rustad jr. reaction dynamics, molecular clusters, and aqueous geochemistry. annu. rev. earth planet. sci. 35: 21-46, 2007. centeno md, persoone g, goyvaerts m. cystbased toxicity tests. ix. the potential of thamnocephalus platyurus as test species in comparison with streptocephalus proboscideus (crustacea, branchiopoda, anostraca). environ. toxicol. water qual. 10: 275-282, 1995. christian pc, von der kammer f, baalousha m, hofmann t. nanoparticles: structure, properties, preparation and behaviour in environmental media. ecotoxicology, 17: 326343, 2008. chun ke p, qiao r. carbon nanomaterials in biological systems. j. phys: condens. matter 19: 373101, 2007. crane m, handy rd, garrod j, owen r. ecotoxicity test methods and environmental hazard assessment for engineered nanoparticles. ecotoxicology 17: 421-437, 2008. cui d, tian f, ozkan cs, wang m, gao h. effect of single wall carbon nanotubes on human hek293 cells. toxicol. lett. 155:73-85, 2005. curl rf jr. dawn of the fullerenes: experiment and conjecture. nobel lecture chemistry, 1-32, 1996. 92 davies wj, freeman sj. the hydra attentuata assay. meth. mol. biol. 43: 321-326, 1995. deguchi s, alargova rg, tsujii k. stable dispersions of fullerenes, c60 and c70, in water. preparation and characterization. langmuir 17: 6013-6017, 2001. diegoli s, manciulea al, begum s, jones jp, lead jr, preece ja. interaction between manufactured gold nanoparticles and naturally occurring organic macromolecules. sci. total environ. 402: 51-61, 2008. di gioacchino m, verna n, gornati r, sabbioni e, bernardini g. metal nanoparticle health risk assessment. [in press]. domínguez j, velando a, aira m, monroy f. uniparental reproduction of eisenia fetida and e. andrei (oligochaeta: lumbricidae): evidence of self-insemination. pedobiologia 47: 530-534, 2003. drobne d, jemec a, pipan tkalec z. in vivo screening to determine hazards of nanoparticles: nanosized tio2. environ. pollut. 157: 1157-1164, 2009. duffin r, mills nl, donaldson k. nanoparticles: a thoracic toxicology perspective. yonsei med. j. 48: 561-72, 2007. dunphy guzman ka, finnegan mp, banfield jf. influence of surface potential on aggregation and transport of titania nanoparticles. environ. sci. technol. 40: 7688-7693, 2006a. dunphy guzman ka, taylor mr, banfield jf. environmental risks of nanotechnology: national nanotechnology initiative funding, 2000-2004. environ. sci. technol. 40: 14011407, 2006b. ebert d. ecology, epidemiology and evolution of parasitism in daphnia. national library of medicine (us), ncbi, bethesda, usa, 2005. (http://www.ncbi.nlm.nih.gov). ecetoc. aquatic hazard assessment ii. brussels, 2003. egeler p, gilberg d, scheffczyk a, moser t, römbke j. validation of a soil bioaccumulation test with terrestrial oligochaetes by an international ring test. technical report: oecd/fkz: 204 67 458, 2009. (http://www.oecd.org/dataoecd/12/20/42552727 .pdf). emerich df, thanos cg. the pinpoint promise of nanoparticle-based drug delivery and molecular diagnosis. biomol. eng. 23: 171184, 2006. fang j, lyon dy, wiesner mr, dong j, alvarez pj. effect of a fullerene water suspension on bacterial phospholipids and membrane phase behavior. environ. sci. technol. 41: 2636-2642, 2007. farré m, gajda-schrantz k, kantiani l, barceló d. ecotoxicity and analysis of nanomaterials in the aquatic environment. anal. bioanal. chem. 393: 81-95, 2009. filella m, rellstab c, chanudet v, spaak p. effect of the filter feeder daphnia on the particle size distribution of inorganic colloids in freshwaters. water res. 42: 1919-1924, 2008. fortner ld, lyon dy, sayes cm, boyd am, falkner jc, hotze em, et al. c60 in water: nanocrystal formation and microbial response. environ. sci. technol. 39: 4307-4316, 2005. gagné f, auclair j, turcotte p, fournier m, gagnona c, sauvé s, et al. ecotoxicity of cdte quantum dots to freshwater mussels: impacts on immune system, oxidative stress and genotoxicity. aquatic toxicol. 86: 333-340, 2008. glenn jc. nanotechnology: future military environmental health considerations. technol. forecast. soc. change 73: 128-137, 2006. gornati r, papis e, di gioacchino m, sabbioni e, dalle donne i, milzani a, et al. in vivo and in vitro models for nanotoxicology testing [in press]. gourmelon a, ahtiainen j. developing test guidelines on invertebrate development and reproduction for the assessment of chemicals, including potential endocrine active substances the oecd perspective. ecotoxicology 16:161-167, 2007. griffitt rj, luo j, gao j, bonzongo jc, barber ds. effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. environ. toxicol. chem. 27: 19721978, 2008. handy rd, owen r, valsami-jones e. the ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs. ecotoxicology 17: 315-325, 2008a. handy rd, von der kammer f, lead jr, hassellöv m, owen r, crane m. the ecotoxicology and chemistry of manufactured nanoparticles. ecotoxicology 17: 287-314, 2008b. hardman r. a toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. environ. health perspect. 114: 165-172, 2006. hart ac. behavior. in: the c. elegans research community (ed), wormbook, massachusetts general hospital cancer center, charlestown, ma, usa, pp 1-62, 2006.,doi/10.1895/wormbook.1.87.1, http://www.wormbook.org. hassellöv m, readman jw, ranville jf, tiede k. nanoparticle analysis and characterization methodologies in environmental risk assessment of engineered nanoparticles. ecotoxicology 17: 344-361, 2008. havens ke. littoral zooplankton responses to acid and aluminum stress during short-term laboratory bioassays. environ. pollut. 73:71-84, 1991. heeger a. semiconducting and metallic polymers: the fourth generation of polymeric materials. nobel lecture chemistry, 380-417, 2000. heinlaan m, ivask a, blinova i, dubourguier hc, kahru a. toxicity of nanosized and bulk zno, cuo and tio2 to bacteria vibrio fischeri and crustaceans daphnia magna and thamnocephalus platyurus. chemosphere 71: 1308-1316, 2008. heymann d. solubility of c60 and c70 in seven normal alcohols and their deduced solubility in water. fullerene sci. technol. 4, 509-515, 1996. 93 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=books hines a, yeung wh, craft j, brown m, kennedy j, bignell j, et al. comparison of histological, genetic, metabolomics, and lipid-based methods for sex determination in marine mussels. anal. biochem. 369: 175-186, 2007. holbrook rd, murphy ke, morrow jb, cole kd. trophic transfer of nanoparticles in a simplified invertebrate food web. nat. nanotechnol. 3: 352-355, 2008. holdway da. hydra population reproduction toxicity test method (chapter 11). in: blaise c, férard j-f (eds), small-scale freshwater toxicity investigations. vol. 1: toxicity tests methods, springer verlag, dordrecht, nl, pp 395-411, 2005. hund-rinke k, simon m. ecotoxic effect of photocatalytic active nanoparticles (tio2) on algae and daphnids. environ. sci. pollut. res. int. 13: 225-232, 2006. iguchi t, watanabe h, katsu y. toxicogenomics and ecotoxicogenomics for studying endocrine disruption and basic biology. gen. comp. endocrinol. 153: 25-29, 2007. ingle tm, alexander r, bouldin j, buchanan ra. absorption of semiconductor nanocrystals by the aquatic invertebrate ceriodaphnia dubia. bull. environ. contam. toxicol. 81: 249-252, 2008. jackson bp, pace he, lanzirotti a, smith r, ranville jf. synchrotron x-ray 2d and 3d elemental imaging of cdse/zns quantum dot nanoparticles in daphnia magna. anal. bioanal. chem. 394: 911-917, 2009. jemec a, drobne d, remškar m, sepčić k, tišler t. effects of ingested nano-sized titanium dioxide on terrestrial isopods porcellio scaber. environ. toxicol. chem. 27: 1904-1914, 2008. joncxyk e, gilron g. acute and chronic toxicity testing on daphnia sp. (chapter 10). in: blaise c, férard j-f (eds), small-scale freshwater toxicity investigations, vol. 1: toxicity tests methods, springer verlag, , dordrecht, nl, pp 337-394, 2005. kandlikar m, ramachandran g, maynard a, murdock b, toscano wa. health risk assessment for nanoparticles: a case for using expert judgment. j. nanoparticle res. 9: 137156, 2007. kennedy aj, hull ms, steevens ja, dontsova km, chappell ma, gunter jc, et al. factors influencing the partitioning and toxicity of nanotubes in the aquatic environment. environ. toxicol. chem. 27: 1932-1941, 2008. kim j, takahashi m, shimizu t, shirasawa t, kajita m, kanayama a, et al. effects of a potent antioxidant, platinum nanoparticle, on the lifespan of caenorhabditis elegans. mech. ageing devel. 129: 322–331, 2008. king-heiden tc, wiecinski pn, mangham an, metz km, nesbit d, pedersen ja, et al. quantum dot nanotoxicity assessment using the zebrafish embryo. environ. sci. technol. 43:1605-11, 2009. klaper r, crago j, barr j, arndt d, setyowati k, chen j. toxicity biomarker expression in daphnids exposed to manufactured nanoparticles: changes in toxicity with functionalization. environ. pollut. 157: 11521156, 2009. koehler a, marx u, broeg k, bahns s, bressling j. effects of nanoparticles in mytilus edulis gills and hepatopancreas a new threat to marine life? mar. environ. res. 66: 12–14, 2008. kroto hw. symmetry, space, stars and c60. nobel lecture chemistry, 44-79, 1996. kumar j, singh rk, samanta sb,1 rastogi rc, singh r. single-step magnetic patterning of iron nanoparticlesin a semiconducting polymer matrix. macromol. chem. phys. 207: 15841588, 2006. lauterwasser c. small sizes that matter: opportunities and risks of nanotechnologies. allianz center for technology, report in cooperation with the oecd international futures programme (2003-2015), 2003. lazorchak jm, smith me, haring hj. development and validation of a daphnia magna four-day survival and growth test method. environ. toxicol. chem. doi: 10.1897/08-296.1, 2008. leeuw tk, reith m, simonette ra, harden me, cherukuri p, tsyboulski da, et al. single-walled carbon nanotubes in the intact organism: nearir imaging and biocompatibility studies in drosophila. nano lett. 7: 2650-2654, 2007. lim sf, riehn r, ryu ws, khanarian n, tung c-k, tank d, et al. microscopy imaging of upconverting nanophosphors in caenorhabditis elegans. nano lett. 6: 169-174, 2006. loux nt, savage n. an assessment of the fate of metal oxide nanomaterials in porous media. water air soil pollut. 194: 227-241. 2008. lovern sb, klaper r. daphnia magna mortality when exposed to titanium dioxide and fullerene (c60) nanoparticles. environ. toxicol. chem. 25: 1132-1137, 2006. lovern sb, strickler jr, klaper r. behavioral and physiological changes in daphnia magna when exposed to nanoparticle suspensions (titanium dioxide, nano-c60, and c60hxc70hx). environ. sci. technol. 41: 4465-4470, 2007. macdiarmid ag. “synthetic metals”: a novel role for organic polymers. nobel lecture – chemistry, 427-447, 2000. malvindi am, carbone l, quarta a, tino a, manna l, pellegrino t, et al. rod-shaped nanocrystals elicit neuronal activity in vivo. small 4: 17471755, 2008. martínez de. mortality patterns suggest lack of senescence in hydra. exper. gerontol. 33: 217225, 1998. meulenkamp ea. size dependence of the dissolution of zno nanoparticles. j. phys. chem. b, 102: 7764-7769, 1998. moore mn. do nanoparticles present ecotoxicological risks for the health of the aquatic environment? environ. int. 32: 967-976, 2006. moore mn, lowe dm, soverchia c, haigh sd, hales sg. uptake of a noncalorific, edible sucrose polyester oil and olive oil by marine mussels and their influence on uptake and effects of anthracene. aquat. toxicol. 39: 307320, 1997. 94 mueller nc, nowack b. exposure modeling of engineered nanoparticles in the environment. environ. sci. technol. 42: 4447-4453, 2008. naha pc, casey a, tenuta t, lynch i, dawson ka, byrne hj, et al. preparation, characterization of nipam and nipam/bam copolymer nanoparticles and their acute toxicity testing using an aquatic test battery. aquat. toxicol. 92: 146-154, 2009. nowack b, bucheli td. occurrence, behavior and effects of nanoparticles in the environment. environ. pollut. 150: 5-22, 2007. oberdörster e. manufactured nanomaterials (fullerenes, c60) induce oxidative stress in the brain of juvenile largemouth bass. environ. health perspect. 112: 1058-1062, 2004. oberdörster e, mcclellan-green p, haasch m. ecotoxicity of engineered nanomaterials. in: kumar cssr (ed), nanomaterials: toxicity, health and environmental issues, wiley-vch, weinheim, germany, pp 35-50, 2006a. oberdörster e, zhu sq, blickley tm, mcclellangreen p, haasch ml. ecotoxicology of carbonbased engineered nanoparticles: effects of fullerene (c60) on aquatic organisms. carbon 44: 1112-1120, 2006b. oberdörster g, oberdörster e, oberdörster j. concept of nanoparticle dose metric and response metric. environ. health perspect. 115: a290, 2007. oberdörster g, oberdörster e, oberdörster j. nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. environ. health perspect.113: 823-839, 2005. oughton dh, hertel-aas t, pellicer e, mendoza e, joner ej. neutron activation of engineered nanoparticles as a tool for tracing their environmental fate and uptake in organisms. environ. toxicol. chem. 27: 1883-1887, 2008. paradise j, diliberto gm, tisdale aw, kokkoli e. exploring emerging nanobiotechnology drugs and medical devices. food drug law j. 63: 407-420, 2008. pauluhn j. pulmonary toxicity and fate of agglomerated 10 nm and 40 nm aluminum oxyhydroxides (alooh) following 4-week inhalation exposure of rats: toxic effects are determined by agglomerated, not primary particle size. toxicol. sci., doi:10.1093/toxsci/kfp046, 2009. persoone, g, blaise c, snell t, janssen c, van steertegem m. cyst-based toxicity tests ii. report on an international intercalibration exercise with three cost-effective toxkits. z. angewandte zool. 79 1: 17-36, 1993. persoone g, janssen c, de coen w. cyst-based toxicity tests. x. comparison of the sensitivity of the acute daphnia magna test and two crustacean microbiotests for chemicals and wastes. chemosphere 29: 2701-2710, 1994. petersen ej, huang q, weber wj jr. bioaccumulation of radio-labeled carbon nanotubes by eisenia foetida. environ. sci. technol. 42: 3090-3095, 2008a. petersen ej, huang q, weber wj jr. ecological uptake and depuration of carbon nanotubes by lumbriculus variegatus. environ. health perspect. 116: 496-500, 2008b. peyrot c, gagnon c, gagné f, willkinson kj, turcotte p, sauvé s. effects of cadmium telluride quantum dots on cadmium bioaccumulation and metallothionein production to the freshwater mussel, elliptio complanata. comp. biochem. physiol. c toxicol. pharmacol., [epub ahead of print], 2009. pieters bj, bosman-meijerman d, steenbergen e, van den brandhof ej, vanbeelen p, van der grinten e, et al. ecological quality assessment of dutch surface waters using a new bioassay with the cladoceran chydorus sphaericus. proc. neth. entomol. soc. meet. 19: 157-164, 2008. poynton hc, varshavsky jr, chang b, cavigiolio g, chan s, holman ps, et al. daphnia magna ecotoxicogenomics provides mechanistic insights into metal toxicity. environ. sci. technol. 41: 1044-1050, 2007. raisuddin s, kwok kw, leung km, schlenk d, lee js. the copepod tigriopus: a promising marine model organism for ecotoxicology and environmental genomics. aquat. toxicol. 83:161-173, 2007. ralston-hooper k, hopf a, oh c, zhang x, adamec j, sepùlveda ms. development of gcxgc/tof-ms metabolomics for use in ecotoxicological studies with invertebrates. aquat. toxicol. 88: 48–52, 2008. rickerby dg, morrison m. nanotechnology and the environment: a european perspective. sci. technol. adv. mat. 8: 19–24, 2007. roberts ap, mount as, seda b, souther j, qiao r, lin s, et al. in vivo biomodification of lipidcoated carbon nanotubes by daphnia magna. environ. sci. technol. 41: 3025-3029, 2007. rocha s, thünemann af, pereira mc, coelho man, möhwald h, brezesinski g. the conformation of b18 peptide in the presence of fluorinated and alkylated nanoparticles. chem. bio. chem. 6: 280-283, 2005. ruck jc. toxkit technology and new zealand water quality guidelines. environ toxicol. water qual. 13: 337-345, 1998. ruffin, p.b. nanotechnology for missiles. proc. spie 5359: 177-187, 2004. sarkar s, sharma c, yog r, periakaruppan a, jejelowo o, thomas r, et al. analysis of stress responsive genes induced by single-walled carbon nanotubes in bj foreskin cells. j. nanosci. nanotechnol. 7: 584-592, 2007. scott-fordsmand jj, krogh ph, schaefer m, johansen a. the toxicity testing of doublewalled nanotubes-contaminated food to eisenia veneta earthworms. ecotoxicol. environ. saf. 71: 616619, 2008. service rf. nanotoxicology. nanotechnology grows up. science 304: 1732-1734, 2004. shirakawa h. the discovery of polyacetylene film: the dawning of an era of conducting polymers. nobel lecture chemistry, 453-462, 2000. shvedova aa, kisin er, porter d, schulte p, kagan ve, fadeel b, et al. mechanisms of pulmonary toxicity and medical applications of carbon nanotubes: two faces of janus? pharmacol. ther. 121: 192-204, 2009. 95 smalley re. discovering the fullerenes. nobel lecture chemistry, 89-103, 1996. snape jr, maundb sj, pickford db, hutchinson th. ecotoxicogenomics: the challenge of integrating genomics into aquatic and terrestrial ecotoxicology. aquat. toxicol. 67: 143–154, 2004. snell tw. rotifer ingestion test for rapid assessment of toxicity (chapter 9). in: blaise c, férard j-f (eds), small-scale freshwater toxicity investigations, vol. 1: toxicity tests methods, springer verlag, dordrecht, nl, pp 323-336, 2005. soetaert a, van der ven k, moens ln, vandenbrouck t, van remortel p, de coen wm. daphnia magna and ecotoxicogenomics: gene expression profiles of the antiecdysteroidal fungicide fenarimol using energy-, moltingand life stage-related cdna libraries. chemosphere 67: 60-71, 2007. spohn p, hirsch c, hasler f, bruinink a, krug hf, wick p. c60 fullerene: a powerful antioxidant or a damaging agent? the importance of an in-depth material characterization prior to toxicity assays. environ pollut. 157: 11341139, 2009. steinberg ce, stürzenbaum sr, menzel r. genes and environment striking the fine balance between sophisticated biomonitoring and true functional environmental genomics. sci. total environ. 400: 142-161, 2008. stoeger t, reinhard c, takenaka s, schroeppel a, karg e, ritter b, et al. installation of six different ultrafine carbon particles indicate a surface area threshold dose for acute lung inflammation in mice. environ. health perspect. 114: 328-333, 2006. stoeger t, schmid o, takenaka s, schulz h. inflammatory response to tio2 and carbonaceous particles scales best with bet surface area. environ. health perspect. 115: a290-291, 2007. sun h, zhang x, zhang z, chen y, crittenden jc. influence of titanium dioxide nanoparticles on speciation and bioavailability of arsenite. environ. pollut. 157: 1165-70, 2009. tang m, wang m, xing t, zeng j, wang h, ruan dy. mechanisms of unmodified cdse quantum dot-induced elevation of cytoplasmic calcium levels in primary cultures of rat hippocampal neurons. biomaterials 29: 438391, 2008. tedesco s, doyle h, redmond g, sheehan d. gold nanoparticles and oxidative stress in mytilus edulis. mar. environ. res. 66: 131133, 2008. teeguarden jg, hinderliter pm, orr g, thrall bd, pounds jg. particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments. toxicol. sci. 95: 300-312, 2007. templeton rc, ferguson pl, washburn km, scrivens wa, chandler gt. life-cycle effects of single-walled carbon nanotubes (swnts) on an estuarine meiobenthic copepod. environ. sci. technol. 40: 7387-7393, 2006. tenne r, seifert g. recent progress in the study of inorganic nanotubes and fullerene-like structures. annu. rev. mater. res. 39: 2.12.27, 2009. the c. elegans sequencing consortium. genome sequence of the nematode c. elegans: a platform for investigating biology. science 282: 2012-2018, 1998. tiede k, boxall ba, tear sp, lewis j, david h, hassellöv m. detection and characterization of engineered nanoparticles in food and the environment. food add. contam. 25: 795-821, 2008. tiede k, hassellöv m, breitbarth e, chaudhry q, boxall ba. considerations for environmental fate and ecotoxicity testing to support environmental risk assessments for engineered nanoparticles. j. chromatog. a 1216: 503-509, 2009. tin-tin-win-s, mitsushima d, yamamoto s, fukushima a, funabashi t, kobayashi t, et al. changes in neurotransmitter levels and proinflammatory cytokine mrna expressions in the mice olfactory bulb following nanoparticle exposure. toxicol. appl. pharmacol. 226: 192198, 2008 tortiglione c, quarta a, tino a, manna l, cingolati r, pellegrino t. synthesis and biological assay of gsh functionalized fluorescent quantum dots for staining hydra vulgaris. bioconjug. chem. 18: 829-835, 2007. tungittiplakorn w, cohen c, lion lw. engineered polymeric nanoparticles for bioremediation of hydrophobic contaminants. environ. sci. technol. 39: 1354-1358, 2005. velzeboer i, hendriks j, ragas mj, van de meent d. nanomaterials in the environment: aquatic ecotoxicity of some nanomaterials. environ. toxicol. chem. 27: 1942-1947, 2008. vogelsberger v. thermodynamic and kinetic considerations of the formation and the dissolution of nanoparticles of substances having low solubility. j. phys. chem. b 107: 9669-9676, 2003. wang h, wick rl, xing b. toxicity of nanoparticulate and bulk zno, al2o3 and tio2 to the nematode caenorhabditis elegans. environ. pollut. 157: 1171-1177, 2009. warheit db, hoke ra, finlay c, donner em, reed kl, sayes cm. development of a base set of toxicity tests using ultrafine tio2 particles as a component of nanoparticle risk management. toxicol. lett. 171: 99-110, 2007. wells ml, goldberg ed. occurrence of small colloids in sea water. nature 353: 342-344, 1991. williams ta. limb morphogenesis in the branchiopod crustacean, thamnocephalus platyurus, and the evolution of proximal limb lobes within anostraca. j. zool. syst. evol. res. 45: 191-201, 2007. wittmaack k. in search of the most relevant parameter for quantifying lung inflammatory response to nanoparticle exposure: particle number, surface area, or what? environ. health perspect. 114: 187-194, 2006. wittmaack k. dose and response metrics in nanotoxicology. environ. health perspect. 115: a291-292, 2007. 96 zakharenko lp, zakharov, ik, vasiunina ea, karamysheva tv, danilenko am, nikiforov aa. determination of the genotoxicity of fullerene c60 and fullerol using the method of somatic mosaics on cells of drosophila melanogaster wing and sos-chromotest. genetika 33: 405409,1997. zhang, x., sun h, zhang z, niu q, chen y, crittenden jc. enhanced bioaccumulation of cadmium in carp in the presence of titanium dioxide nanoparticles. chemosphere 67: 160166, 2007. zhu s, oberdörster e, haasch ml. toxicity of an engineered nanoparticle (fullerene, c60) in two aquatic species, daphnia and fathead minnow. mar. environ. res. 62 (suppl): s5-9, 2006. zhu x, zhu l, chen y, tian s. acute toxicities of six manufactured nanomaterial suspensions to daphnia magna. j. nanopart. res. 11: 67-75, 2009. 97 1 department of biotechnology and molecular sciences (dbms), university of insubria, varese, italy corresponding author: anna giulia cattaneo department of biotechnology and molecular sciences (dbms) university of insubria via j-h dunant 3, 21100 varese, italy davies wj, freeman sj. the hydra attentuata assay. meth. mol. biol. 43: 321-326, 1995. wound healing in annelids isj 7: 192-197, 2010 issn 1824-307x minireview mechanism of wound healing in annelids m grdisa division of molecular medicine, rudjer boskovic institute, zagreb, bijenicka 54, croatia accepted september 15, 2010 abstract all animals possess some type of tissue repair mechanism. in some species, the capacity to repair tissues is limited to the healing of wounds, but others posses a striking repair capability to replace the entire organs. it has been reported that some mechanisms, namely extracellular matrix remodeling, appear to occur in most repair processes. however, it remains unclear to what extent the process of wound healing is similar to organ regeneration. key words: annelid; wound healing; mechanism introduction the phylum annelid (13,000 living species) is a line of invertebrate life dating back 540 million years. their elongated, segmentated body plan and other body features have allowed annelids to specialize at burrowing through substrates, and to radiate into other ecological niches (class polychaeta segmentated worms in marine environments; class oligochaeta in fresh water and on land; class hirudina leeches parasites). animal species possess different capability to replace lost body parts through regeneration (herlant-meewis, 1964; thouveny and tassava, 1998; brusca and brusca, 2003). some species such as planaria, hydra or starfish can readily regenerate lost organs or body parts. in contrast, other species, such as most vertebrates have limited regenerative capacities. but, all species have some capacity to heal wounds produced by external factors during their existence. many researchers attempted to find the relationship between the process of wound healing and regeneration. there are some indications that these processes might differ at the cellular and molecular levels. in see urchins has been suggested that healing of broken spines occurs by a morphallactic mechanism involving recruitment of differentiated cells, while regeneration of removed spines and pedicellaria occurs by an epimorphic process involving undifferentiated precursors (dubois and ameye, 2001). also in zebra fish mutant (dobdevoid of blastema) was found that it failed to regenerate the ___________________________________________________________________________ corresponding author: mira grdisa division of molecular medicine, rudjer boskovic institute, bijenicka 54, 10 000 zagreb, croatia e-mail: grdisa@irb.hr fin but the response in wound healing was normal (whitehead et al., 2005). recently, it has been shown that both wound healing and regeneration in axolotl are dependent on epithelial/mesenchymal interactions, the formation of the wound epidermis, the restructuring of the extracellular matrix and other cellular/molecular events (roy and levesque, 2006). to see if the cellular mechanisms during wound healing and organ regeneration are similar, the sea cucumber holothuria glaberrima was investigated (miguel-ruiz and garcia-arraras, 2007). animals were lesioned and damaged tissue included muscle, nerve, water canal and dermis. the authors founded that cellular events associated with wound healing correspond to those occurred during organ regeneration. these include: an increase in the number of spherule-containing cells, remodeling of extracellular matrix, formation of spindle-structures that signal dedifferentiation of muscle cells and intense cellular division. thus, it is possible that regenerative limitations in some organisms are due to the absence of particular mechanisms associated with repair or the inability of activating the repair process in some tissues or stages. annelids: wound healing and regeneration annelids have a reputation for impressive regenerative abilities, but this ability varies widely. manny annelid worms are limited in their ability to regenerate anterior body parts, whereas posterior segment regeneration is much more common (bely, 2006). in addition to their ability to regenerate body segments, annelids generally have a marked capacity for wound healing (zoran and martinez, 2009). the remarkable ability of some annelids to reconstruct their entire body require coordinated 192 mailto:grdisa@irb.hr activation of multiple developmental, regenerative and wound healing processes in response to injury. on the other hand, leeches are incapable to regenerate lost segments, whereas they generate an effective response to injury by assembling an extracellular scaffold of proteins that facilitates the restoration of traumatized structures (tettamanti et al., 2004). wound healing in the earthworms could be used as a biomarker for assessing chemical toxicity. wound healing in earthworms is continuing process from open to healed wound with different stages and activities (cooper and roch, 1986). it involves the inflammatory response and various cell types, including immunoactive macrophage-like celomocytes. also the influence on cell membranes, cell division, energy production or use, synthesis of dna or rna and enzymatic pathways should be sufficient to interfere with the complex processes required to heal damaged tissue. wound healing in earthworms lumbricus terrestris (cikutovic et al., 1999) was monitored after cutting of three-sided patch of integument of l. terrestris during 5 days, after exposure to variety chloride compounds. the authors found that both, concentration and duration of exposure significantly reduced wound healing. similar finding was observed earlier (cooper and roch, 1992; ville et al., 1995). suppression of healing could be the consequence of interfering chloride compounds with the membranes of macrophages and other cells that function in healing process. it was found (ville et al., 1995; goven et al., 1993, giggleman et al., 1998) that some organics (polychlorinated biphenyl, pentachlorophenol, chlordane) suppress phagocytosis in earthworm celomocytes, probably by affecting their cell membranes. another chlorinated pesticide, lindane, was reported to reduce rna synthesis in mammals (lewis and adams, 1985; thomas and faith, 1985), which could also interfere with tissue repair during wound healing. cell division, which is also important in the wound healing process, might be affected with pollution in soil (cikutovic et al., 1993). heavy metals, such cd2+ or cu2+ may affect enzyme activity necessary for wound healing. it was shown (chen et al., 2001) that cu2+ interferes with an enzymatic pathway in celomocytes leading to production of superoxide (o2-), which is responsible for killing phagocytosed microorganisms in many animal species. it has been shown that exposure to cd2+ in mammals increased infections (lawrence, 1985), inhibited rna and dna synthesis (daun et al., 1993) and atp utilization (graham et al., 1975), whereas in l. terrestris celomocytes suppressed phagocytosis (roy and levesque, 2006). suppression of the healing process portends pathological effects in wildlife exposed to environmental toxicant if they are wounded during natural activities. healing of lacerations, punctures and abrasions of the integument or digestive tract may be compromised, resulting in microbial infection or parasite infestation. injured animals may be even more susceptible to pathogens if the chemicals that suppress the healing process also affect immunoactive cells responsible for phagocytizing and killing microorganisms. during wound healing the extracellular matrix (ecm) is produced, as well as its structural component, collagen. collagen also plays a major role in the modulation of several cell functions, including adhesion, migration, growth and differentiation (hay, 1991; birk and zycband, 1994; lim et al., 1994). in addition, fibrillar collagens are also involved in numerous processes, including stabilization of tissue shape and form during both vertebrate development and tissue regeneration (birk and trelstad, 1984; ingberg, 1994; fraizer et al., 1996; kletsas et al., 2000; badylak, 2002). collagen fibrils are present in both vertebrates and in lower invertebrates (bradbury, 1958; matsudanakagava and nicholls, 1991; sicot et al., 1997). previously was demonstrated that hirudo medicinalis (annelida, hirudinea) could be a very good animal model for investigation of tissue repair and wound healing (de eguileor et al. 2001, 2004; grimaldi et al. 2010). the body of leech has a simple organization. tissue repair in leeches shows a high degree of similarity to wound healing in vertebrates in biochemical and structural-functional points of view. the wound healing process in leeches can be divided into three stages: inflammation, granulation tissue and scar tissue remodeling. granulation stage in leeches is characterized by re-epithelization, angiogenesis and fibroplasias. during these steps occurs the formation of new epithelium, followed by the blood vessels and then by connective tissues (de eguileor et al. 2004; tettamanti et al. 2004) reorganization of collagen was studying in leech wound healing (tettamanti et al., 2004). after surgical lesion of the leeches it was shown that newly synthesized collagen acting as an extracellular scaffold. it directs and organizes the outgrowth of new vessels and the migration of immune cells towards the tissue lesions. in these animals, the collagen fibrils generated during tissue regeneration, showed similarities to both the structural pattern of collagen bundles and assembly processes observed in several vertebrate systems (fish scales, amphibian skin, human cornea). thus, leeches respond to surgical lesions with the same sequence of wound healing and tissue regeneration events as that described for vertebrate (tettamanti et al., 2005). it was found that the general architecture of leech collagen fibril organization and bundle orientation is identical with the structural pattern of collagen bundles observed in vertebrate cornea (birk and trelstad, 1984). thus, it could be hypothesized that collagen structures, characterized by a striking structural complexity and multifunctional purposes, are anatomical system highly conserved throughout evolution. to support the observation that “nature has followed economic and conservative strategies based on the conservation of a lot of molecules and related functions” (ottaviani et al., 2004), probably, evolution preserved the primitive models because of their excellent functional utility and effectiveness. the phases which could be involved in annelid wound healing is depicted on figure 1. if the cellular mechanisms during wound healing are similar to those occurred during organ regeneration, these report could be expanded with additional data. regeneration of annelids was studied 193 fig. 1 phase during wound healing in annelids in few groups, even at molecular level, with identification of the genes involved in the regeneration process (bely and sikes, 2010). in review bely (2006) has described that the most annelids have the ability to regenerate posteriorly. the ability to regenerate anteriorly is common but less widespread. the molecular mechanisms for regeneration process have not been yet completed. some annelids exhibit regenerative abilities very similar to planarians (bode et al., 1973; newmark et al., 2000), which can completely regenerate a new organism from small body fragment. however, the regeneration mechanisms are thought to be quite different between planarians and annelids. the planarians regenerate via totipotent stem cells (neoblasts) that are widely distributed throughout their bodies (redien and alvaro, 2004). the annelids regeneration is thought to occur primarily by cellular dedifferentiation and redifferentiation, without the contribution of totipotent cells (thouveny and tassava, 1998), but lately more date point on the involvement of stem cells (totipotent cells) in regeneration process (grimaldi et al. 2008, 2010). to better understanding the regeneration mechanisms in annelids, the genes that are expressed specifically during the course of regeneration, were identified on model animal enchytraeus japonensis (myohara et al., 2006). besides the known genes which play the roles in development (e.g genes for ecm, glutamine synthetase, nice-5, glucosidase), the new genes ejrup1-5 upregulated during regeneration, were isolated. after structural analyses of the products of these genes, a variety of putative functions that can be associated with their protein products were noticed. these functions include transportation and binding, transcriptional regulation, protein interaction and cell adhesion and perhaps some of them play an important role in regeneration. using in situ hybridization (niv et al., 2008), a strong expression of glutamine synthetase gene occurs in the blastemal regions of regenerating e. japonensis. strong expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. thus, according the results the authors suggested that e. japonensis glutamine synthetase may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis and gametogenesis. regenerative phenomenon has been explored also on the echinoderms and many details are shown in the reviews (candia-carnevali, 2006; kondo and akasaka, 2010). most explored model in echinoderm regeneration studies is the process of arm regeneration after lost following traumatic or self-induced amputation (candia-carnevali and bonasoro, 1994, 2001; bonasoro et al. 1995, 1998; candia-carnevali et al. 1995, 1998; patruno et al. 2001) using the feather star (crinoid antedon mediterranea). in this process new structures develop from migratory actively proliferating cells. different type of cells is involved in regeneration, including those that are considered to be stem cells. during regeneration, coelomocytes from coelomic canal and amoebocytes from brachial nerve, migrate to the distal wound area and are involved in regenerative process. from migratory amebocytes is formed a blastema. on the other hand, migratory coelomocytes contribute to regenerate the celomic system. cells proliferate at the blastema, coelomic canals and brachial nerve. since the migrating cells differentiate into new structure of the arm, they are presumably undifferentiated multipotent stem cells. but the knowledge about stem cells in crinoids 194 would be further support with molecular analyses. recently, similar results have been reported on the study of wound healing and arm regeneration in ophiderma longicaudum and amphiura filiformis (biressi et al. 2010). the earthworm provides a unique and valuable model to investigate the mechanism of regeneration because this process is rapid and it regeneration of a complete head and tail requires the reformation of various tissues and organs. to study the head and tail regeneration on annelid perionyx excavatus, the expression pattern of three labial genes (pex-la-01, pex-lab02, pex-lab03) was monitored (cho et al., 2009). the results indicated that these genes were expressed only in the head-regenerating tissues. also, the authors found that the expression of pexlab01 and pex-lab02 was up-regulated, and this indicated their involvement in wound healing and the blastema formation process during early head regeneration. on the other hand, the leeches do not possess ability to regenerate segments posteriorly or anteriorly (hyman, 1940). only some leeches can wound heal (le gore et al., 1971; huguet and molinas, 1996) and undergo limited nervous system repair (von bernhardi and muller, 1995), without any tissue or segment regeneration. data about wound healing in annelids are very obscure. the annelids have a big potential for regeneration of different body part, because they produce unique and potent molecules. probably, it is a reason that they are also investigated as a wound healing agent (matausic-pisl et al., 2010). the elucidation of the annelid regeneration mechanisms is thus expected to provide valuable information that may allow us in the future to explore strategies to enhance the regenerative capabilities in vertebrates. the cellular events that occur during wound healing, namely the pattern of cell division, ecm remodeling, and muscle dedifferentiation are similar to those that take place during regeneration of complex structures in the animals that posses a high regeneration capacity. to date a few molecular studies have been carried out to the understanding the mechanisms of annelids wound healing and regeneration. additional studies should be conducted to shed more light on this subject. references badylak sf. the extracellular matrix as a scaffold for tissue reconstruction. sem. cell dev. biol. 13: 377-383, 2002. bely ae. distribution of segment regeneration ability in the annelida. integr. & comp. biol. 46: 508518, 2006. bely ae, sikes jm. latent regeneration abilities persist following recent evolutionary loss in asexual annelids. proc. natl. acad. sci. usa 107: 1464-1469, 2010. biressi acm, zou t, dupont s, dahlberg c, di benedetto c, bonasoro f, et al. wound healing and arm regeneration in ophiderma longicaudum and amphiura filiformis (ophiuroidea, echinodermata): comparative morphogenesis and histogenesis. zoomorphology 129: 1-19, 2010. birk de, trelstad rl. extracellular compartments in matrix morphogenesis: collagen fibril, bundle and tamellar formation by corneal fibroblasts. j. cell. biol. 99: 2024-2033, 1984. birk de, zycband e. assembly of the tendom extracellular matrix during development. j. anat. 184: 457-463, 1994. bode h, berking s, david cn, gierer a, schaller h, trenkner e. quantitative analysis of cell types during growth and morphogenesis in hydra. roux's arch. 171: 269-285, 1973. bradbury s. a cytological and hystochemical study of the connective tissue fiber of the leech, hirude medicinalis. q. j. microsc. sci. 99: 131142, 1958. brusca rc, brusca gj. invertebrates. sinaur associates, sunderland, ma, 2003. carnevali mdc, bonasoro f, lucca e, thprndyke mc. pattern of cell proliferation in the early stages of arm regeneration in the feather star antedon mediterranea. j. exp. zool. 272: 464474, 1995. carnevali mdc. regeneration in echinoderms: repair, regrowth, cloning. inv. surv. j. 3: 64-76, 2006. carnevali mdc, bonasoro f, patruno m, thorndyke mc. cellular and molecular mechanisms of arm regeneration in crinoid echinoderms: the potential of arm explants. dev. genes evol. 208: 421-430, 1998. carnevali mdc, galassi s, bonasoro f, patruno m, thorndyke mc. regenerative response and endocrine disrupters in crinoid echinoderms: arm regeneration in antedon mediterranea after experimental exposure to polychlorinated biphenyls. j. exp. biol. 204: 835-842, 2001. chen sc, fitzpatrck lc, goven aj, venables bj, cooper el. nitroblue tetrazolium dye reduction by earthworm lumbricus terrestris coelomocytes: an enzyme assay for nonspecific immunotoxicity of xenobiotics. environ. toxicol. chem. 10: 1037-1043, 2001. cho aj, koh ks, lee e, park sc. differntial expression of three labial genes during earthworm regeneration. biosci. biotechnol. biochem. 73: 2609-2614, 2009. cikutovic ma, fitzpatrck lc, goven aj, venables bj, giggleman ma, cooper el. wound healing in earthworm lumbricus terrestris: a cellularbased biomarker for assessing sublethal chemical toxicity. bull. environ. contam. toxicol. 62: 508-514, 1999. cikutovic ma, fitzpatrck lc, venables bj, goven aj. sperm count in earthworm (lumbricus terrestris) as a biomarker for enviromental toxicology: effect of cadmium and chlordane. environ. pollut. 81: 123-125, 1993. cooper el, roch p. second-set allograft response in the earthworm lumbricus terrestris: kinetics and characteristics. transplantation 41: 514520, 1986. cooper el, roch p. the capacities of earthworms to heal wounds and to destroy allografts are modified by polychlorinated biphenyls (pcbs). j. inverteber. pathol. 60: 59-63, 1992. daum jr, shepherd dm, noelle rj. immunotoxicology of cadmium and mercury on 195 b-lymphocytes: i. effects on lymphocytes function. int. j. immunopharmacol. 15: 383394, 1993. de eguileor m, grimaldi a, tettamanti g, ferrarese r, congiu t, protasoni m, et al. hirudo medicinalis: a new model system for testing activators and inhibitors of angiogenesis. angiogenesis 4: 299-313, 2001. de eguileor m, tettamanti g, grimaldi a, perletti g, congiu t, rinaldi l, et al. hirudo medicinalis: avascular tissues for clear-cut angiogenesis studies? curr. pharm. des. 10: 1979-1988, 2004. dubois p, ameye l. regeneration of spines and pedicellarieae in echinoderms: a review. microsc. res. tech. 55: 427-437, 2001. fraizer k, wiliams s, kothapalli d, klapper h, grotendorst gr. stimulation of fibroblast cell growth, matrix production and granulation tissue formation by connective tissue growth factor. j. invest. dermatol. 107: 404-411, 1996. giggleman m, fitzpatrick lc, goven aj, venables bj. effect of pentachloraphenol (pcp) on survival of earthworms lumbricus terrestris and on phagocytosis by their immunoactive coelomocytes. environ. toxicol. chem. 17: 2391-2394, 1998. goven aj, eyambe g, fitzpatrick lc, venables bj. cellular biomarkers for measuring toxicity of xenobotics: effects of polychlorinated biphenyls an earthworm lumbricus terrestris coelomocytes. environ. toxicol. chem. 12: 863870, 1993. graham ja, gardner de, waters md, cogffin dl. effect of trace metals on phagocytosis of alveolar macrophages. infect. immun. 11: 1278-1283, 1975. grimaldi a, bianchi c, greco g. tettamanti g, noonan dm, valvassori r, et al. in vivo isolation and characterization of stem cells with diverse phenotypes using growth factor impregnated biomatrices. plos one 3(4):e1910. doi:10.1371, 2008. grimaldi a, banfi s, bianchi c, greco g. tettamanti g, noonan dm, et al. the leech: a novel invertebrate model for studying muscle regeneration and dseases. curr. pharm. des. 16: 968-977, 2010. hay e. collagen and other matrix glycoproteins in embriogenesis. in: hay e (ed), cell biology of extracellular matrix, plenum publishing, new yorg, ny, pp 419-462, 1991. herlant-meewis h. regeneration in annelids. adv. morphog. 4: 155-215, 1964. huguet g, molinas m. myofibroblast-like cells and wound contaction in leech wound healing. j. exp. zool. 275: 308-316, 1996. hyman lh. aspects of regeneration in annelids. am. nat. 74: 513-527, 1940. ingberg d. cellular tensegrety: exploring how mechanical changes in the cytosceleton regulate cell growth, migration and tissue pattern during morphogenesis. int. rev. cytol. 150: 173-224, 1994. kletsas d, pratsinis h, zervolea i, handris p, sevaslidou e, ottaviani e, et al. fibroblast responses to exogenous and autocrine growth factors relevant to tissue repair. the effect of aging. ann. ny acad. sci. 908: 155-166, 2000. kondo m, akasaka k. regeneration in crinoids. dev. growth differ. 52: 57-68, 2010. lawrence da. immunotoxicity of heavy metals. in: dean jh, luster mi, munson ae, amos h (eds), immunotoxicology and immunopharmacology, raven press, new york, ny, pp 341-353, 1985. le gore rs, sparks ak. repair of body wall incision in the rhynchobdellid leech pisscicol salmositica. j. invertebr. pathol. 18: 40-45, 1971. lewis jg, adams do. the mononuclear phagocytic system and its interactions with xenobiotics. in: dean jh, luster mi, munson ae, amos h (eds), immunotoxicology and immunopharmacology, raven press, new york, ny, pp 23-44, 1985. lim a, greenspan ds, smith bd. expression of α2 type i collagen in w8 cells increases cell adhesion and dereases colony formation in soft agar. matrix biol. 14: 21-30, 1994. masuda-nakagava lm, nicholls jg. extracellular matrix molecules in development and regeneration of the leech cns. philos. trans. r. soc. lond. 331: 323-335, 1991. matausic-pisl m, cupic h, kasuba v, mikecin am, grdisa m. tissue extract from eisenia foetida as a wound-healing agent. eur. rev. med. pharmacol. sci. 14: 177-184, 2010. miguel-ruiz jes, garcia-arraras je. common cellular events occur during wound healing and organ regeneration in the sea cucumber holothuria glaberrina. bmc dev. biol. 7: 115134, 2007. myohara m, niva cc, lee jm. molecular approach to annelid regeneration: cdna subtraction cloning reveals various novel genes that are upregulated during the large-scale regeneration of the oligochaete, enchytraeus japonensis. dev. dyn. 235: 2051-2070, 2006. newmark pa, sanchez alvardo a. bromodeoxiuridine specifically labels the regenerative stem cells of planarians. dev. biol. 220: 142-153, 2000. niv cc, lee jm, myohara m. glutamine synthetase gene expression during the regeneration of the annekid enchytraeus japonensis. dev. genes evol. 218: 39-46, 2008. ottaviani e, franchini a, kletsas d. platel-derived growth factor and transforming growth factor-β in invertebrate immune and neuroendocrine interactions: another sign of conservation in evolution. comp. biochem. physiol. 129c: 295306, 2001. patruno m, thorndyke mc, carnevali mdc, bonasoro f, beesley pw. growth factors, heatshock proteins and regeneration in echinoderms. j. exp. biol. 843-848, 2001. reddien pw, alvardo as. fundamentals of palnarian regeneration. ann. rev. cell dev. biol. 20: 725-757, 2004. roy s, levesque m. limb regeneration in axolotl: is it superhealing? tsw dev. embryol. 1(s1): 1215, 2006. 196 sicot fx, exposito jy, masselot m, garrone r, deutsch j, gaill f. cloning of an annelid fibrillar-collagen gene and phylogenetic analysis of vertebrate and invertebrate collagen. eur. j. biochem. 246: 50-58, 1997. tettamanti g, grimaldi a, rinaldi l, arnaboldi f, congiu t, valvassori r, et al. the multifunctional role of fibroblasts during wound healing in hirudo medicinalis (annelida, hirudinea). biol. cell 96: 443-455, 2004. tettamanti g, grimaldi a, rinaldi l, congiu t, perletti g, raspanti m, et al. collagen reorganization in leech wound healing. biol. cell 97: 557-568, 2005. thomas pt, faith re. adult and perinatal immunotoxicity induced by halogenated aromatic hydrocarbons. in: dean jh, luster mi, munson ae, amos h (eds), immunotoxicology and immunopharmacology, raven press, new york, ny, pp 305-313, 1985. thouveny y, tassava ra. regeneration through phylogenesis. in: ferretti p, geraudie j (eds), cellular and molecular basis of regeneration: from invertebrates to humans. john wiley & sons, new york, pp 9-43, 1998. ville p, roch p, cooper el, masson p, narbone jf. pcbs increase molecular-related activities (lysozyme antibacterial, hemolysis, proteases) but inhibit mecrophage-related functions (phagocytosis, wound healing) in earthworms. j. inverteber. pathol. 65: 217-224, 1995. von bernhardi r, muller kj. repair of the cental nervous system: lessons from lesions in leeches. j. neurobiol. 27: 353-366, 1995. whitehead gg, makino s, lien c-l, keating mt. fgf20 is essential for initiating zebrafish fin regeneration. science 310: 1957-1960, 2005. zoran mj, martinez vg. lumbriculus variegatus and the need for speed: a model system for rapid escape, regeneration and asexual reproduction. in: annelids in modern biol. shain dh (ed), john wiley & sons inc. new york, n y, pp 185204, 2009 197 isj 4: xx-yy, 2007 isj 4: 86-91, 2007 issn 1824-307x minireview a review on nutritive effect of mulberry leaves enrichment with vitamins on economic traits and biological parameters of silkworm bombyx mori l. r rajabi kanafi1, r ebadi1, sz mirhosseini2, ar seidavi3*, m zolfaghari4, k etebari5 1plant protection department, agriculture college, isfahan university of technology, isfahan, iran 2animal science department, agriculture college, guilan university, rasht, iran 3islamic azad university, science and research branch, teheran, iran 4plant protection student, agriculture college, bu-ali sina university, hamedan, iran 5sericulture department, natural resources college, university of guilan, iran accepted august 02, 2007 abstract sericulture depends on rearing of silkworm on mulberry leaves; for this reason, silk production has direct relationship with larval growth on mulberry. the quality and quantity of mulberry leaves change due to climatic conditions and field practices. one of the alternative ways of improvement of larval feeding is enrichment of mulberry leaves with supplementary nutrients such as vitamins. many studies are accomplished on the effects of mulberry leaves enrichment with vitamin on economic traits and biological parameters, and this review is the first trial for organization of all available data related to vitamins for elucidation of this topic of science. key words: silkworm; mulberry leaves; enrichment; vitamin; economic traits introduction nutrition plays an important role in improving the growth and development of the silkworm, bombyx mori l. like other organisms. legay (1958) stated that silk production is dependent on the larval nutrition and nutritive value of mulberry leaves plays a very effective role in producing good quality cocoons. seki and oshikane (1959) observed better growth and development of silkworm larvae as well as good quality cocoons when fed on nutritionally enriched leaves. silkworm obtains its entire nutritional requirement from mulberry leaves because this insect is monophagous and can complete the life cycle on mulberry leaves exclusively. studies of ito (1978) determined that generally vitamins present in the mulberry leaves satisfy minimum needs of silkworm but the amount of vitamins present in mulberry leaves varies on the basis of environmental conditions, usage of fertilizers in field and mulberry varieties and other field practices. sengupta et al. (1972) showed that b. mori requires specific essential sugars, amino acids, proteins and vitamins for its normal growth, ___________________________________________________________________________ corresponding author: alireza seidavi islamic azad university, science and research branch, teheran, iran e-mail: alirezaseidavi@yahoo.com survival and also for the silkgland growth. akhtar and asghar (1972) found that vitamins and mineral salts played an important role in the nutrition of silkworm. the effect of vitamin supplementation on the growth of b. mori have been investigated by many researchers (majumdar and medda, 1975; bhattacharyya and medda, 1981; das and medda, 1988; faruki, 1990, 1998, 2005; babu et al., 1992; faruki et al., 1992; khan and saha, 1996; nirwani and kaliwal, 1996, 1998; mosallanejad, 2002; etebari et al., 2004; rajabi et al., 2006a,b). keeping the importance of vitamins on silkworm nutrition in mind, following review was accomplished in order to determinate enrichment efficacy of mulberry leaves by vitamins. a list of vitamin quoted in international research papers are showed in table 1. water soluble vitamins water-soluble vitamins consist of members of the vitamin b complex and the vitamin c. ascorbic acid (vitamin c) ascorbic acid has many important functions in the animal body. it is a powerful antioxidant, protecting against oxidative damage to dna, membrane lipids and proteins. antioxidant activity of 86 mailto:alirezaseidavi@yahoo.com table 1 list of used vitamins and its derivation associated with related location. references location derivative vitamin etebari et al. (2004) iran vitamin c sarkar et al. (1995) bangladesh el-karaksy and idriss (1990) egypt chauhan and singh (1992) babu et al. (1992) gomma et al. (1977) india das and medda (1998) majumdar and medda (1975) bhattacharya and medda (1981) india cyanocobalmin (b12) rajabi et al. (2006b) iran pyridoxine (b6) faruki (2005) bangladesh rajabi et al. (2006a) iran riboflavin (b2) nirwani and kaliwal (1998) khan and saha (2003) india thiamine (b1) faruki (1998) bangladesh thianomin nirwani and kaliwal (1996) india folic acid (b9) faruki and khan (1992) khan and faruki (1990) bangladesh paba khan and saha (1996) bangladesh fe-plus etebari and matindoost (2004) saha and khan (1996) iran niacin (b3) etebari and matindoost (2005) iran multi-vitamin compounds muniandy et al. (1995) india saha and khan (1996) bangladesh evangelista et al. (1997) brazil sarkar et al. (1995) bangladesh b-complex mosallanejad (2002) iran vitamin e ascorbic acid decreases reactive oxygen species and oxidative pressure, and, as a result, the absorption of nutritious substances in the midgut would increase (felton and summers, 1993). ascorbic acid shows a particular behaviour as it is very susceptible to degradation, especially when in solution, and/or exposed to light, oxygen, and free radicals. ito (1961) recorded relationship of ascorbic acid supplementation and growth of silkworm. the absence of ascorbic acid in the diet of first and second instar larvae postponed growth and development of silkworm. there is enough vitamin c in mulberry leaves and ascorbic acid content of growing larvae is dependent on amount of this vitamin in diet. supplementation of mulberry leaves more than any other vitamin ascorbic acid has been used (etebari et al., 2004). several research demonstrated phagostimulatory effect of ascorbic acid for insects (ito, 1978; dobzhenok, 1974). in silkworm a gustatory stimulating activity have been observed to some extent (ito, 1961). gomma et al. (1977) observed that ascorbic acid significantly increased the weight of silkworm larvae. babu et al. (1992) observed that the first and second instar larvae reared on 1.5 % ascorbic acid enriched mulberry leaves resulted in higher silk filament length, weight and denier values. sengupta et al. (1972) reported that silk production increased with 1% ascorbic acid in the diet of silkworm. etebari et al. (2004) demonstrated that feeding on mulberry leaves enriched with ascorbic acid at 3% concentration decreased larval weight due to hypervitaminosis. chauhan and singh (1992) showed that 1% concentration of ascorbic acid could increase the number of eggs in the silkworm. although its lower concentration the leaves in the first and second generation also did not have positive effects on the fecundity in the silkworm. vitamin b complex the vitamin b complex is traditionally made up of 10 members (listed below) that differ in their biological actions, although many participate in energy production from carbohydrates and fats. the optimal levels of essential vitamins such as biotin, 87 table 2 comparison of quantitative requirements for bvitamins of the silkworm with the amounts in mulberry leaves (from ito, 1978) amount in mulberry leaves mg/g of dry matter minimum amount required mg/g of dry diet vitamin 0.2-0.8 1 biotin(b8) 930-1550 750 choline 4000 1000 inositol 69-99 20 nicotinic acid(b3) 16-35 20 pantothenic acid(b5) 43-50 5 pyridoxine(b6) 13-21 5 riboflavin(b2) 6.7 0.5 thiamine(b1) choline, pyridoxine, panthotinate, inositol, riboflavin, thiamine, nicotinic acid have been determined by horie and ito (1963, 1965) (table 2). riboflavin (b2) riboflavin is important in promoting the release of energy from carbohydrates, fats and proteins “i.e. in the metabolic pathway for atp production”. the enrichment of mulberry leaves with riboflavin at 77 ppm enhanced certain economic characters of silkworm, and improved silk production in north climatic conditions of iran (rajabi et al., 2006a). male cocoon weight (1.195 g) was greater at 77 ppm while female cocoon weight (1.622 g) was greater at 127ppm. maximum male pupal weight was recorded at 37 ppm (0.895 g) compared to 127 ppm for the female (1.169 g). male and female shell weight (0.311 and 0.318 g) had significant increase at 77 ppm compared to control (0.276 and 0.277 g). male and female cocoon shell percentage reached their maximum at 77 ppm treatment, which were 26.06 % and 21.46 % respectively. average of 50 egg weight, number of eggs for every female and hatchability percentage did not show significant difference among the treatments and the control. similar improvements were not obtained in natanz, in the center of iran, place which has dry climatic conditions. folic acid (b9) folic acid plays a major role in cellular metabolism including the synthesis of some of the components of dna and pigment precursor (national research council (u.s), 1987, chapman, 1998). yosuhiro and sholchi (1971) noted that the silkworm growth decreased when folic acid was eliminated from artificial diet. nirwani and kaliwal (1996) determined that folic acid has phagostimulatory effects with significant increase in female and male cocoon weight and shell weight. para-amino benzoic acid (paba) is a growth regulator and represents one of the forms of folic acid. paba is one of the substances belonging to the vitamin b-complex group and supports vital function in insects and especially in silkworm (pai et al., 1988). paba supplementation has no significant effects on adult weight whereas it caused deleterious effects on their length (p<0.001) and wing-span (p<0.001) (faruki and khan, 1992). feplus® (ferrus fumarate+folic acid) supplementation significantly increased larval, pupal and adult weight in comparison with controls with lowest and highest growths obtained at the concentrations of 0.32 and 0.64 %, respectively (khan and saha, 1996). larval and pupal periods decreased at lower doses (0.08 and 0.16 %) while they increased after exposure to higher doses (0.32 and 0.64 %). fertility increased significantly in all treatments when compared to control except for 0.64 % concentration (khan and saha, 1996). pyridoxine (b6) pyridoxine is necessary for the proper functioning of over 60 enzymes that participate in amino acid metabolism. it is also involved in carbohydrate and fat metabolism. without pyridoxine or its derivatives no larva reached the third instar under aseptic condition. pyridoxine is important in protein metabolism and its deficiency in mammals results in decrease in phosphorylases (national research council (u.s), 1987). faruki (2005) reported that mulberry leaf fortification by pyrol® (pyridoxine hydrochloride hcl.25) in various concentrations from the third instar significantly reduced the fecundity. in this experiment it was found the lowest number of eggs was produced at the lowest concentration (10 µg/ml) and at higher concentrations it remains lower than in the control. banerjee and khan (1992) observed that vitamin b6 enhances the oviposition of the silkworm infected by bacillus thuringiensis var. kurstaki but the rate was lower than the control. faruki (2005) reported that higher concentration of vitamins reduced the fecundity and fertility of silkworm. further, the percent reproduction control (prc) in lower concentrations was more than higher concentrations (faruki, 2005). the reproductive success in lower concentrations was prominent with compared with the higher concentrations (faruki, 2005; etebari and matindoost, 2005). rajabi et al (2006b) showed that in north climatic conditions of iran, larval weight reached a maximum 2.601 g at the end of 5th instar. effective rate of rearing (err) was higher (75.33 %) at 100 ppm concentration compared with other concentrations (10, 500 and 1000 ppm). larval and silk indices reached their maximum at 100 ppm concentration while pupal and adult indices reached their maximum at 1000 ppm concentration in male and female. larval duration was longer (622.5 h) in 88 treatments against control (604.5 h). treatment differences were recorded also in respect of other economic characteristics (rajabi et al., 2006b). nicotinic acid (b3, niacin) niacin is important for the release of energy from carbohydrates and fats, the metabolism of proteins and production of several hormones (national research council (u.s), 1987). horie and ito (1965) showed the required level of niacin for silkworm is highly regulated to the most appropriate level of 33 μg/l of dry weight and the increase of niacin reduced the larval weight. horie (1995) showed a reduction of requirement pattern with increasing larval weight. niacin caused significant deleterious effects on larval growth. cocoon parameters such as cocoon weight, pupal weight and cocoon shell weight also showed significant decrease in all treatments (etebari and matindoost, 2004). furthermore, mulberry leaf enrichment with nicotinamide (10, 20 and 30 g/l) from first instar caused intensive mortality in the larval growth and only 1.2% larvae could reach the 5th instar. high doses (20 and 30 g/l) of nicotinamide killed all larvae before entering the 5th instar (etebari and matindoost, 2004). horie and ito (1965) reported that the analogues of niacin, 4-acetyl pyridine interrupts the larval growth when added to mulberry leaves and acted as an antimetabolite. niacin with 0.5 g/l acted as an antifeedant for silkworm larvae and decreased their metabolism (etebari and matindoost, 2004). thiamine (b1) thiamine is important for energy metabolism (national research council (u.s), 1987). nirwani and kaliwal (1998) reported that the weight of larvae and silk glands in all the thiamine fed groups had not shown any significant changes. on the other hand, larval duration, cocoon weight, shell weight and fecundity increased significantly. in opposition with what observed for ascorbic acid and folic acid, it has been found that thiamine has no phagostimulatory effect on silkworm (horie and ito, 1963; nirwani and kaliwal, 1996). faruki (1998) reported that the thiamine derivative thianomin enhanced the growth of silkworm larvae, pupae and adults at all concentrations used (50, 100, 500 and 1000 ppm). mulberry leaf enrichment with thianomin increased the growth indices such as larval, pupal and adult weight. silk index increased too in all treatments. thianomin increased cocoon characters such as cocoon weight, shell weight, cocoon shell length and breadth significantly (p< 0.05). cyanocobalamin (b12, cobalamin) cyanocobalamin plays important role in silkworm because it is a co-factor of propionate metabolism which is important substrate for biosynthesis of juvenile hormone in insects (halarnkar and blomquist, 1989). vitamin b12 does not occur in mulberry leaves but considerable amount of this vitamin was observed in larvae and pupae. it is believed that actinomycetes in the gut lumen produce vitamin b12. das and medda (1998) reported that supplementation of mulberry leaves with b12 vitamin could increase the synthesis of nucleic acid and proteins in the silkglands of the silkworm. pantothenic acid (b5) pantothenic acid is the precursor of coenzyme a that is vital for the metabolism of carbohydrates, the synthesis and degradation of fats, the synthesis of sterols and the resultant steroid hormones, and the synthesis of many other important compounds (national research council (u.s), 1987). biotin (b8, vitamin h) biotin has an important role in carbohydrate and fat metabolism. it has been showed that biotin is one of the essential vitamins for the silkworm b. mori (horie et al., 1966). it has important role in the synthesis of fatty acids in the silkworm and it is confirmed that minimal optimal level of biotin for growth and survival of the silkworm was much lower than those of other vitamins including nicotinic acid, pantothenic acid and pyridoxine (horie and ito, 1965). it is identical with the minimal threshold for alteration of fatty acid composition (horie and nakasone, 1968). however, to the best of our knowledge, research reports on enrichment mulberry leaves with biotin are not available. choline and inositol choline is traditionally not a vitamin; however it was identified as part of vitamin b complex. inositol is an important part of signaling mechanism that transmits information from outside to the inside of cells. it is generally considered dispensable in insect diets. choline and inositol are required by silkworm in higher level because they are lipogen substrate, also involved in the production of cell membranes. however, research reports on enrichment mulberry leaves with choline and inositol do not seem to be available. multi-vitamin compounds saha and khan (1996) described the extensive effects of multi-vitamin compounds as diet factors on growth interruption and the decrease of cocoon economical characteristics. it is showed that multivitamin and mineral compounds could increase the food intake, growth and conversion efficiency of silkworm (muniandy et al., 1995). evangelista et al. (1997) reported that the larval and cocoon weight increase under multi-vitamin compound treatment, but did not have any positive effects on cocoon shell weight. feeding with multi-vitamins in the larval stage adversely affected the hatchability of eggs. multi-vitamins even though could increase some biological characteristics in silkworm, did not influence the economical and yield contributing parameters. etebari and matindoost (2005) reported that feeding of silkworm on mulberry leaves enriched by with multi-vitamins from 4th instar increased female cocoon shell weight in 2.5% 89 concentration, while female pupal weight increased in 1% concentration. male and female shell ratio did not increase compared to controls. fat-soluble vitamins fat-soluble vitamins consist of the a, d, e and k vitamins. among these, enrichment of mulberry leaves with vitamin a, d or k for silkworm do not seem to have been studied. vitamin e α-tocopherol is slightly effective in increasing the number of eggs laid by moths and β-carotene has also some growth-promoting effect (ito, 1978). enrichment of mulberry leaves with e vitamin did not have significant effect on food consumption in silkworm larvae (mosallanejad, 2002). concluding remarks this review summarizes data showing that enrichment of mulberry leaves with various vitamins have different effects on economic traits and biological parameters of the silkworm. however, reported effects depend also on weather condition, larval stage treated, type of vitamin, varieties of mulberry and silkworm race studied. this possibly indicates the need for elaborating comprehensive studies on the subject. it is advisable that we keep in mind the negative effects of enrichment beside its positive effects on economic traits and biological parameters. moreover, each intervention on the natural content of silkworm food should take into account also costs, environmental safety and large scale feasibility. acknowledgements our studies cited in this paper were supported by the biotechnology institute and iran silkworm research center. references akhtar m, asghar a. nutritional requirement of silkworm bombyx mori. pakistan j. zool. 4: 101-107, 1972. babu m, swamy mt, rao pk, rao ms. effect of ascorbic acid-enriched mulberry leaves on rearing of bombyx mori l. indian j. seric. 31: 11-114, 1992. banerjee sk, khan ar. effect of vitamin b6 on the fecundity of bacillus thuringiensis var. kurstaki treated bombyx mori l. bangladesh j. zool. 20: 361-362, 1992. bhattacharyya a, medda ak. effect of cyanocobalmine and cobalt chloride on glycogen of silkgland of bombyx mori l.nistari race. sci. cult. 77: 268-270, 1981. chapman, r. f. the insect structure and function, cambridge university press, cambridge. 1998. chauhan t, singh k. studies on the effect of ascorbic acid on the fecundity in the mulberry silkworm. sericologia 32: 567-574, 1992. das s, medda a. effect of cyanocobalamine on protein and nucleic acid contents of ovary of silkworm, bombyx mori l., during larval, pupal and adult stages of development. insect sci. appl. 9: 641-646, 1988. dobzhenok nv. the effect of ascorbic acid on the physiological condition of the codling moth and its resistance to fungus and bacterial infection. zakhist roslin. 19: 3-7, 1974. el-karaksy, i. r. and m. idriss. ascorbic acid enhances the silk yield of the mulberry silkworm, bombyx mori l. j. appl. entomol. 109: 81-86, 1990. etebari k, ebadi r, matindoost l. effect of feeding mulberry’s enriched leaves with ascorbic acid on some biological, biochemical and economical characteristics of silkworm bombyx mori l. int. j. indust. entomol. 8: 81– 87, 2004. etebari k, matindoost l. application of multivitamins as nutrients on biological and economical characteristics of silkworm bombyx mori l. j. asia-pacefic entomol. 8: 16, 2005. etebari k, matindoost l. effects of hypervitaminosis of vitamin b3 on silkworm biology. j. biosci. 29: 417–422, 2004. evangelista a., carvalho ad, takahashi r, de carvalho ad. performance of silkworm (bombyx mori l.) fed with vitamin and mineral supplement. rev. agricul. pirac. 72: 199-204, 1997. faruki si, khan ar, mannan a. fecundity and fertility of the silkworm, bombyx mori l. fed on mulberry leaves supplemented with paraamino benzoic acid. bangladesh j. zool. 20: 351-353, 1992. faruki si. effect of pyridoxine on the reproduction of the mulberry silkworm, bombyx mori l. (lepidoptera: bombycidae). inv. surv. j. 2: 2831, 2005. faruki si. nutritive effects of thianomin® enriched mulberry leaves on the silkworm, bombyx mori l. univ. j. zool. rajshahi univ. 17: 39-44, 1998. faruki si. the effect of para-amino benzoic acid on bombyx mori l. (lepidpotera; bombycidae) m.sc. thesis, rajshahi university, bangladesh, 1990. felton gw, summers cb. potential role of ascorbic oxidase as a plant defense protein against insect herbivory. j. chem. ecol. 19: 1553-1568, 1993. gomma aa, el-shaaraway mf, salam ys, rizk ma. effect of dietary constituents on the biology of silkworm, bombyx mori l. ii. vitamins. z. ang. ent. 64: 231-240, 1977. halarnkar pp, blomquist g. comparative aspects of propionate metabolism. comp. biochem. physiol. 92b: 227-231, 1989. horie y, ito t. nutrition of the silkworm, bombyx mori-x. vitamin b requirments and the effects of several analogues. j. insect physiol. 11: 1585–1593, 1965. horie y, ito t. vitamin requirements of the silkworm. nature 197: 98-99, 1963. horie y, nakasone s. effect of dietary biotin on fatty acid composition of the silkworm, bombyx mori l. j. insect. physiol, 14: 13811387, 1968. 90 horie y, watanabe k, ito t. nutrition of silkworm, bombyx mori l. xiv. further studies on the requirements for b vitamins. bull. seric. exp. stn japan, 20393-409, 1966. horie y. recent advances of nutritional physiology and artificial diet of the silkworm, in japan. korean j. seri. sci. 37: 235–243, 1995. ito t. effect of dietary ascorbic acid on the silkworm, bombyx mori l. nature, 192: 951-952, 1961. ito t. silkworm nutrition; in the silkworm an important labratoary tool. tazima, y. (ed), pp. 121-157, kodansha ltd, tokyo, 1978. khan ar, faruki si. growth and development of bombyx mori l. on feed supplemented with para-amino benzoic acid. univ. j. zool. rajshahi univ. 9: 47-53, 1990. khan ar, saha bn. nutritive effects of fe-plus® (ferrus fumarate + acid folic) on the silkworm, bombyx mori l. bangladesh j. zool. 24: 199203, 1996. khan ar, saha bn. nutritive effects of thiamine on the silkworm, bombyx mori. bangladesh. j. zool. 31: 169-176, 2003. legay jm. recent advances in silkworm nutrition. ann. rev. ent. 3: 75-86, 1958. majumdar ac, medda ak. studies on the thyroxine and vitamin b12 induced changes in the life cycles of silkworms. indian j. physiol. appl. sci. 29: 1-13, 1975. mosallanejad h, bagheri zonus e, nouzari j, talebi, m. effect of feeding the first to third instar larvae of silkworm (bombyx mori) with mulberry leaves enriched with vitamin e on some reproductive characteristics. proceeding of 15th iranian plant protection congress. september, 2002. kermanshah. p. 167, 2002. muniandy s, sheela m, nirmala s. effect of vitamins and minerals (filibon) on food intake, growth and conversion efficiency in bombyx mori. environ. ecol. 13: 433-435, 1995. national research council (u.s). vitamin tolerance of animals. national research council (u.s.). subcommittee on vitamin tolerance.; netlibrary, inc.national academy press, washington dc, pp 96, 1987. nirwani rb, kaliwal bb. effect of folic acid on economic traits and the change of some metabolic substances of bivoltine silkworm, bombyx mori l. korean j. seric. sci. 38: 118123, 1996. nirwani rb, kaliwal bb. effect of thiamine on commercial traits and biochemical contents of the fat body and haemolymph in the silkworm bombyx mori l. sericologia 38: 639-646, 1998. pai ik, hedge sn, krishnamurthy nb. paba induced genotoxic effects in silkworm, bombyx mori. proc. int. sym. on nature of genetic variation in man (xii ann. conf. of emsi), hydarabad, p. 54, 1988. rajabi r, ebadi r, fazilati m, mirhoseini sz. nutritive effects of mulberry leaves enrichment with riboflavin vitamin on bio-economic characters of silkworm, bombyx mori l. 9th arab congress of plant protection. 19-23 november, damascus, syria, 2006a. rajabi r, ebadi r, fazilati m, mirhoseini sz. the effects of mulberry leaves enrichment with pyridoxine-hcl on economic traits and biological parameters of silkworm bombyx mori l. 17th iranian plant protection congress, december 2006, teheran, p391, 2006b. saha bn, khan ar. the growth and development of the silkworm, bombyx mori l. on feed supplemented with nicotinic acid. bangladesh j. life sci. 1: 103-109, 1996. sarkar a, haque m, rab m, absar n. effect of feeding mulberry (morus sp.) leaves supplemented with different nutrients to silkworm, bombyx mori l. curr. sci. 69: 185188, 1995. seki k, oshikane k. res. reports. fac. textile and sericulture, shinshu university, 1959. sengupta k, singh bd, mustafi jc. nutrition of silkworm, bombyx mori l. i. studies on the enrichment of mulberry leaf with various sugars, proteins, amino acids and vitamins for vigorous growth of the worm and increased cocoon crop protection. indian j. seric. 11: 1127, 1972. yasuhiro h, sholchi n. effects of levels of biosynthesis of fatty acids and carbohydrates in a diet on the biosynthesis of fatty acids in larvae of silkworm. j. insect physiol. 19: 14411450, 1971. 91 http://worldcat.org/search?q=au%3anational+research+council+%28u.s.%29.+subcommittee+on+vitamin+tolerance.&qt=hot_author http://worldcat.org/search?q=au%3anational+research+council+%28u.s.%29.+subcommittee+on+vitamin+tolerance.&qt=hot_author http://worldcat.org/search?q=au%3anetlibrary%2c+inc.&qt=hot_author << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice review isj 7: 228-238, 2010 issn 1824-307x review insect immunity and its signalling: an overview s tsakas, vj marmaras department of biology, university of patras, 26500 patras, greece accepted october 21, 2010 abstract the innate immunity is the immediate and sole response of invertebrates for the protection against foreign substances and pathogens. in insects, it relies on both humoral and cellular responses that are mediated via certain recognizing receptors and activation of several signalling pathways. fat body and hemocytes are the origins for the production and secretion of antimicrobial agents and activators/regulators of cellular response, while cell mediated immunity in insects is performed by hemocytes. in the last years, research has focused on the mechanisms of microbial recognition and activation of intracellular signalling molecules in response to invaders. in this review, we summarize the mechanisms of the innate immunity in insects and refer to potential interactions between humoral and cellular responses, combined with the involving signalling pathways and their cross talk. key words: insects; innate immunity; signalling pathways introduction living creatures are surrounded by a basically hostile environment. in order to survive, they have developed several defense mechanisms, including the immune system. these mechanisms protect organisms against foreign substances and pathogen invasion. in case of such an invasion, the first line of defense is available immediately and involves mechanisms, either humoral or cellular, that are non specific. the discrimination between humoral and cellular responses is, up to a point, arbitrary, since they all share same signalling pathway that are activated by different stimuli (lavine and strand, 2002; marmaras and lampropoulou, 2009).these mechanisms are embraced under the term innate immunity, which is the sole immune response in invertebrates. vertebrates have developed a second line of defense, the acquired immunity, which is highly specific and contains mechanisms targeting to a particular threat, each time. insects, the most widespread metazoans on earth, have a well-developed innate immune system that allows general and rapid responses to infectious agents while they lack an acquired immune system. the protection against pathogens begins primarily with certain barriers such as cuticle, gut and trachea, tissues that are difficult to be penetrated, while immune response is originated by ___________________________________________________________________________ corresponding author: vassilis j marmaras department of biology, university of patras, 26500 patras, greece e-mail address: marmaras@upatras.gr the fat body and the hemocytes. fat body is the largest organ of the hemocel, the insect body cavity, and is a major site for the production and secretion of antimicrobial peptides (hoffmann, 2003). hemocytes circulate in insect hemolymph. they derive from stem cells that differentiate into specific lineages. however, certain hemocyte types are not common in all insects and differ among species (charalambidis et al., 1995; meister and lagueux, 2003). the humoral immune response is based on the products of characterized immune genes induced by microbial infection and encode antimicrobial peptides, which are synthesized predominantly in fat body and released into hemolymph (hoffmann, 1995; gillespie et al., 1997; nakatogawa et al., 2009; shia et al., 2009). hemocytes and epithelial layers of the integument and the gut are also sites for the synthesis of such molecules. these genes are either not expressed or are constitutively expressed at a low rate prior to infection (hoffmann, 1995; engström, 1998). in addition, humoral immune responses include activation of enzymic cascades that regulate coagulation and melanization of hemolymph, and production of reactive oxygen and nitrogen species (ros-rns) (gilespie et al., 1997; bogdan et al., 2000; nappi and vass, 2001; hoffmann, 2003; mavrouli et al., 2005). cellular responses are performed by hemocytes and include phagocytosis, nodulation and encapsulation (schmidt et al., 2001; nappi et al., 2004; lamprou et al., 2005; mavrouli et al., 2005; sideri et al., 2007). 228 mailto:marmaras@upatras.gr there are a lot of important review papers in the literature which present, in details, specific groups of signalling pathways or mechanisms of innate immunity in insects. this paper is an overview of these mechanisms, describing, in general, humoral and cellular responses along with major signalling transduction pathways, emphasizing on their cross talk. origins of innate immunity in insects fat body the larval fat body is the major site of the intermediate metabolism of insects and plays functions analogous to those of the vertebrate liver. it consists of thin layers or strings, generally one or two cells thick, or small nodules suspended in the hemocel and distributed throughout insect body (roma et al., 2010). the majority of proteins of the hemolymph are synthesized in this tissue, which also serves as lipid, carbohydrate and protein storage. the fat body is a target tissue for all principal insect hormones such as neural hormones, juvenile hormone and ecdysone (keeley, 1985) and is also a site of response to microbial infection. characterized immune genes, in the fat body, are induced by microbial infection and encode antimicrobial peptides which are then released into the hemolymph (hoffmann, 1995; engström, 1998). in drosophila, seven antibacterial peptides have been characterised namely, cecropin, attacin, defensin, drosocin, diptericin, metchnikowin and also an antifungal peptide called drosomycin (lemaitre and hoffmann, 2007). in addition, lepidopreran fat body synthesizes and releases several other proteins, such as pattern recognition protein hemolin and two immulectins serine proteinases: prophenoloxidase activating proteinase and a serine proteinase inhibitor from the serpin family (zhu et al., 2003). hemocytes in insects, there are no blood vessels. blood and interstitial fluid are indistinguishable and are collectively referred as hemolymph which bathes all internal tissues, organs and hemocytes, and facilitates the transport of nutrients, waste products and metabolites. the most common types of circulating hemocytes, in the hemolymph of lepidoptera (manduca sexta, bombyx mori) and diptera (drosophila melanogaster, ceratitis capitata) are granulocytes and plasmatocytes (lavine and strand, 2002; kanost et al., 2004). however, these haemocyte types are not common in all insect species (lavine and strand 2002; meister and lagueux, 2003; lamprou et al., 2007). in addition, the terminology used to designate each haemocyte type is often different from one insect species to another (ribeiro and brehelin, 2006), although, there have functional similarities among different insect species. in drosophila, plasmatocytes are professional phagocytes and are the equivalent of mammalian cells from the monocyte/macrophage lineage. phagocytosis permits rapid removal of dead cells, during embryogenesis and metamorphosis and pathogens during infections. plasmatocytes also synthesize and secrete antimicrobial peptides and signal to the larval fat body, the functional equivalent of the mammalian liver, in response to an infection (agaisse et al., 2003). based on morphological criteria, hemocyte types similar to drosophila have been recognised and classified in c. capitata larvae, although they may differ significantly in function. medfly plasmatocytes, besides phagocytic activity, are involved in nodule formation and melanization, as they contain the precursors of the prophenoloxidase cascade (mavrouli et al., 2005; sideri et al., 2007; marmaras and lampropoulou, 2009). pattern recognition proteins/receptors the first step for the initiation of immune response, either humoral or cellular, is the recognition of the pathogen. this is achieved by the pattern recognition proteins/receptors (prps), that recognize and bind conserved domains (patterns) located on the pathogen surface, which are called pathogen-associated molecular patterns, (pamps) (medzhitov and janeway, 1997) the most characterized prps are the type c lectins, the peptidoglycan recognizing proteins, the β-1,3-glucan proteins, the hemolin and the integrins (bettencourt et al., 1997; michel et al., 2001; bettencourt et al., 2004). these proteins are present on the plasma membrane of fat body cells and hemocytes or they are soluble in the hemolymph. they bind on lipids and carbohydrates which are synthesized by microorganisms and are exposed on their surface, such as lipopolysaccharites (lps) of gram negative bacteria, lipoteichoic acids and peptidoglycans of gram positive bacteria and β-1,3-glucans of fungi (nappi et al., 2000). insights on the characterization of prps have been obtained mainly from studies in drosophila. certain hemocyte protein recognition receptors appear to be unique to drosophila whereas others have direct homologues to other insect species or even mammals (marmaras and lampropoulou, 2009). the binding of invaders’ pamps on prps induces the synthesis of antimicrobial proteins or initiates the proteolytic activation of phenoloxidase cascade or activates cellular immune response, leading to phagocytosis, nodule formation and encapsulation of the invaders (yu xq et al., 2002; marmaras and lampropoulou, 2009). immunolectins lectins are sugar recognition molecules and play an important role in immune-related reactions enabling an organism to distinguish self from nonself or modified-self determinants. they are characterized by a wide range of binding activities. nineteen genes were originally identified in drosophila as members of the c-type lectin family, although the distinct function, for each one of them, had not been clarified (theopold et al., 1999). ctype lectins had been classified into seven groups based on their overall domain structure. analyses of the superfamily representation in several completely sequenced genomes have added 10 new groups (zelensky and gready, 2005). in lepidopterans, immunolectins are involved in prophenoloxidase 229 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-4899v7y-3&_user=83471&_coverdate=05%2f31%2f2003&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=c000059670&_version=1&_urlversion=0&_userid=83471&md5=78b2191eae38a0987be919748d9327b8#bib22#bib22 activation, phagocytosis and nodule formation (yu et al., 2003; yu and kanost, 2004). peptidoglycan recognizing proteins peptidoglycan (pgn) consists of a sugar backbone and a stem-peptide of three to five amino acids, found mostly on the surface of gram positive bacteria (kaneko and silverman, 2005; little and cobbe, 2005). the peptidoglycan recognizing proteins (pgrps) are small extracellular proteins (20 kda) which are synthesized and secreted by the fat body, integument, gut, and in a minor degree by the hemocytes (kaneko and silverman, 2005). they are defined by a domain with homology to an enzyme called amidase. several pgrps, from flies and mammals, present amidase activity, and several others are predicted to have amidase activity. in addition, some pgrps lack a critical cysteine required for the active site, and are therefore thought to function only as recognition proteins without enzymic activity (mellroth et al., 2003). recognition of pgns on gram-positive or gram-negative bacteria, by circulating pgrps, activates the toll or imd intracellular signalling pathways, respectively, leading to the nuclear translocation of two nf-κb/rel proteins and drives anti-bacterial peptide gene expression. the details of these intracellular signalling pathway have been reviewed previously (silverman and maniatis, 2001; aggrawal and silverman, 2007). β-1,3-glucan recognising proteins in drosophila there is a gram-negative bacteria-binding protein (dgnbp) family, (kim et al., 2000). dgnbp-1 exists in both soluble and glycosylphosphatidylinositol-anchored membrane and functions as a pattern recognition receptor for lps from gram-negative bacteria and β-1,3-glucan from fungi and mediates innate immune signalling for the induction of antimicrobial peptide gene induction in cultured drosophila immune cells. (kim et al., 2000). two biosensor for fungal and bacterial infection, called β-1,3-glucan recognition proteins (βgrps), are present in the hemolymph of m. sexta (wang and jiang, 2010). both βgrps specifically recognize soluble or insoluble β-1,3-glucan and lps, bind onto a hemolymph proteinase-14 precursor (prohp14) through specific protein– protein interactions and initiate propo activation system.(wang and jiang, 2010). similar activation of propo system occurs in the beetle tenebrio molitor (zhao et al., 2007). hemolin the hemolin is a member of the immunoglobin superfamily and is synthesized by the fat body. hemolin has been found in the hemolymph of two lepidopteran species hyalophora cecropia and m. sexta and its concentration increases 20-fold, upon bacterial infection, although it has no direct antimicrobial activity (bettencourt et al., 1997; eleftherianos et al., 2007). in m. sexta, hemolin recognizes and binds lps on gram negative bacteria and lipoteichoic acid on gram positives bacteria, leading to their aggregation. (daffre and faye, 1997; yu and kanost, 2002). it must be noted that lps and lipoteichoic acid bind on the same site on the hemolin molecule. hemolin may also bind on glycolipids of the bacterial wall, showing that it acts as a wide range prp against infection. in h. cecropia, hemolin binds on bacterial lps and then binds on hemocytes, in a calcium dependent manner, thus activating protein kinase c, which initiates phagocytosis (bettencourt et al., 1997; daffre and faye, 1997). integrins integrins are surface proteins, widely expressed in metazoans (sponges to humans), and participate in adhesion, migration and tissue organization (hughes, 2001). integrins recognize and bind rgd motifs (amino-acid triplet arg-gly-asp) in specific cell-surface, or extracellular matrix (ecm) or soluble proteins (collagen, laminins, fibronectins) (hynes, 2002; humphries et al., 2004). integrins are primary molecules for the recognition of foreign agents and the initiation of immune response. in the medfly c. capitata, they are involved in bacteria (grampositive and gram-negative) phagocytosis by plasmatocytes, but not in lps or abiotic targets uptake (lamprou et al., 2007; mamali et al., 2009). in m. sexta, integrins play a key role in stimulating hemocytes adhesion leading to encapsulation (zhuang et al., 2008). humoral responses the recognition of invading pathogen either as bacteria or fungi or even viruses is followed by the immediate de novo synthesis of antimicrobial peptides (amps) and their secretion into the hemolymph (zasloff, 2002; bulet et al., 2004). these peptides are mainly synthesized by the fat body and in a lesser degree by the hemocytes, integument, gut, salivary glands and reproductive structures (nappi and ottaviani, 2000). antimicrobial peptides over 150 antimicrobial peptides (amps) have been isolated and characterized in insects. these molecules are small, 12-50 amino acids, cationic peptides, which bind anionic bacterial or fungal membranes leading to disruption and cell death (zasloff, 2002; yount and yeaman, 2006). although they have different structure and target organisms (bacteria or fungi), the amps are classified in four groups; a) cecropins, b) cysteine-rich peptides, c) proline-rich peptides, and d) glycine-rich peptides. cecropins were firstly isolated in h. cecropia after injection with bacteria (hultmark et al., 1980; steiner et al., 1981). these peptides are produced in response to septic injury by either gram positive or gram negative bacteria bacteria and affect on cellular proliferation by inhibiting the synthesis of proteins of the cell membrane. defensins and drosomycin are cysteine-rich peptides. defensins, destroy mostly gram-positive peptides by forming channels in the plasma membrane which leads to cell lysis, while drosomycin has an antifungal activity. diptericin is an antibacterial peptide that has been found only in diptera species and is induced upon gram negative bacteria infection in a way similar to attacines (nappi and ottaviani, 2000). lysozymes are enzymes that 230 http://www3.interscience.wiley.com/cgi-bin/fulltext/118714469/main.html,ftx_abs#b3#b3 degrade peptidoglycans of the bacterial cell wall. they are also found in other animals, plants, fungi and bacteriophages (bulet et al., 1999). enzymic cascades coagulation of hemolymph insects have developed mechanisms for the coagulation of hemolymph, in case of wounding, to prevent loss of body fluids (theopold et al., 2002).in the cockroach leucophaea maderae, hemocytes secrete a calcium dependent transglutaminase that catalyzes the polymerization of lipophorins and vitellogenin-like proteins. these last proteins have a domain homologous to the von willebrand clotting factor in mammals (βohn et al., 1994). the most characterized mechanism is the one in lymulus polyphemus, which appears to be similar in drosophila (vimlos and kurucz, 1998). according to this, lps and β-1,3-glucan trigger a serine protease chain reaction, finally leading to the coagulation of the hemolymph. in addition, serine protease activates melanization cascade (nappi et , al., 1995; mavrouli et al., 2005; sideri et al., 2007). it must be noted the dual role of serine protease in the insect immunity since intermediate metabolites of these two cascades, preclotting enzymes, melanin derivatives and reactive oxygen species, are toxic invading pathogens. melanization of hemolymph melanization, the pathway leading to melanin formation, has a central role in defense against a wide range of pathogens and participates in wound healing as well as in nodule and capsule formation in some lepidopteran and dipteran insects, (lavine and strand, 2001; lavine and strand, 2003; mavrouli et al., 2005). melanization depends on tyrosine metabolism. briefly, tyrosine is converted to dopa, an important branch point substrate, by activated phenoloxidase (po). dopa may be either decarboxylated by dopa decarboxylase (ddc) to dopamine or oxidised by po to dopaquinone. dopamine is also an important branch point substrate, because dopamine-derived metabolites either via po or through other enzymes are used in several metabolic pathways, participating in neurotransmission, cuticular sclerotization, crosslinking of cuticular components via quinone intermediates, phagocytosis, wound healing and melanization in immune reactive insects (fearon, 1997; aderem and underhill, 1999; ling and yu, 2005; marmaras and lampropoulou, 2009). cellular responses hemocytes are responsible for a number of defense responses in insects, among which phagocytosis, nodulation, encapsulation and melanization have been documented. these processes appear to be discrete immune responses in terms of gene expression and outcome. however, these certain immune responses share a number of common elements that function in concert to clear pathogens from the hemolymph. below we have outlined the current data on these defense responses and their relationships. phagocytosis phagocytosis initiates with the recognition of the invading pathogens, engulfment and is completed with their intracellular destruction, by individual hemocytes. in insects, phagocytosis is achieved mainly by the circulating plasmatocytes or granulocytes, in the hemolymph (gillespie et al., 1997; meister and lagueux, 2003; lamprou et al., 2005, 2007). the uptake of a microbe by a phagocytic cell is an extremely complex and diverse process which requires multiple successive interactions between the phagocyte and the pathogen as well as sequential signal transduction events. phagocytosis is induced when phagocyte surface receptors, are activated by target cells. it must be noted that the hemocyte response to various bacteria differs. for example, in a. aegypti hemocytes respond to e. coli with phagocytosis, whereas to micrococcus luteus with melanization (hernandez-martinez et al., 2002; hillyer and schmidt 2003a, b). furthermore, differences exist in the efficiency and speed of phagocytosis among different bacteria. it has been shown that e. coli is more readily phagocytosed than s. aureus, in a. gambiae as well as in isolated medfly hemocytes (levashina et al., 2001; moita et al., 2006; lamprou et al., 2007). these results strongly suggest that several distinct molecular mechanisms regulate phagocytosis in insects. nodulation nodulation refers to multicellular hemocytic aggregates that entrap a large number of bacteria. melanized or non-melanized nodules are formed in response to a number of invaders. nodule formation appears to be related with eicosanoids in many insect species (miller et al., 1999) or prophenoloxidase (po) and dopa decarboxylase (ddc) in medfly hemocytes (sideri et al., 2007). encapsulation encapsulation refers to the binding of hemocytes to larger targets, such as parasites, protozoa, and nematodes. encapsulation can be observed when parasitoid wasps lay their eggs in the hemocel of drosophila larvae. hemocytes after binding to their target they form a multilayer capsule around the invader, which is ultimately accompanied by melanization. within the capsule the invader is killed, by the local production of cytotoxic free radicals ros and rns, or by asphyxiation (nappi et al., 1995; nappi and ottaviani, 2000). antiviral response viruses are intracellular pathogens that infect all forms of life. the first potent antiviral defense mechanism was identified in plants, through rna silencing (ding and voinnet, 2007). recently, rnai was found to play an important role in the control of viral infection in drosophila. this mechanism of gene silencing depends upon small rnas that are 21-30 nucleotides. central to the rnai mechanism are the slicing enzymes of the argonaute (ago) family, which mediate highly specific cleavage of target rna molecules. the specificity of ago enzymes is achieved by their association with small rnas, 231 which guide them to complementary sequences. three rnai pathways, involving different members of the ago family, have been defined in drosophila: first, the small interfering (si)rna pathway involves ago-2, and is activated by double-stranded (ds)rna. sirnas are produced by the rnaseiii enzyme dicer-2, which forms a complex with the dsrna-binding protein (dsrbp) protein r2d2; second, the micro (mi)rna pathway involves ago1, dicer-1, and its dsrbd cofactor r3d1, and regulates expression of drosophila genes, in particular during development; third, the piwiassociated rna (pirna) pathway involves the three other ago proteins encoded by the drosophila genome, namely piwi, aubergine, and ago3. pirnas are involved in the control of mobile genetic elements, including the retrovirus gypsy, in the germ-line (brennecke et al., 2007; ding and voinnet, 2007; kemp and imler, 2007). signalling pathways in innate immunity from the overview of the mechanisms concerning the innate immunity, three major responses can be summarized. the production of antimicrobial peptides due to specific receptors, either soluble or membrane, the internalizationphagocytosis, which follows the attachment of bacteria on the cell membrane and the role of rna interference in the antiviral immunity. the hallmark of the drosophila humoral immune response is the production of antimicrobial peptides in the fat body and their release into the circulation (aggarwal, and silverman, 2008; feldhaar and gross, 2008). two recognition and signalling cascades regulate expression of these antimicrobial peptide genes. the toll pathway is activated by fungal and many gram-positive bacterial infections, whereas the immune deficiency (imd) pathway responds to gram-negative bacteria. both of these are initiated by peptidoglycan recognition proteins (pgrps) and complete their action via the conserved nf-κb signalling cascades for the control of immune-induced gene expression (aggarwal and silverman, 2008). phagocytosis is triggered by certain transmembrane proteins on the hemocyte surface. the most common classes of such receptors in insect plasmatocytes are the scavenger receptors, the egf-like-repeat-containing receptors, the integrins and the pgrps (feldhaar and gross, 2008; marmaras and lamproulou, 2009). the key intracellular molecules that promote signals from pathogens that attach on cell-surface receptors, are the scaffold and adaptor proteins. scaffold proteins are proteins that bind other proteins that usually function in sequence. adaptor proteins are proteins that augment cellular responses by recruiting other proteins to a complex. these molecules function as organizing platforms that bring together both the enzymes and the substrate proteins, in the same complex (marmaras and lampropoulou, 2009). we show that antimicrobial peptide synthesis and bacterial internalization share a lot of signalling molecules and pathways. antiviral response, although it consists of a totally different procedure targeting to the degradation of viral nucleic acids by rna interference it includes three classical immune signalling pathways (toll, imd, and jak-stat) responsive to infection by different viruses (kemp and imler, 2009; sabin et al., 2010). the toll pathway insects respond to gram-positive bacterial and fungal infections via the toll pathway. its basic component is the transmembrane receptor toll and the intracellular adaptors tube and myd88 (lemaitre and hoffmann, 2007; aggarwal and silverman, 2008), toll is not a pattern recognition receptor since it does not bind pathogens or pathogen-derived compounds, directly and is activated by the extracellular cytokine spätzle. to activate toll pathway, microbial recognition must be preceded. the detection of gram-positive bacterial peptidoglycans and fungal betaglucans by specific pgrps and gnbps, respectively, activate serine protease cascades from the fat body that culminate in spätzle cleavage, thus, liberating the c-terminal 106 amino acids of spätzle, the mature toll ligand. the cleaved spätzle binds the toll receptor which recruits the tube/myd88 complex, followed by the kinase pelle activation. pelle kinase triggers an intracellular signalling cascade involving several factors resulting in the activation of the transactivator proteins dorsal and dif belonging to the nf-kb family. after their translocation in the nucleus they induce transcription of the respective genes encoding for instance defensins, drosomycin, cecropins. in addition to dorsal and dif a drosophila ikb homolog called cactus is activated which is an inhibitory factor that negatively modulates the tollmediated immune response (feldhaar and gross, 2008). the imd pathway the gram-negative bacteria activate antimicrobial peptide synthesis via the imd pathway (nappi et al., 2004; lemaitre and hoffmann, 2007; aggarwal and silverman, 2008). this pathway was initially defined by the identification of a mutation named immune deficiency (imd) that impaired the expression of several antibacterial peptide genes (lemaitre and hoffmann, 2007). the bind of bacterial monomeric or polymeric dap-type pgn on the single-pass transmembrane cell surface receptor pgrp-lc, results the recruiting of the intracellular adaptor imd (aggarwal and silverman 2008). signal transduction leads to relish cleavage and the rel domain translocates to the nucleus, whereas the inhibitory domain remains stable in the cytoplasm. diptericin gene is an imd target in response to injection of e. coli (gram-negative bacteria). the jak/stat pathway the jak/stat pathway, has three main cellular components: the receptor domeless, the janus kinase (jak), and the stat transcription factor (lemaitre and hoffmann, 2007). bacterial 232 fig. 1 humoral immune response in insect fat body. secreted cytokines as well as pathogens, either bacteria or fungi, bind on several immune-related receptors in a non specific way, among insect species. this leads to the expression of antimicrobial protein genes and secretion of their respective peptides, via certain cytoplasmic pathways either specific (jak/stat for domeless or imd for peptidoglycan recognizing proteins-pgrp) or non specific (toll receptor) for each receptor. infections induce hemocyte to produce cytokine unpaired-3 (upd3), which is the ligand of domeless. the result of this pathway, after immune challenge, is the stat protein accumulation in the nucleus and the activation of gene expression. the transcriptional regulation is complex, with additional inputs from both the imd and mapk (mitogenactivated protein kinase) pathways (aggarwal and silverman, 2008). rna interference pathway rna interference (rnai) has been found to play an important role in the control of viral infection in drosophila (kemp and imler, 2009). central to the rnai mechanism are the slicing enzymes of the argonaute (ago) family, which mediate highly specific cleavage of target rna molecules. these enzymes associate with small rnas, which guide them to complementary sequences. three rnai pathways, involving different members of the ago family, have been defined in drosophila: • the small interfering (si)rna pathway involves ago-2, and is activated by double-stranded (ds)rna. sirnas are produced by the rnaseiii enzyme dicer-2, which forms a complex with the dsrna-binding protein (dsrbp) protein r2d2 (ding and voinnet, 2007) • the micro (mi)rna pathway involves ago-1, dicer-1, and its dsrbd cofactor, and regulates expression of drosophila genes • the piwi-associated rna (pirna) pathway involves the three other ago proteins encoded by the drosophila genes, namely piwi, aubergine, and ago3. pirnas are involved in the control of mobile genetic elements, including the retrovirus gypsy, in the germ-line (brennecke et al., 2007) demonstration of the critical role of rnai as a potent antiviral mechanism in drosophila is based on three lines of evidence: genetic data indicating that rnai pathway mutants are hypersensitive to rna virus infections, identification of viral suppressors of rnai (vsrs), which counteract the immune defense of the fly, and the presence of sirnas of viral origin in infected cells/flies (kemp and imler, 2009). integrin pathway integrins are heterodimeric transmembrane receptors consisting by an α and a β subunit. integrins recognize and bind rgd motifs (aminoacid triplet arg-gly-asp) in specific cell-surface, or extracellular matrix (ecm) or soluble proteins such as collagen, laminin and fibronectin (hynes, 2002; humphries et al., 2004). this ability leads to intracellular signal transduction (outside-in signalling) via activation fak/src pathways and mapk (lamprou et al., 2007; marmaras and lampropoulou, 2009). in the medfly c. capitata, integrins are involved in phagocytosis of bacteria, but not lps, by hemocytes (lamprou et al., 2005; lamprou et al., 2007; mamali et al., 2009), due to the activation of p38 via ras/rho/actin remodelling pathway, while in m. sexta, they lead to stimulate encapsulation by the stimulation of hemocyte adhesion (zhuang et al., 2008). pathway cross talk signal transduction is based on several pathways which form a complicated network, cross talking to each other in order to lead to the appropriate response, due to extracellular stimuli. such interactions may appear in every level of these pathways either in recognition (fig. 1) or signal transduction or even the final response (fig. 2) (garcia-lara et al., 2005). 233 fig. 2 humoral and cellular immune response in insect hemocytes. the bind of bacteria on a β-integrin transmembrane subunit, triggers cytoplasmic signalling via focal adhesion kinase (fak) and mitogen activated protein kinases (mapks) activation, major key point pathways. this leads to cellular responses such as phagocytosis, nodulation and encapsulation. the activation of fak and mapks may also lead to humoral response such as melanisation and wound healing, through the activation of cell surface prophenoloxidase (propo). different peptidoglycan recognition proteins (pgrps) show strong preference, but not exclusivity, towards specific pathogen–associated molecular patterns (pamps) and on the other hand these pathogens may be concerted with other protein recognition patterns (prrs). in drosophila hemolymph, certain soluble pgrp, which recognizes not only a pgn, common to s. aureus and other gram-positive bacteria but even a gnbp1, activate the toll signal transduction pathway (michel et al., 2001; gobert et al., 2003). the result is the synthesis of antimicrobial peptides (amps), such as drosomycin. on the other hand, a membrane pgrp (choe et al., 2002; gottar et al., 2002; ramet et al., 2002) and a soluble pgrp recognize peptidoglycans and activate the imd pathway (lemaitre et al., 1997; takehana et al., 2002). other pgrps may also weakly recognize gram-positive-type pgn or can bind with low affinity gram-negative-type pgn as well as lipopolysaccharide (lps) and lipotteichoic acid (lta) (dziarski, 2004). it becomes obvious that an initial signal does not guarantee a specific outcome, since there is no exclusivity for the activated receptor. in addition, the transduction of the signal, from the cell membrane into the cytoplasm, does not necessarily follow a single pathway but it may change course to another, by unspecific intracellular pathways such as these of src family or mapks namely, erk, p38 and jnk (garcia-lara et al., 2005). these enzymes appear to have overlapping and complementary functions in many pathways. perhaps the function of these enzymes is to modulate the overall intracellular signalling network in the fat body and hemocytes, rather than operating as exclusive signalling switches for defined pathways. thus, the final product differs among species and tissues. drosophila responds to gram-positive bacteria by the induction of drosomycin synthesis, through the toll pathway, while in gram-negatives by the induction of diptericin, attacin and cecropin through the imd pathway (leclerc and reichhart, 2004). however, it has also been reported that s. aureus, may induce the production of cecropins, while e. coli may induce the expression of drosomycin, giving additional evidence for a cross-talk between the two pathways (hedengren-olcott et al., 2004). the jak/stat pathway is activated in response to gram-negative bacteria and appears to branch out from the imd pathway in fruit flies and mosquitoes (agaisse and perrimon, 2004). the production of amps is not the only final response to the same initial stimulus. other humoral as well as cellular responses may be triggered, indicating an extracellular response network of innate immunity, too. immune response cross talk fat body and hemocytes are the major components of the innate immune response in insects. they possess a diverse repertoire of receptors that allow cells to respond to external stimuli such as cytokines and pathogen-associated molecules. signals resulting from these stimuli 234 activate the synthesis of antiviral peptides and the synthesis and secretion of antimicrobial peptides by the fat body. the functional responses of hemocytes are adhesion, cytokine release, melanization, phagocytosis, nodule formation and encapsulation. hemocyte challenging, by a pathogen, triggers all these humoral and cellular responses, which do not function separately, but they seem to cooperate with each other, in order to block pathogen invasion (fig. 2). in medfly and mosquito, phagocytosis begins with the binding of e. coli on an integrin β-subunit of the hemocyte surface (humphries et al., 2004; mavrouli et al., 2005; moita et al., 2006; mamali et al., 2009). integrins transmit signal to focal adhesion kinase/sarcoma (fak/src) and mitogen activated protein kinase pathways (mavrouli et al., 2005). this signal transduction leads to the secretion of serine proteases which convert the surface inactive prophenoloxidase to the active phenoloxidase and initiate melanization. in parallel, phagocytosis and nodule formation are triggered. although these responses seem to be distinct, they appear to cooperate since blockade of one of them inhibits the other (sideri et al., 2007). abiotic latex beads and lipopolysaccharide (lps) phagocytosis do not depend on propo activation (mavrouli et al., 2005; lamprou et al., 2007). the propo activation system is composed of proteins recognizing several pattern-recognition proteins, serine proteases, propo, as well as proteinase inhibitors that function as regulatory factors (cerenius and söderhall, 2004). propo is synthesized in hemocytes and appears to be distributed ubiquitously in the cytoplasm as well as on the surface of hemocytes (ling and yu, 2005; mavrouli et al., 2005). the propo activation system is triggered by several microbial components, such as lps and peptidoglycans. activated po catalyses the hydroxylation of tyrosine to 3,4-dihydroxyphenyl-alanine (dopa). dopa can be oxidized by po to dopaquinone, which, via po and the dopachrome conversion enzyme, ultimately result in melanin. dopa may also be decarboxylated by dopa decarboxylase (ddc) to form dopamine (marmaras and lampropoulou, 2009). ddc is involved in wound healing, parasite defense, cuticle hardening and melanisation (hodgetts and o’keefe, 2006). a pobased oxidation of dopamine leads to dopaminequinone and finally the cross-linking and melanization of proteins. the expression of ddc mrna in the hemocytes of pseudaletia separata was enhanced by injection of an insect cytokine, growth-blocking peptide (noguchi et al., 2003). melanization is the process that leads to melanin formation in both hemocyte-free hemolymph as well as on hemocyte surface after wounding or upon invasion with pathogens. ddc is a key enzyme between melanization and phagocytosis, two unrelated procedures that are linked and facilitate each other. the activity of ddc is elevated during melanotic responses in drosophila and in the mosquito armigeres subalbatus (nappi et al., 1992; huang et al., 2005). melanization is also a critical process in defense against bacteria, and several reports link components of the melanization process with phagocytosis (johansson and söderhall, 1996; hillyer et al., 2004; mavrouli et al., 2005) it has been proposed that pathogens might be killed by toxic reactive oxygen metabolites produced in the process of melanisation (nappi et al., 1995). however, ddc based melanization, appears to be distinct from the pathway leading to phagocytosis (sideri et al., 2007). these two unrelated procedures share a number of substrates (tyrosine, dopa, dopamine) and enzymes (po, ddc). nodulation, as stated in the introduction, refers to multicellular hemocyte aggregates that entrap a large number of bacteria, and po and ddc are key enzymes in this process (mavrouli et al., 2005). nodules may be attached to tissue or surrounded by hemocytes. nodule formation has not been fully characterized, although it is known that it is lectinmediated. melanization and nodulation are two distinct pathways which share a number of substrates and enzymes. phagocytosis and nodulation processes are distinct from the melanisation process and questions have been raised whether branch-point substrates exist to differentiate phagocytosis from nodulation or whether they are processes in sequence (sideri et al., 2007). conclusion innate immunity is an interesting and exciting field for research. its evolutionary conserved mechanisms make insects first line tools for investigation. the study of immune responses and the relative signalling pathways have revealed their cooperation and the key components. insect innate immunity appears to be an easy to handle tool to study how different stimuli activate the same receptor and how this receptor activates different pathways, leading to the same or different response. references aderem a, underhill dm. mechanisms of phagocytosis in macrophages. annu. rev. immunol. 17: 593-623, 1999. agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell. 5: 441-450, 2003. aggrawal k, silverman n. peptidoglycan recognition in drosophila. biochem. soc. trans. 35: 14961500, 2007. aggarwal k, silverman n. positive and negative regulation of the drosophila immune response. bmb rep. 41: 267-277, 2008. bettencourt r, asha h, dearolf c, ip yt. hemolymph-dependent and -independent responses in drosophila immune tissue. j. cell. biochem. 92: 849-863, 2004. bettencourt r, lanz-mendoza h, lindquist kr, faye i. cell adhesion properties of hemolin, an insect immune protein in the ig superfamily. eur. j. biochem. 250: 630-637, 1997. bettencourt r, lanz-mendoza h, lindquist kr, faye i. cell adhesion properties of hemolin, an insect immune protein in the ig superfamily. eur. j. biochem. 250: 630-637, 1997. 235 http://www.ncbi.nlm.nih.gov/pubmed/10358769 http://www.ncbi.nlm.nih.gov/pubmed/10358769 http://www.ncbi.nlm.nih.gov/pubmed/12967563 http://www.ncbi.nlm.nih.gov/pubmed/12967563 http://www.ncbi.nlm.nih.gov/pubmed/12967563 http://www.ncbi.nlm.nih.gov/pubmed/15211580 http://www.ncbi.nlm.nih.gov/pubmed/15211580 http://www.ncbi.nlm.nih.gov/pubmed?term=%22bettencourt%20r%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22lanz-mendoza%20h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22lindquist%20kr%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22faye%20i%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'eur%20j%20biochem.'); bogdan c, rollinghoff m, diefenbach a. reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. curr. opin. immunol. 12: 64-76, 2000. brennecke j, aravin aa, stark a, dus m, kellis m, sachidanandam r, et al. discrete small rnagenerating loci as master regulators of transposon activity in drosophila. cell 128: 1089-1103, 2007. bulet p, hetru c, diamrco jl, hoffmann d. antimicrobial peptides in insect; structure and function. dev. comp. immunol. 23: 329-344, 1999. bulet, p, stocklin, r, menin, l. anti-microbial peptides: from invertebrates to vertebrates. immunol. rev. 198: 169-184. 2004. cerenius l, söderhall k. the prophenoloxidaseactivating system in invertebrates. immunol. rev. 198: 116-126, 2004. charalambidis nd, zervas cg, lambropoulou m, katsoris pg, marmaras vj. lipopolysaccharide-stimulated exocytosis of nonself recognition protein from insect hemocytes depend on protein tyrosine phosphorylation. eur. j. cell. biol. 67: 32-41, 1995. daffre s, faye i. lipopolysaccharide interaction with hemolin, an insect member of the igsuperfamily. febs lett. 408: 127-130, 1997. ding sw, voinnet o. antiviral immunity directed by small rnas. cell 130: 413-426, 2007. eleftherianos i, gökçen f, felföldi g, millichap pj, trenczek te, ffrench-constant rh, et al. the immunoglobulin family protein hemolin mediates cellular immune responses to bacteria in the insect manduca sexta. cell. microbiol. 9: 1137-1147, 2007. engström, yi. insect immune gene regulation. in: brey p, hultmark d (ed), molecular mechanisms of immune responses in insects, chapman & hall, london, uk, pp 211-244, 1998. fearon dt. seeking wisdom in innate immunity. nature 388: 323-324, 1997. feldhaar h, gross r. immune reactions of insects on bacterial pathogens and mutualists. microbes infect. 10: 1082-1088, 2008. garcia-lara j, needham aj, foster sj. invertebrates as animal models for staphylococcus aureus pathogenesis: a window into host-pathogen interaction. fems immunol. med. microbiol. 43: 311-23, 2005. gillespie jp, kanost mr, trenczek t. biological mediators of insect immunity. annu. rev. entomol. 42:611-643, 1997. hernandez-martinez s, lanz h, rodriguez mh, gonzalez-ceron l, tsutsumi v. cellularmediated reactions to foreign organisms inoculated into the hemocoel of anopheles albimanus (diptera: culicidae). j. med. entomol. 39: 61-69, 2002. hillyer jf, schmidt sl, christensen bm. hemocyte-mediated phagocytosis and melanization in the mosquito armigeres subalbatus following immune challenge by bacteria. cell tissue res. 313: 117-127, 2003a. hillyer jf, schmidt sl, christensen bm. rapid phagocytosis and melanization of bacteria and plasmodium sporozoites by hemocytes of the mosquito aedes aegypti. j. parasitol. 89: 6269, 2003b. hoffmann ja. the immune response of drosophila. nature 426: 33-38, 2003. hoffmann, ja. innate immunity of insects. curr. opin. immunol. 7: 4-10, 1995. hughes al. evolution of the integrin alpha and beta protein families. j. mol. evol. 52: 63-72, 2001. hultmark d, steiner h, rasmuson t, boman hg. insect immunity. purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of hyalophora cecropia. eur. j. biochem. 106: 7-16, 1980. humphries mj, travis ma, clark k, mould ap. mechanisms of integration of cells and extracellular matrices by integrins. biochem. soc. trans. 32: 822-825, 2004. hynes ro. integrins: bidirectional, allosteric signaling machines. cell 110: 673-687, 2002. johansson mw, söderhall k. the prophenoloxidase activating system and associated proteins in invertebrates. prog. mol. subcell. biol. 15: 4666, 1996. kaneko t, silverman n. bacterial recognition and signalling by the drosophila imd pathway. cell. microbiol. 7: 461-469, 2005. keeley ll. physiology and biochemistry of the fat body. in: kerkut ga, gilbert li (eds), comprehensive insect physiology, biochemistry and pharmacology. pergamon press, oxford, uk, pp 211-248, 1985. kemp c, imler j-l. antiviral immunity in drosophila. curr. opin. immunol. 21: 3-9, 2009. kim ys, ryu jh, han sj, choi kh, nam kb, jang ih, et al. gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in drosophila melanogaster cells. j. biol. chem. 275: 3272132727, 2000. lamprou i, mamali i, dallas k, fertakis v, lampropoulou m, marmaras vj. distinct signalling pathways promote phagocytosis of bacteria, latex beads and lipopolysaccharide in medfly haemocytes. immunology 121: 314-327, 2007. lamprou i, tsakas s, theodorou gl, karakantza m, lampropoulou m, marmaras vj. uptake of lps/e. coli/latex beads via distinct signalling pathways in medfly hemocytes: the role of map kinases activation and protein secretion. biochim. biophys. acta 1744: 1-10, 2005. lavine md, strand mr. insect hemocytes and their role in immunity. insect biochem. mol. biol. 32: 1295-1309, 2002. lavine md, strand mr. surface characteristics of foreign targets that elicit an encapsulation response by the moth pseudoplusia includens. j. insect physiol. 2001 47: 965-974, 2001. lavine md, strand mr. haemocytes from pseudoplusia includens express multiple alpha and beta integrin subunits. insect mol. biol. 12: 441-452, 2003. 236 http://www.ncbi.nlm.nih.gov/pubmed/10679404 http://www.ncbi.nlm.nih.gov/pubmed/10679404 http://www.ncbi.nlm.nih.gov/pubmed/10679404 http://www.ncbi.nlm.nih.gov/pubmed/15199959 http://www.ncbi.nlm.nih.gov/pubmed/15199959 http://www.ncbi.nlm.nih.gov/pubmed/7641728 http://www.ncbi.nlm.nih.gov/pubmed/7641728 http://www.ncbi.nlm.nih.gov/pubmed/7641728 http://www.ncbi.nlm.nih.gov/pubmed/7641728 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-4899v7y-3&_user=83471&_coverdate=05%2f31%2f2003&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=c000059670&_version=1&_urlversion=0&_userid=83471&md5=78b2191eae38a0987be919748d9327b8#bbib22#bbib22 http://www.ncbi.nlm.nih.gov/pubmed/9237746 http://www.ncbi.nlm.nih.gov/pubmed/9017902 http://www.ncbi.nlm.nih.gov/pubmed/9017902 http://www.ncbi.nlm.nih.gov/pubmed/11931273 http://www.ncbi.nlm.nih.gov/pubmed/11931273 http://www.ncbi.nlm.nih.gov/pubmed/11931273 http://www.ncbi.nlm.nih.gov/pubmed/11931273 http://www.ncbi.nlm.nih.gov/pubmed/12838409 http://www.ncbi.nlm.nih.gov/pubmed/12838409 http://www.ncbi.nlm.nih.gov/pubmed/12838409 http://www.ncbi.nlm.nih.gov/pubmed/12838409 http://www.ncbi.nlm.nih.gov/pubmed/12659304 http://www.ncbi.nlm.nih.gov/pubmed/12659304 http://www.ncbi.nlm.nih.gov/pubmed/12659304 http://www.ncbi.nlm.nih.gov/pubmed/12659304 http://www.ncbi.nlm.nih.gov/pubmed?term=%22hultmark%20d%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22steiner%20h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22rasmuson%20t%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22boman%20hg%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'prog%20mol%20subcell%20biol.'); http://www.ncbi.nlm.nih.gov/pubmed/17376199 http://www.ncbi.nlm.nih.gov/pubmed/17376199 http://www.ncbi.nlm.nih.gov/pubmed/17376199 http://www.ncbi.nlm.nih.gov/pubmed/17376199 http://www.ncbi.nlm.nih.gov/pubmed/15878392 http://www.ncbi.nlm.nih.gov/pubmed/15878392 http://www.ncbi.nlm.nih.gov/pubmed/15878392 http://www.ncbi.nlm.nih.gov/pubmed/15878392 http://www.ncbi.nlm.nih.gov/pubmed?term=%22lavine%20md%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22strand%20mr%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'j%20insect%20physiol.'); http://www.ncbi.nlm.nih.gov/pubmed?term=%22lavine%20md%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22strand%20mr%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'insect%20mol%20biol.'); lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. levashina ea, moita lf, blandin s, vriend g, lagueux m, kafatos fc. conserved role of a complement-like protein in phagocytosis revealed by dsrna knockout in cultured cells of the mosquito, anopheles gambiae. cell 104: 709-718, 2001. ling e, yu xq. prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in manduca sexta. insect biochem. mol. biol. 35: 1356-1366, 2005. little tj, cobbe n the evolution of immune-related genes from disease carrying mosquitoes: diversity in a peptidoglycanand a thioesterrecognizing protein. insect mol. biol. 14: 599605, 2005. mamali i, lamprou i, karagiannis f, karakantza m, lampropoulou m, marmaras vj. a beta integrin subunit regulates bacterial phagocytosis in medfly haemocytes. dev. comp. immunol. 33: 858-866, 2009. marmaras vj, lampropoulou m. regulators and signalling in insect haemocyte immunity. cell signal. 21: 186-95, 2009. mavrouli md, tsakas s, theodorou gl, lampropoulou m, marmaras vj. map kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes. biochim. biophys. acta 1744: 145156, 2005. medzhitov r, janeway ca jr. innate immunity: impact on the adaptive immune response. curr. opin. immunol. 9: 4-9, 1997. meister m, lagueux m. drosophila blood cells. cell. microbiol. 5: 573-580, 2003. mellroth p, karlsson j, steiner h. a scavenger function for a drosophila peptidoglycan recognition protein. j. biol. chem. 278: 70597064, 2003. michel t, reichhart jm, hoffmann ja, royet j. drosophila toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. nature 414: 756-759, 2001. miller js, howard rw, rana rl, tunaz h, stanley dw. eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, gryllus assimilis. j. insect physiol. 45: 75-83, 1999. moita lf, vriend g, mahairaki v, louis c, kafatos fc. integrins of anopheles gambiae and a putative role of a new beta integrin, bint2, in phagocytosis of e. coli. insect biochem. mol. biol. 36: 282-290, 2006. nakatogawa s, oda y, kamiya m, kamijima t, aizawa t, clark kd, et al. a novel peptide mediates aggregation and migration of hemocytes from an insect. curr. biol. 19: 779785, 2009. nappi aj, kohler l, mastore m. signaling pathways implicated in the cellular innate immune responses of drosophila. inv. surv. j. 1: 5-33, 2004. nappi aj, ottaviani e. cytotoxicity and cytotoxic molecules in invertebrates. bioessays 22: 469480, 2000. nappi aj, vass e. cytotoxic reactions associated with insect immunity. adv. exp. med. biol. 484: 329-348, 2001. nappi aj, vass e, frey f, carton y. nitric oxide involvement in drosophila immunity. nitric oxide 4: 423-430, 2000. nappi aj, vass e, frey f, carton y. superoxide anion generation in drosophila during melanotic encapsulation of parasites. eur. j. cell biol. 68: 450-456, 1995. ribeiro c, brehelin m. insect haemocytes: what type of cell is that? j. insect physiol. 52: 417-429, 2006. roma gc, bueno oc, camargo-mathias mi. morpho-physiological analysis of the insect fat body: a review. micron 41: 395-401, 2010. sabin lr, hanna sl, cherry s. innate antiviral immunity in drosophila. curr. opin. immunol. 22: 4–9, 2010. schmidt o, theopold, u, strand m. innate immunity and its evasion and suppression by hymenopteran endoparasitoids. bioessays 23: 344-351, 2001. shia ak, glittenberg m, thompson g, weber an, reichhart jm, ligoxygakis p. toll-dependent antimicrobial responses in drosophila larval fat body require spätzle secreted by haemocytes. j. cell sci. 122: 4505-4515, 2009. sideri m, tsakas s, markoutsa e, lampropoulou m, marmaras vj. innate immunity in insects: surface-associated dopa decarboxylasedependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes. immunology 123: 528-537, 2008. silverman n, maniatis t. nf-kappab signaling pathways in mammalian and insect innate immunity. genes dev. 15: 2321-2342, 2001. silverman n, zhou r, erlich rl, hunter m, bernstein e, schneider d, et al. immune activation of nf-kappab and jnk requires drosophila tak1. j. biol. chem. 278: 4892848934, 2003. steiner h, hultmark d, engström a, bennich h, boman hg. sequence and specificity of two antibacterial proteins involved in insect immunity. nature 292: 246-248, 1981. theopold u, rissler m, fabbri m, schmidt o, natori s. insect glycobiology: a lectin multigene family in drosophila melanogaster. biochem. biophys. res. commun. 261: 923-927, 1999. vilmos p, kurucz e. insect immunity: evolutionary roots of the mammalian innate immune system. immunol. lett. 62: 59-66,1998. wang y, jiang h. binding properties of the regulatory domains in manduca sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase activation system. dev. comp. immunol. 34: 316-322, 2010. yount ny, yeaman mr. multidimensional signatures in antimicrobial peptides. proc. natl. acad. sci. usa 101: 7363-7368, 2004. yu xq, kanost mr. manduca sexta lipopolysaccharide-specific immulectin-2 237 http://www.ncbi.nlm.nih.gov/pubmed/16291091 http://www.ncbi.nlm.nih.gov/pubmed/16291091 http://www.ncbi.nlm.nih.gov/pubmed/16291091 http://www.ncbi.nlm.nih.gov/pubmed/18790716 http://www.ncbi.nlm.nih.gov/pubmed/18790716 http://www.ncbi.nlm.nih.gov/pubmed/12925127 http://www.ncbi.nlm.nih.gov/pubmed/12770398 http://www.ncbi.nlm.nih.gov/pubmed/12770398 http://www.ncbi.nlm.nih.gov/pubmed/12770398 http://www.ncbi.nlm.nih.gov/pubmed/19375321 http://www.ncbi.nlm.nih.gov/pubmed/19375321 http://www.ncbi.nlm.nih.gov/pubmed/19375321 http://www.isj.unimo.it/articoli/isj003.pdf http://www.isj.unimo.it/articoli/isj003.pdf http://www.isj.unimo.it/articoli/isj003.pdf http://www.ncbi.nlm.nih.gov/pubmed?term=%22nappi%20aj%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ottaviani%20e%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'bioessays.'); http://www.ncbi.nlm.nih.gov/pubmed/11419001 http://www.ncbi.nlm.nih.gov/pubmed/11419001 http://www.ncbi.nlm.nih.gov/pubmed/8690025 http://www.ncbi.nlm.nih.gov/pubmed/8690025 http://www.ncbi.nlm.nih.gov/pubmed/8690025 http://www.ncbi.nlm.nih.gov/pubmed/16527302 http://www.ncbi.nlm.nih.gov/pubmed/16527302 http://www.ncbi.nlm.nih.gov/pubmed?term=%22roma%20gc%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22bueno%20oc%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22camargo-mathias%20mi%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'micron.'); http://www.ncbi.nlm.nih.gov/pubmed/11268040 http://www.ncbi.nlm.nih.gov/pubmed/11268040 http://www.ncbi.nlm.nih.gov/pubmed/11268040 http://www.ncbi.nlm.nih.gov/pubmed?term=%22shia%20ak%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22glittenberg%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22thompson%20g%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22weber%20an%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22reichhart%20jm%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ligoxygakis%20p%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'j%20cell%20sci.'); http://www.ncbi.nlm.nih.gov/pubmed/17983437 http://www.ncbi.nlm.nih.gov/pubmed/17983437 http://www.ncbi.nlm.nih.gov/pubmed/17983437 http://www.ncbi.nlm.nih.gov/pubmed/17983437 http://www.ncbi.nlm.nih.gov/pubmed/17983437 http://www.ncbi.nlm.nih.gov/pubmed/19900472 http://www.ncbi.nlm.nih.gov/pubmed/19900472 http://www.ncbi.nlm.nih.gov/pubmed/19900472 http://www.ncbi.nlm.nih.gov/pubmed/19900472 http://www.ncbi.nlm.nih.gov/pubmed/12590970 http://www.ncbi.nlm.nih.gov/pubmed/12590970 protects larvae from bacterial infection. dev. comp. immunol. 27: 189-196, 2003. zelensky an, gready je. the c-type lectin-like domain superfamily. febs j. 272: 6179-6217, 2005. yu xq, kanost mr. immulectin-2, a pattern recognition receptor that stimulates hemocyte encapsulation and melanization in the tobacco hornworm, manduca sexta. dev. comp. immunol. 28: 891-900, 2004. zhao p, li j, wang y, jiang h. broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by manduca sexta phenoloxidase. insect biochem. mol. biol. 37: 952-959, 2007.yu xq, kanost mr. binding of hemolin to bacterial lipopolysaccharide and lipoteichoic acid. an immunoglobulin superfamily member from insects as a pattern-recognition receptor. eur. j. biochem. 269: 1827-1834, 2002. zhu y, johnson tj, myers aa, kanost mr. identification by subtractive suppression hybridization of bacteria-induced genes expressed in manduca sexta fat body. insect biochem. mol. biol. 33: 541-559, 2003. yu xq, zhu yf, ma c, fabrick ja, kanost mr. pattern recognition proteins in manduca sexta plasma. insect biochem. mol. biol. 32: 12871293, 2002. zhuang s, kelo l, nardi jb, kanost mr. multiple alpha subunits of integrin are involved in cellmediated responses of the manduca immune system. dev. comp. immunol. 32: 365-379, 2008. zasloff m. antimicrobial peptides of multicellular organisms. nature 415: 389-395, 2002. 238 http://www.ncbi.nlm.nih.gov/pubmed/12590970 http://www.ncbi.nlm.nih.gov/pubmed?term=%22zelensky%20an%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22gready%20je%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'febs%20j.'); http://www.sciencedirect.com/science/journal/09651748 http://www.sciencedirect.com/science/journal/09651748 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235053%232003%23999669994%23418957%23fla%23&_cdi=5053&_pubtype=j&view=c&_auth=y&_acct=c000059670&_version=1&_urlversion=0&_userid=83471&md5=65ed6b099f154363a090bfeb4ca63bde http://www.ncbi.nlm.nih.gov/pubmed?term=%22zhuang%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22kelo%20l%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22nardi%20jb%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22kanost%20mr%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); humoral responses the recognition of invading pathogen either as bacteria or fungi or even viruses is followed by the immediate de novo synthesis of antimicrobial peptides (amps) and their secretion into the hemolymph (zasloff, 2002; bulet et al., 2004). these peptides are mainly synthesized by the fat body and in a lesser degree by the hemocytes, integument, gut, salivary glands and reproductive structures (nappi and ottaviani, 2000). johansson mw, söderhall k. the prophenoloxidase activating system and associated proteins in invertebrates. prog. mol. subcell. biol. 15: 46-66, 1996. shia ak, glittenberg m, thompson g, weber an, reichhart jm, ligoxygakis p. toll-dependent antimicrobial responses in drosophila larval fat body require spätzle secreted by haemocytes. j. cell sci. 122: 4505-4515, 2009. zelensky an, gready je. the c-type lectin-like domain superfamily. febs j. 272: 6179-6217, 2005. 52 isj 15: 52-60, 2018 issn 1824-307x research report molecular cloning and characterization of rheb from white shrimp (litopenaeus vannamei) x liu1,2,3, m wang1,2, j shao1,2,3, b wang1,2, k jiang1,2, m liu1,2*, l wang1,2* 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory of marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237 3university of chinese academy of sciences, beijing 100049, china accepted february 11, 2018 abstract the ras family gtpase rheb, a gtp-binding protein, binds specifically to the mtor catalytic domain and induces activation of the mtor catalytic function. furthermore, rheb is related to a stable modification of the configuration of mtorc1 that increases access of substrates to their binding site on the raptor polypeptide. in the present research, a cdna of 898 bp for the litopenaeus vannamei rheb was cloned via rapid amplification of cdna ends (race) technique. the complete cdna sequence of rheb contained an open reading frame (orf) of 549 bp, which encoded a protein of 182 amino acids. the amino acid sequence of rheb shared more than 60% similarity with other identified rheb proteins. a ras domain (from p3 to a169) was found in the amino acid sequence of rheb that can react selectively and non-covalently with gtp. the mrna transcripts of rheb were consistently expressed in all the tested tissues, including muscle, gill, hepatopancreas, eyestalk, intestine and stomach. the mrna expression profiles of rheb in muscle after the stimulation with rapamycin were promoted, which further proved that rheb protein could be a feedback regulator to mtor signaling pathway. furthermore, the results of the present study indicated that rheb played an important role in the regulation of mtor signaling pathway during the stimulation of dietary restriction, amino acid supplementation and rapamycin stimulation in shrimp. key words: litopenaeus vannamei; molecular cloning; mtor; rheb introduction mechanistic target of rapamycin (mtor) is a fundamental regulator of cell growth and proliferation in all eukaryotes (wullschleger et al., 2006). mtor can sense stress, oxygen, amino acids, energy levels and growth factors to perform cell function (laplante et al., 2012). the functions of mtor were performed by two independent complexes. the mtor complex 1 (mtorc1) is comprised of mtor ___________________________________________________________________________ corresponding authors: mei liu key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: liumei@qdio.ac.cn lei wang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn in association with raptor, lst8, deptor, ttl1/tel2 and ppras40, which could be inhibited by rapamycin (rapa) (kunz et al., 1993; hara et al., 2002; kim et al., 2002; loewith et al., 2002). the mtor complex 2 (mtorc2) shares include mtor, mlst8, deptor, and tti1/tel2 with mtorc1. in addition, rictor, msin1, and ppr5/protor are specific for mtorc2 (loewith et al., 2002; jacinto et al., 2004; sarbassov et al., 2004). its output is insensitive to rapa. in mammalian cells, mtorc1 output is sensitive to amino acid (especially leucine and arginine) sufficiency (long et al., 2005a). amino acid withdrawal can lead to the inhibition of mtorc1 signaling (demetriades et al., 2014). the overexpression of rheb can rescue mtor from inactivation in vivo caused by amino-acid withdrawal (long et al., 2005a). what’s more, genetic evidence from drosophila indicated that the rheb is an indispensable activator of mtorc1 in the insulin/igf-i receptor pathway. the studies indicate that rheb is a key regulator of the output of mtorc1. mailto:liumei@qdio.ac.cn mailto:wanglei@qdio.ac.cn 53 rapa is a specific inhibitor of mtorc1 and the effects of rapa on mtor signaling are complicated. many mtorc1 functions are highly sensitive to rapa, for example, mtor-mediated protein synthesis, lipogenesis, energy metabolism, and lysosome biogenesis (thomas et al., 1997; schmelzle et al., 2000; brugarolas et al., 2003; laplante et al., 2009; settembre et al., 2012). rapa forms a complex with the intracellular 12-kda fk506-binding protein (fkbp12) (brown et al., 1994; sabatini et al., 1994). this complex directly combines with and inhibits mtorc1. rheb can relieve the inhibition of the mtorc1 (ma et al., 2008; sun et al., 2008). the influence and mechanism of rheb to control the mtorc1 are as yet unknown. rheb can bind specifically to the mtor catalytic domain in vivo and in vitro (avruch et al., 2014). the mtor–rheb interaction can promote the activation of mtor kinase (long et al., 2005a). amino acids appear to control the efficacy of rheb-gtp towards mtorc1 (avruch et al., 2009). besides, rheb promotes a reconfiguration of the mtorc1 complex that enhances the accession and binding of substrates to raptor. a previous research showed that the mtorc1 substrates s6k1 and 4e-bp bound directly to raptor (hara et al., 2002). the process can promote the phosphorylation of translational regulators eukaryotic translation initiation factor 4e (eif4e)-binding protein 1 (4e-bp1) and s6 kinase 1 (s6k1), which, in turn, promote protein synthesis (ma et al., 2009). however, further work is needed to elucidate the mechanisms of rheb–mtor interaction that promotes mtor kinase activity. the researches about rheb functions concentrates upon mammals and some insects. su et al. found that rheb mrna expression was increased in wssv-infected shrimp (su et al., 2014). beyond that, very few studies have been done on its functions in crustaceans. what’s more, a relatively small number of studies explore the interaction between rheb and mtorc1. so, the mechanism of activation is not fully understood. therefore, studies to elucidate the rheb-mtor signaling pathway are of great importance. litopenaeus vannamei has proven to be a useful decapod crustacean model system for the study of evolution, in addition to its importance as a food source (sakthivel, 2014). the research about mtor signaling pathway in l. vannamei can deepen our understanding of the important signaling pathway that regulate growth and metabolism in eukaryotes and reveal the evolutionary traces of mtor signaling pathway. meanwhile, the studies about growth mechanism in shrimp can lead to more reliable way to the selection of the new breed and optimizing the feed formula. materials and methods experimental animals litopenaeus vannamei (5 ± 0.5 g) from the qingdao ruizi aquaculture base (shandong, china) were used in this study and were stocked in two tanks at a density of 100 shrimps per tank (1000 l) at 30‰ salinity. shrimp were maintained for 1 week and fed commercial pellets (42.3% protein, 7.2% fat, 11.6% water, and 15.5% ash, supplied by da le co., ltd, yantai, china). the water quality parameters were evaluated 2−3 times per week and maintained at ph 7.5−8.2, temperature 25−29 °c, dissolved oxygen 5.0−6.5 mgl-1 during the trial. rapamycin injection in the first tank, we injected shrimps on the sixth uromere with 100 μl rapa (500 mm), and shrimps in the other tank were injected with 100 μl dmso diluted by phosphate-buffered saline (pbs) (0.01 m, ph 7.4) on the ratio of 1:1000 (the solvent of rapa). shrimps were dissected at different time-points (0, 0.5, 1, 2, 4 and 6 h) after injection with rapa (experimental group) or dmso diluent (control group) to obtain muscle tissue. the control and experimental groups (n = 9 shrimps/group, conducted in triplicate) obtained at each time-point were used to obtain muscular tissue for real-time (rt)-pcr analysis. the muscle tissue was preserved in rna store solution (beijing comwin biotech co., ltd., beijing, china). amino acid injection the shrimps were divided into six groups. the shrimps in the first group injected with 100 μl pbs (0.01 m, ph 7.4) were the control group. the last group srhimps were injected with 100 µl rapa (500 mm). the remaining 4 groups were starved for 3 days. after the dietary restriction (dr), shrimps in the second group were injected with 100 μl pbs (0.01 m, ph 7.4). the shrimps in the third and fourth groups were injected with 100 μl 0.1 m leucine or 100 μl 0.1 m arginine, respectively. the shrimps in the fifth group were injected with 100 μl leucine (0.1 m) and 100 μl rapa (500 mm). the control and experimental groups (n = 9 shrimps/group, conducted in triplicate) were used to obtain muscular tissue for rt-pcr analysis 30 min after injection. the muscle tissue was preserved in rna store solution (beijing comwin biotech co., ltd., beijing, china). rna preparation and cdna synthesis total rna was extracted using a minibest universal rna extraction kit (takara, dalian, china) according to the manufacturer’s instructions. rna degradation and contamination was monitored on 1% agarose gels. the cdna synthesis was carried out by transscript ii one-step gdna removal and cdna synthesis supermix (ah311-02, transgen biotech, china). the reactions were performed at 42 °c for 30 min, terminated by heating at 85 °c for 5 min and then stored at -80 °c. cloning the cdna of rheb the partial sequence of rheb cdna was obtained from the transcriptome database of l. vannamei. standard procedures were used for cdna cloning. two pairs of gene-specific primers, 3r1/2 and 5r1/2, were designed based on this partial sequence to clone the 3’ end and 5’ end of rheb cdna by rapid-amplification of cdna ends (race) technique. the full-length cdna of the rheb from l. vannamei was amplified by pcr using the primers rheb3'-f and rheb5'-r. the primers were designed by ncbi primer blast and were given in table 1. all pcr amplification was performed in a mj 54 table 1 oligonucleotide primers used in the current experiments. name sequence (5’-3’) brief information 3r1 agcgtgggcaaatcctcctt gene specific primer for race 3r2 atgccccgaccatcgagaac gene specific primer for race 5r1 ccctcgggactgatgttgcc gene specific primer for race 5r2 gttgcccaccaagacaatggg gene specific primer for race qrhe-f aggaaagtggccgttatggg gene specific primer for real-time pcr qrheb-r taccagctccaggccatact gene specific primer for real-time pcr qβ-actin-f gcccatctacgagggata internal control for real-time pcr qβ-actin-r ggtggtcgtgaaggtgtaa internal control for real-time pcr rheb3'-f tgtctctcccttcccttcgg gene specific primers used to amplify full-length rheb rheb5'-r aaggtccatcctataacccagg gene specific primers used to amplify full-length rheb nup aagcagtggtatcaacgcagagt universal primers for race upml ctaatacgactcactatagggcaagcagtggtatcaacgcagagt universal primers for race upms ctaatacgactcactatagggc universal primers for race 3’cds aagcagtggtatcaacgcagagtac(t)30v n oligo (dt) for cdna synthesizing 5’cds aagcagtggtatcaacgcagagtgggggggggghn anchor primer for 5’ race m13f tgtaaaacgacggccagt vector primer for sequencing m13r caggaaacagctatgacc vector primer for sequencing mini personal thermal cycler (bio-rad, usa), and the pcr products were purified using dna gel extraction kit (dp210, tiangen, china) and cloned into the peasy-t1 cloning vector (ct101, transgen biotech, china). after being transformed into the trans5α chemically competent cell (cd201, transgen biotech, china), the positive recombinants were identified via anti-ampicillin selection. sequence characterization and multiple sequence alignment the protein sequence similarities were discovered by protein blast at the national center for biotechnology information (ncbi). the physicochemical property of protein rheb were analyzed by protparam tool (https://www.expasy.ch/tools/protparam.html). signalp 4.1 program was utilized to predict the presence and location of signal peptide (http://www.cbs.dtu.dk/services/signalp/). the protein domain features of rheb were predicted by simple modular architecture research tool (smart) 7.0 (http://smart.emblheidelberg.de/). multiple sequence alignment of rheb and other rhebs was performed with clustalw multiple alignment program 2.1 (http://www.ch.embnet.org/software/clustalw.html) and multiple alignment show program 2.0 (http://www.bioinformatics.org/sms2/color_align_con s.html). a neighbor-joining (nj) phylogenic tree of rheb was constructed with mega 6.0 software package. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. real-time pcr analysis of rheb mrna expression the mrna transcripts of rheb in muscle, gill, hepatopancreas, eyestalk, intestine and stomach were quantified by rt-pcr, and its temporal expression profiles in muscle of l. vannamei stimulated with rapa were determined by rt-pcr. pcr amplification was performed using the following cycling conditions: denaturation for 30 s at 94 °c, followed by 40 cycles of 5 s at 94 °c, and 30 s at 60 °c. to confirm that only one pcr product was amplified and measured, dissociation curve analysis was performed at the end of each pcr. all rt-pcr was performed with the sybr premix ex taq kit (takara biotechnology co., dalian, china). the information of all primers used in this assay was shown in table 1. the primers were designed by ncbi primer blast. the expression of rheb was normalized to the expression of β-actin gene for each sample. the comparative ct method (2-δδct) was used to analyse the expression level of rheb (schmittgen et al., 2008). statistical analysis results are expressed as means ± sd. the statistical analysis were performed by one-way analysis of variance (one-way anova) using spss software to detect significant intergroup differences. the p values less than 0.05 were considered statistically significant. results the molecular features, sequence alignment and phylogeny relationship of rheb a nucleotide sequence from the l. vannamei transcriptome is homologous to rheb identified previously confirmed by sequencing and blast analysis. based on this fragment, a fragment was amplified with nested primers upm/3r1 and nup/3r2 to obtain the 3’ end of the sequence. a fragment was amplified with nested primers upm/5r1 and nup/5r2 to obtain the 5’ end of the sequence. a 898 bp nucleotide sequence representing the complete cdna sequence of rheb 55 fig. 1 nucleotide and deduced amino acid sequences of rheb. the nucleotides and deduced amino acids are numbered along the left margin. the ras domain was in shade. conserved amino acids involved in mtor binding activity are boxed. conserved amino acids involved in guanyl nucleotides binding activity are circled. the asterisk indicated the stop codon. the amino acid sequences of rheb has been submitted to genbank and the accession number is mg696863. of l. vannamei was assembled. the complete cdna sequence of rheb contains a 208 bp 5’ untranslated region, a 141 bp 3’ untranslated region with a poly (a) tail and the complete sequence of an open reading frame (orf) of 549 bp (fig. 1). the orf encoded a polypeptide of 182 amino acid residues with a calculated molecular mass of approximately 20.55 kda. the theoretical isoelectric point is 5.67. no signal peptide was predicted in the deduced amino acid sequence of rheb by signalp program. a ras domain (from p3 to a169) was found in the amino acid sequence of rheb. the deduced amino acid sequences of the six crustacean rheb proteins were highly conserved, showing high identity and similarity to each other (fig. 2). sequence identity was particularly high within the ‘g box’ motifs (g1–g5) in all the rheb proteins, particularly in the g2 motifs (fig. 2). the effect domain (switch i) is highly conservative region too. the deduced amino acid sequence of rheb exhibited high similarity with other reported rhebs, such as 91% with that from homarus americanus (adv76255), 86% with that from carcinus maenas (adv76253) and 66% with that from homo sapiens (np_005605) (fig. 3). the nucleotide sequence of rheb has been submitted to genbank and the accession number is mg696863. the tissue distribution of rheb mrna the rt-pcr analysis was employed to detect the tissue distribution of the rheb mrna in different tissues and the β-actin gene as internal control. the lowest expression level of rheb transcripts was present in intestine. in other detected tissues, the rheb mrna transcripts were significantly higher 56 fig. 2 multiple alignments of deduced amino acid sequences for rheb proteins. the black shadow region indicated positions where all sequences share the same amino acid residue. similar amino acids are shaded in grey. g boxes and effector domain (switch i region) has been marked in the figure. the effector switch i region is responsible for interactions with the mtor protein, fkbp38 and other proteins (aspuria and tamanoi, 2004; ma et al., 2008). species and gene accession numbers are as follows: litopenaeus vannamei (mg696863), homarus americanus (adv76255), carcinus maenas (adv76253), zootermopsis nevadensis (xp_021934312), danio rerio (np_957023) and homo sapiens (np_005605). fig. 3 neighbor-joining (nj) phylogenic tree of rheb constructed using mega 6.0 software package based on the amino acid sequences of rhebs from different organisms. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. species and protein sequences id are as follows: litopenaeus vannamei (mg696863), drosophila melanogaster (np_730950), danio rerio (np_957023), homo sapiens (np_005605), mus musculus (np_444305), homarus americanus (adv76255), carcinus maenas (adv76253), cimex lectularius (xp_014260514), zootermopsis nevadensis (xp_021934312), xenopus laevis (np_001080494), taeniopygia guttata (np_001232539), the rheb protein of l. vannamei is indicated with a black triangle. the numbers at the forks indicated the bootstrap value. the scale bar represents the proportion of amino acid differences between sequences based on nucleotide substitutions per site. than those in intestine. what’s more, the expression level of rheb among muscle, gill, hepatopancreas, eyestalk, intestine and stomach showed no significant difference (fig. 4). the temporal expression profile of rheb mrna post rapa stimulation the temporal mrna expression profile of rheb in muscles after rapa stimulation was examined via rt-pcr. the mrna expression of rheb in muscles increased significantly during 0.5 h-6 h (p < 0.05) after the stimulation of rapa. the mrna transcripts increased to the peak level at 1 h post stimulation (19.53-fold, p < 0.05). in the control group, no significant change of rheb mrna expression was observed after dmso injection during the whole experiment (fig. 5). the temporal expression profile of rheb mrna post dietary restriction, amino acid and rapa stimulation rt-pcr analysis was employed to examine the mrna expression profile of rheb in muscles after dietary restriction and at 30 min after injection of amino acid and rapa. as shown in fig. 6, the mrna 57 fig. 4 tissue distribution of rheb mrna transcripts detected by rt-pcr technology. β-actin gene was used as an internal control. rheb mrna transcripts come from stomach, eyestalk, hepatopancreas, muscle, intestine and gill of five adult shrimp. vertical bars represented mean ± s.d. (n = 5), and bars with different characters indicated significantly different (p < 0.05). transcripts of rheb in muscles increased after the shrimps deprived of feed for 3 days (3.2-fold compared with the origin level, p < 0.05). the leucine and arginine injections can relieve the influence caused by hunger and make the expression of rheb decrease to the original expression level. the injection of rapa can hinder the ability of leucine injection to relieve the increase expression level of rheb due to dietary restriction. there was still a significant 3.0-fold increase in rheb mrna in the group injected with leucine and rapa compared to control group (p < 0.05) (fig. 6). discussion the small gtpase rheb protein that can bind directly to mtorc1 and play a positive role in the regulation of mtor signaling pathway is involved in the activation of protein synthesis and growth (sato et al., 2008). in the present study, the full-length cdna of rheb was cloned from l. vannamei. the deduced polypeptide of rheb consisted of 182 amino acids, and its calculated molecular weight was 20.55 kda, which was close to those from vertebrate and invertebrate (fig. 1). the amino acid sequence of rheb shared over 60% similarities with other identified rhebs (fig. 2). moreover, a typical ras domain (fig. 1) was found which can react selectively and non-covalently with gtp (akashi et al., 2007). the switch i domain (switch i) is a highly conservative region that is crucial for the direct bond between mtor and rheb (long et al., 2007). our work also verified the viewpoint that the decapod crustacean rheb contained the five highly conserved g boxes which are related to gtp binding and gtpase activity (maclea et al., 2012). as shown in fig. 3, the rheb is a very conservative protein. the amino acid sequence of rheb in l. vannamei shared more than 60% similarity with other identified rheb proteins. to investigate the function of rheb in controlling cell function of shrimp, the distribution of its mrna in different tissues was detected by rt-pcr technique. the rheb mrna transcripts were observed consistently expressed in all the detected tissues. the analogous expression of rheb in the tissues indicated that it played vital role in the regulation of cell function. the reason for lower expression of rheb in intestine maybe was that intestine is the main tissue absorbing amino acids that makes the concentration of amino acids in intestine is higher than other tissues. in order to prevent the irrational regulation of mtor signaling pathway caused by amino acids, the low expression of rheb could be essential for the cell function regulation in intestine. to further understand the regulative roles of rheb in mtor signaling pathway, we depressed the mtor signaling pathway by rapa. the temporal expression profile in muscle post rapa stimulation was detected by rt-pcr technique. we found the expression of rheb increased when the mtor signaling pathway was depressed by rapa. the highest expression was found when we injected rapa into shrimps for 1 h. which was 19.53-fold (p < 0.05) of that in control group. previous studies found that rheb overexpression activates torc1 signaling (sun et al., 2008). ma et al. (2008) identified that rheb binds specifically to fkbp38 58 fig. 5 temporal mrna expression profiles of rheb detected by rt-pcr in shrimp muscle at 0, 0.5, 1, 2, 4 and 6 h post rapa stimulation. the shrimps injected with dmso were employed as control groups. β-actin gene was used as an internal control to calibrate the cdna template for all the samples. each value was shown as mean ± s.d. (n = 5), and bars with different characters indicated significantly different (p < 0.05). (highly similar to fkbp12) in a gtp-dependent manner through its switch 1 region in hek293 cells. the combination between rheb and fkbp38 can displace fkbp38 (an mtor inhibitor) from the fkbp/rapa-binding domain and contributes to mtorc1 activation (avruch et al., 2009). our work suggested that the feedback regulations between rheb and mtor were existing in shrimp. the depression of mtor signaling pathway caused by rapa can promote the expression of rheb. rheb overexpression might be able to activate torc1 signaling in shrimp, too. however, as yet unknown and the considerable additional work is needed to elucidate the feed-back mechanisms by which the rheb–tor interaction promotes tor kinase activity. mtor output is sensitive to amino-acid (especially leucine and arginine) (long et al., 2005a). furthermore, kimball et al. found leucine caused the most obvious stimulation of the tor signaling pathway compared with other amino acids (jefferson et al., 2001). so, we injected shrimps which were deprived of food for three days with leucine, arginine or rapamycin alone or leucine and rapamycin combination to explore the regulation of rheb expression related to mtor pathway under these circumstances. previous works have found dietary restriction (dr) can reduces mtorc1 activity (mejia et al., 2015; garratt et al., 2016). in our work, the dr group expression level of rheb was obviously increased. this result further proved the existence of feedback regulation between rheb and mtor. previous research has proved the inhibition of mtorc1 signaling path caused by leucine withdrawal can be completely reversed by overexpression of rheb (avruch et al., 2009). in our work, the adding of leucine and arginine to shrimp deprived food for three days can relieve the accelerated expression of rheb. the result proved that rheb was regulated by amino acids, and the regulation play an important role in mtor signaling path in shrimp. in “leu + rapa” group, we injected leucine and rapa into shrimps. we found that rapa can totally hinder the regulation of leucine to rheb compared with leucine group in shrimp. long x et al. found that rheb-mtor interaction can activate mtorc1 activity in vitro and the interaction appears to be regulated by amino acid (long et al., 2005a; long et al., 2005b). some work has been proved that rheb can relieves the inhibition mtor signaling path caused by rapa (ma et al., 2008). our results are consistent with these findings. so, we suspect that the inhibitory effect of rapa-fkbp12 complex and the positive impact of rheb on mtor signaling pathway are mutually inhibited in shrimp. the dominant rapa hamper the activation of mtor caused by amino acid. so, the rheb expression of “leu + rapa” group don’t have obvious change compared with dr group. and it was shown that the dr and rapa injection exhibited a similar effect on the expression of rheb (fig. 6). 59 fig. 6 the mrna expression profiles of rheb detected by rt-pcr post dietary restriction (dr), amino acid and rapa stimulation. the shrimps injected with pbs was the control group. the expression profiling of second group was done 3 days after dr and 30 min after injection with pbs. the mrna expression profiles of other groups were done 30 min after injection with leucine (leu), arginine (arg), rapamycin (rapa) and “leucine + rapamycin” (leu + rapa). β-actin gene was used as an internal control to calibrate the cdna template for all the samples. each value was shown as mean ± s.d. (n = 5), and bars with different characters indicated significantly different (p < 0.05). acknowledgement this research was supported by the national natural science foundation of china (41406151), and the chinese academy of sciences sts major deployment project (kfzd-sw-106). we are grateful to all the laboratory members for their technical advice and helpful discussion. competing financial interests the authors declare no competing financial interests. reference akashi s, shirouzu m, yokoyama s, takio k. detection of molecular ions of non-covalent complexes of rasgdp and rasgppnp by maldi-tofms. j. mass. spectrom. soc. jpn. 44: 269-277, 2007. aspuria pj, tamanoi f. the rheb family of gtp-binding proteins. cell. signalling 16: 1105-1112, 2004. avruch j, long x, lin y, ortiz-vega s, rapley j, papageorgiou a, et al. activation of mtorc1 in two steps: rheb-gtp activation of catalytic function and increased binding of substrates to raptor. biochem. soc. trans. 37: 223-226, 2009. avruch j, long x, lin y, ortiz-vega s, rapley j, papageorgiou a, et al. activation of mtorc1 in two steps: rheb. plos one. 9: e96274-e96274, 2014. brown ej, albers mw, shin tb, ichikawa k, keith ct, lane ws, et al. a mammalian protein targeted by g1arresting rapamycin-receptor complex. nature. 369: 756-758, 1994. brugarolas jb, vazquez f, reddy a, sellers wr, kaelin wg. tsc2 regulates vegf through mtor-dependent and -independent pathways. cancer cell. 4: 147-158, 2003. demetriades c, doumpas n, teleman aa. regulation of torc1 in response to amino acid starvation via lysosomal recruitment of tsc2. cell. 156: 786-799, 2014. garratt m, nakagawa s, simons mjp. comparative idiosyncrasies in life extension by reduced mtor signalling and its distinctiveness from dietary restriction. aging cell. 15: 737-743, 2016. hara k, maruki y, long xm, yoshino k, oshiro n, hidayat s, et al. raptor, a binding partner of target of rapamycin (tor), mediates tor action. cell. 110: 177-189, 2002. jacinto e, loewith r, schmidt a, lin s, ruegg ma, hall a, et al. mammalian tor complex 2 controls the actin cytoskeleton and is rapamycin insensitive. nat. cell biol. 6: 1122-1130, 2004. jefferson ls, kimball sr. amino acid regulation of gene expression. j nutr. 131: 2460s-2466s, 2001. kim dh, sarbassov dd, ali sm, king je, latek rr, erdjument-bromage h, et al. mtor interacts 60 with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. cell. 110: 163-175, 2002. kunz j, henriquez r, schneider u, deuterreinhard m, movva nr, hall mn. target of rapamycin in yeast, tor2, is an essential phosphatidylinositol kinase homolog required for g(1) progression. cell. 73: 585-596, 1993. laplante m, sabatini dm. an emerging role of mtor in lipid biosynthesis. curr biol. 19: r1046-r1052, 2009. laplante m, sabatini dm. mtor signaling in growth control and disease. cell. 149: 274-293, 2012. loewith r, jacinto e, wullschleger s, lorberg a, crespo jl, bonenfant d, et al. two tor complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. mol cell. 10:457-468, 2002. long x, lin y, ortiz-vega s, yonezawa k, avruch j. rheb binds and regulates the mtor kinase. curr biol. 15: 702-713, 2005a. long x, lin y, ortizvega s, busch s, avruch j. the rheb switch 2 segment is critical for signaling to target of rapamycin complex 1. j. biol. chem. 282: 18542-18551, 2007. long xm, ortiz-vega s, lin ys, avruch j. rheb binding to mammalian target of rapamycin (mtor) is regulated by amino acid sufficiency. j. biol. chem. 280: 23433-23436, 2005b. ma d, bai x, guo s, jiang y. the switch i region of rheb is critical for its interaction with fkbp38. j. biol. chem. 283: 25963-25970, 2008. ma xm, blenis j. molecular mechanisms of mtor-mediated translational control. nat. rev. mol. cell. biol. 10: 307-318, 2009. maclea ks, abuhagr am, pitts nl, covi ja, bader bd, chang es, et al. rheb, an activator of target of rapamycin, in the blackback land crab, gecarcinus lateralis: cloning and effects of molting and unweighting on expression in skeletal muscle. j. exp. biol. 215: 590-604, 2012. mejia p, treviño-villarreal jh, hine c, harputlugil e, lang s, calay e, et al. dietary restriction protects against experimental cerebral malaria via leptin modulation and t-cell mtorc1 suppression. nat commun. 6: 6050, 2015. sabatini dm, erdjumentbromage h, lui m, tempst p, snyder sh. raft1: a mammalian protein that binds to fkbp12 in a rapamycin-dependent fashion and is homologous to yeast tors. cell. 78: 35-43, 1994. sakthivel a. effect of mineral deposition on shrimp litopenaeus vannamei in high alkaline water of pennar river, andhra pradesh of southeast coast of india. j. aquac. res. dev. 05, 2014. sarbassov dd, ali sm, kim dh, guertin da, latek rr, erdjument-bromage h, et al. rictor, a novel binding partner of mtor, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. curr biol. 14: 1296-1302, 2004. sato t, umetsu a, tamanoi f. characterization of the rheb-mtor signaling pathway in mammalian cells: constitutive active mutants of rheb and mtor. in: balch, w.e.der, c.j..hall, a. (eds.), small gtpases in disease, part a, pp. 307-320, 2008. schmelzle t, hall mn. tor, a central controller of cell growth. cell. 103: 253-262, 2000. schmittgen td, livak kj. analyzing real-time pcr data by the comparative c-t method. nat. protoc. 3: 1101-1108, 2008. settembre c, zoncu r, medina dl, vetrini f, erdin s, erdin s, et al. a lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mtor and tfeb. embo j. 31: 1095-1108, 2012. su ma, huang yt, chen i, lee dy, hsieh yc, li cy, et al. an invertebrate warburg effect: a shrimp virus achieves successful replication by altering the host metabolome via the pi3k-akt-mtor pathway. plos pathogens. 10: e1004196, 2014. sun y, fang y, yoon ms, zhang c, roccio m, zwartkruis fj, et al. phospholipase d1 is an effector of rheb in the mtor pathway. pnas 105: 8286-8291, 2008. thomas g, hall mn. tor signalling and control of cell growth. curr. opin. cell biol. 9: 782-787, 1997. wullschleger s, loewith r, hall mn. tor signaling in growth and metabolism. cell. 124: 471-484, 2006. accepted february 11, 2018 hypothetical photosensory structure in ciliated protozoan, blepharisma isj 3: 77-83, 2006 issn 1824-307x research report cyst wall formation in the ciliated protozoan colpoda cucullus: cyst wall is not originated from pellicle membranes a kida, t matsuoka institute of biological science, faculty of science, kochi university, kochi 780-8520, japan accepted 18 july 2006 abstract ultrastructural changes during encystment (resting cyst formation) of colpoda cucullus were observed with special reference to cyst wall formation. within 1.5 h after encystment induction, fragmentation of some of the mitochondria occurred, followed by the appearance of a number of net-like globules in the cytoplasm, which were expelled to outside and then involved in cell-to-cell or cell-to-substratum adhesion. the cells were transformed into a spherical shape, and a number of ellipsoidal vacuoles in which ectocyst precursor (amorphous substance) was contained appeared near the cell surface (some of these opened to the outside). in this stage, the ectocyst (outermost layer) was completed. in 3 ~ 10 h toluidine blue-stained substance (tbs), which was probably the precursor for the first synthesized layer of endocyst (en-1), was released from a point near the cell surface and diffused over the cell surface (diffused into between the ectocyst and plasma membrane). thereby, the ectocyst was lined by the en-1. thereafter, several layers of endocyst were periodically formed for 1 ~ 2 weeks. finally a number of reserve grains were accumulated, and cilia were resorbed. key words: colpoda; encystment; cyst wall; ectocyst; endocyst _________________________________________________________________________________________________________ introduction when the protozoans face hazardous environments such as dessication, lack of food organisms and overpopulation, some of them are transformed into resting cysts which can survive hostile environments. the species of genus colpoda (foissner, 1993) are one of the protozoans that have been widely studied with regard to the resting cyst formation (encystment). since the beginning of the last century (for review see corliss and esser, 1974), and especially for the last 50 years, ultrastructural research with ______________________________________________________________________ corresponding author: tatsuomi matsuoka institute of biological science, faculty of science, kochi university, kochi 780-8520, japan email: tmatsuok@cc.kochi-u.ac.jp special reference to the cyst wall formation has been reported (kawakami and yagiu, 1963a, b; tibbs, 1968; janisch, 1980; ruthmann and kuck, 1985; martín-gonzález et al., 1992, 1994; frenkel, 1994; delmonte corrado, 1996; chessa et al., 2002). the knowledge about the structure of the cyst wall of colpodid ciliates and the process of wall formation reported in the previous studies could be summarized as follows: (1) the cyst wall of the resting cyst is composed of a single outermost layer (ectocyst) and several inner layers (endocyst) [classified into ecto-, mesoand endocysts in some reports]; (2) in some studies, it has been suggested that the cyst wall may originate from pellicular membrane (kawakami and yagiu, 1963a; ruthmann and kuck, 1985); (3) in the precystic cells, ellipsoidal vesicles that are believed to be mucocysts 77 (frenkel, 1994) open to the outside, and the their content, which is presumably cyst wall precursor, is excreted (martín-gonzález et al., 1992, 1994; frenkel, 1994). however, we have neither evidence for membrane-derived cyst wall formation nor any images of electron micrographs showing that the ectoand/or endocyst are just being formed from materials excreted from presumed precursor-containing vacuoles. the present study revealed that ectocyst and endocyst of c. cucullus did not originate from pellicle membranes but instead were synthesized with different precursors excreted from the different types of vacuoles. materials and methods colpoda cucullus was cultured in a 0.1 % (w/v) infusion of dried cereal leaves inoculated with bacteria (enterobacter aerogenes) at 23 oc in the dark. bacteria were cultured on agar plates containing 1.5 % agar, 0.5 % polypeptone, 1 % meat extract and 0.5 % nacl. the cultured vegetative cells were collected by centrifugation (1,000 xg, 1 min) and subsequently suspended in a standard saline solution containing 1 mm cacl2, 1 mm kcl and 5 mm tris-hcl (ph 7.2). the cells were rinsed 2-3 times by repeating the sedimentation and suspension in standard saline solution, and finally suspended in the solution to induce encystment. for vital staining of precystic cells with toluidine blue, 0.1 % toluidine blue dissolved in the standard saline solution was added to an equal volume of cell suspension, and kept for 5 ~ 10 min. for prefixation of the precystic cells (0 ~ 1 h after encystment induction), one volume of the suspension of cells was mixed with 6 volumes of a glutaraldehyde (ga) fixative containing 6 % glutaraldehyde, 1 % oso4, 100 mm cacodylate buffer (ph 7.2) and 4 mm sucrose. after 10-min incubation, the prefixed samples were rinsed 5 times in 100 mm cacodylate buffer (ph 7.2) and then postfixed for 2 h in a fixative containing 1 % oso4, 100 mm cacodylate buffer (ph 7.2) and 2 mm sucrose. the precystic cells and mature cysts (1 h ~ 2 weeks after encystment induction) were prefixed with ga fixative without oso4 for 6 h and postfixed with a fixative containing 1 % oso4, 100 mm cacodylate buffer (ph 7.2) and 2 mm sucrose for 1 week. the postfixed samples were rinsed several times in distilled water, dehydrated through a graded ethanol series (30, 40, 50, 60, 70, 80, 90 and 100 % ethanol) for 15 min each and finally suspended in acetone. the dehydrated samples were embedded in spurr’s resin. ultrathin sections were stained with 3 % uranyl acetate and then with lead citrate (10 min each). the sections were observed under a transmission electron microscope (jeol, 1010t). fig. 1 transmission electron micrographs of a vegetative cell of c. cucullus (fig. 1a) and the precystic cell in the early phase of stage 1 (0 ~ 1 h after encystment induction) (figs 1b-d). fig. 1a (inset): a magnified picture of the cortical region, showing the alveolus (al). fig. 1c: arrowheads, mitochondria that has just been torn and fragmented. fig. 1d: fr, mitochondria fragmented into small pieces; ap, autophagosome-like structure. results the morphological events during encystment are described by dividing them into stage 1 ~ 4. the parentheses show the typical terms after encystment induction in which the cytoplasmic events in each stage are observed in most cells. stage 1 (0 ~ 1.5 h after encystment induction). fig. 1a is a longitudinal section of a vegetative cell, showing a number of small-sized vacuoles or vesicles in the cytoplasm and alveoli in the cortical region. within 1 h after encystment induction, some of the mitochondria were fragmented (figs 1b, c, d) and a number of autophagosome-like structures appeared (fig. 1d). during the latter part of this stage, a number of net-like globules that had been described previously (kawakami and yagiu, 1963a) appeared in the cytoplasm (figs 2a, b) and were subsequently released into extracellular space. stage 2 (1.5 ~ 3 h). the precystic cells became round, and cell movement was gradually slower and finally stopped. a number of spherical or ellipsoidal vacuoles, which were possibly similar organelles (mucocysts) previously reported in precystic cells of tillina magna (frenkel, 1994), were localized in the vicinity of the cell surface and opened to the extracellular space to excrete amorphous precursor material (fig. 3b) for an outermost layer (ectocyst) (figs 3a, b). the excreted materials were gradually deposited (fig. 3c) and finally formed an electron-dense single layer (ectocyst) (fig. 3d). stage 3 (3 ~ 10 h). by the time ectocyst completion occurred (figs 4a, b), the cells had ceased swimming, but cells continued to rotate 78 fig. 2 transmission electron micrographs of the precystic cells in the latter phase of stage 1 (1 ~ 1.5 h), showing net-like globules (gl) in the cytoplasm. fig. 2a, a total view of the cell; fig. 2b, a magnified picture and highly magnified one (inset) of net-like globules. for a while (10~30 min) by ciliary movement inside the ectocyst envelope. during this stage, the number of electron-lucent vacuoles was reduced, while numerous endoplasmic reticula and mottled or electron-dense granules appeared (figs 4a, b), which may be responsible for the next event, the synthesis of endocyst. a drastic event was visualized in toluidine blue-stained cells just after the precystic cells ceased to rotate inside the ectocyst envelope (figs 4c, d). an extremely large vacuole (fig. 4c) opened near the cell surface, and substance deeply stained with toluidine blue (tbs) diffused over the entire cell surface within a few minutes (figs 4c, d). the tbs release occurred at 1.5 h in cases when the encystment was most quickly induced but proceeded much more slowly in the case of typical cells. the cells whose cortical region was deeply stained by the diffusion of tbs could hardly be crushed by a mechanical press. in the electron micrograph of this stage, fibrous materials constituting the first layer (en-1) of endocyst were observed between the ectocyst and plasma membrane (fig. 4e), which probably correspond to tbs. fig. 3 transmission electron micrographs of precystic cells in stage 2, showing ectocyst formation. figs 3a-c: ec, ectocyst that has just been formed with amorphous precursor materials (fig. 3b, pr) excreted from spherical or ellipsoidal vacuoles. ma, macronucleus; v, vacuoles containing amorphous materials; ci, cilia; al, alveolus. fig. 3b (inset), a magnified picture of just excreted amorphous materials; fig. 3c (inset), magnified image of ectocyst just being formed. fig. 3d, an image showing a just completed ectocyst envelope (ec). fig. 4 transmission electron micrographs (figs 4a, b, e) of precystic cells and photomicrographs of toluidine blue-stained living cells (figs 4c, d), showing the process of first layer (en-1) formation of endocyst. fig. 4f [stage 4]: a transmission electron micrograph of the cell at 24 h after encystment induction, showing a completed first layer of endocyst (en-1). figs 4b, e, f: er, endoplasmic reticulum; ci, cilia; g, mottled granules; ec, ectocyst; en-1, endocyst-1 (first synthesized endocyst layer); al, alveolus. figs 4c, d: v, vacuole containing presumed precursor for endocyst layer (en-1); arrowhead, a point where toluidine blue-stained substance (tbs) was being released. 79 fig. 5 transmission electron micrographs of an immature cyst in the early phase of stage 4 (2 days after encystment induction), showing a third layer (en-3) of endocyst are being formed. fig. 5a (inset), a magnified picture showing the ectocyst (ec) lined by endocyst-1 (en-1). en-2, second synthesized layer of endocyst; en-3, third synthesized layer of endocyst; v, vacuole containing presumable precursor for endocyst; gl, net-like globule attached to surface of ectocyst; g, electron-dense granule; arrow (fig. 5a), a boundary of two cysts adhered to each other with crushed net-like globules; arrowheads in fig. 5b, vacuoles just beginning to excrete the precursor for endocyst. stage 4 (10 h~ 2 weeks). in most cells, the formation of en-1 was completed within 1 day, and the ectocyst was lined by the en-1 (fig. 4f). during this stage, several layers of endocyst were formed. as shown in fig. 5, in the 2-day-old immature cyst, a third layer (en-3) of endocyst was observed to be just forming. in this case, an extremely large vacuole (figs 5 a, b) associated with many electron-lucent small-sized vacuoles or ducts appeared near the cortical region, some of which opened to the space between the plasma membrane and the already synthesized fig. 6 a photomicrograph (fig. 6c) and transmission electron micrographs (figs 6a, b, d, e) of immature cysts at 1 week after encystment induction and in a 2-week-old mature cyst (fig. 6f). fig. 6a: 1-week-aged cyst. a vacuole (v) containing endocyst precursor opened to the space between the plasma membrane and the already synthesized innermost endocyst layer. fig. 6b: a magnified picture of cyst wall. inset, a highly magnified picture of endocyst showing that endocyst layers are composed of fine fibrous materials. fig. 6c: a photomicrograph of 1-week-aged living cyst. fig. 6d: a section of 1-week-aged cyst showing that cytoplasmic reconstruction rather progressed than the cyst shown in fig. 6a. fig. 6e: a magnified picture of the section shown in fig. 6d. fig. 6f: a section of 2-week-aged mature cyst. figs 6a ~ f: rg, reserve grains; ec, ectocyst; en, endocyst; ci, cilia; gl, net-like globules; gr, small grains surrounding macronucleus; ma, macronucleus; mt, mitochondria; en-3, third synthesized endocyst layer; al, alveolus; arrowhead in fig. 6e, electron-lucent amorphous materials. layer (en-2) of endocyst, and endocyst precursor was excreted (fig. 5b, arrowheads). in addition, a number of electron-dense granules were seen inside and in the vicinity of the large vacuole (figs 5b, g). the complex of the large vacuole and many small-sized vacuoles or ducts leading to the cell surface (fig. 5 b) may correspond to the vacuole (figs 4c, d) observed by light microscope. fig. 6 show a photomicrograph of living cell (fig. 6c) and electron micrographs (figs 6a, b, d, e) of 7-day-old and 2-week-old cysts (fig. 6f). some of the cysts were still immature, and endocyst precursor was observed being released from a large vacuole (fig. 6a) 80 fig. 7 schematic diagrams showing the entire encystment process (fig. 7a) and two different ideas for cyst wall formation (fig. 7b). fig. 7b-1: a hypothesis for ectocyst formation in which it originates pellicle membranes. fig. 7b-2: a schematic diagram showing ectoand endocyst formation based on the present results. ec, ectocyst; en, endocyst; gl, net-like globules; tbs (en), toluidine blue-stained substance (endocyst precursor); rg, reserve grains; al, alveolus, m, plasma membrane; p (ec), precursor for ectocyst. forming the innermost layer of endocyst. during this stage, the compact and narrow layers and broad layers of endocyst were observed to be alternatively formed (figs 6a, b). both of the layers seem to be composed of identical fibrous materials (fig. 6b). the broad layers of endocyst might not be produced by the fixation and dehydration processes for electron microscopy, because a broad space (layer) was seen in a living cyst (fig. 6c). after the endocyst layers were completed, the reserve grains gradually accumulated in the central region (fig. 6d), and 81 the cytoplasm became dappled by the appearance of amorphous electron-lucent structures (figs 6d, e), which seem to develop into the reserve grains. in addition, the macronucleus was surrounded by a number of small grains (fig. 6d). finally, electron-lucent amorphous structures were replaced by a large amount of regularly stacked ellipsoidal reserve grains (fig. 6f), and, as a result, the central region of cytoplasmic space was occupied mainly by the reserve grains and mitochondria located in the peripheral region. in the final stage (figs 6d-f), cilia were resorbed. discussion the outline of the encystment process with special reference to cyst wall formation is schematically summarized in fig. 7. the first drastic events in the stage 1 are the fragmentation of mitochondria and appearance of autophagosome-like structures (figs 1b, c, d, 7a). the fragmented mitochondria are probably digested inside the autophagosomes. the net-like globules appeared and excreted in the latter phase of this stage (figs 2, 7a) are probably involved in a cell-to-cell adhesion (see fig. 5a, arrow) or adhesion of the cells to the substratum. if the ectocyst originated from pellicle membranes, as suggested in the previous reports (kawakami and yagiu, 1963a; ruthmann and kuck, 1985; watoh et al., 2005), alveoli might be fused with one another to produce new plasma membrane and outermost double membranes developing into the ectocyst which are deposited with a precursor substance (fig. 7b-1). although electron microscopic images implying the fusion of alveoli are observed occasionally (watoh et al., 2005), no images showing that the outer double membranes produced by fusion of alveoli further developed into ectocyst or endocyst have been observed in the previous study (watoh et al., 2005). on the other hand, in the present study, we succeeded in obtaining successive images showing that the ectocyst was just being formed with amorphous substance excreted from vacuoles (figs 3a-d, ref. fig. 7b-2). in this process, neither the fusion of alveoli nor the appearance of new alveoli was detected. as a result, we believe that the images of fused alveoli observed in the previous report (watoh et al., 2005) may have been artifacts produced by the fixation and/or dehydration processes. the ectocyst formation is followed by a diffusion of tbs which is probably endocyst precursor (figs 4c, d). in the previous report (watoh et al., 2005), the tbs is suggested to diffuse between the first-formed layer of endocyst (en-1) and the plasma membrane based on the photomicroscopic observation. this idea should be modified, because the present electron microscopy revealed that during this stage, fibrous endocyst materials probably corresponding to tbs were observed in the space between the ectocyst and the plasma membrane (fig. 4e, ref. fig. 7b-2). the formation of endocyst-1 (en-1) is followed by the formation of several layers of endocyst, the precursor of which is probably supplied by a periodic excretion into the space between the plasma membrane and the innermost endocyst layer (figs 5, 6a). in this case, the newly formed endocyst shows the replica-like configuration of the cell surface, i.e. the smooth endocyst layers beneath the smooth cell surface (figs 6a, b) and undulate layers for furrowed cell surface (figs 6d, e). the alternative formation of the narrow and broad layers of endocyst may be responsible for the periodic shrinkage of the cell body. that is, broad endocyst layers may be formed when the endocyst precursor is excreted into the broad space between the plasma membrane and innermost endocyst layer which is produced by the cell shrinkage. every layer of endocyst is composed of fine fibrous materials (fig. 6b), indicating that it is derived from a common precursor material, which is expected to be deeply stained with toluidine blue. in the present study, we defined a “mature cyst” of c. cucullus as a cyst in which every cytoplasmic event (formation of ecto-and endocysts, resorption of cilia, accumulation of reserve grains, aggregation of mitochondria in the peripheral region of cytoplasm) during the resting cyst formation has occurred and further prominent change is not observed (see fig. 6f). it has been reported that in other species of colpoda, the silver impregnation method indicates that some pairs of kinetosomes disappear during resting cyst formation (martín-gonzález et al., 1991). unfortunately, the present microscopy failed to conclude whether the kinetosomal structures completely disappear in the mature cysts, because the cytoplasm of mature cysts is compacted and highly electron-dense. acknowledgement we thank ms watoh t for her technical assistance in electron microscopy. references chessa mg, largana i, trielli f, rosati g, politi h, angelini c, delmonte corrado mu. changes in the ultrastructure and glycoproteins of the cyst wall of colpoda cucullus during resting encystment. eur. j. protist. 38: 373-381, 2002. corliss jo, esser sc. comments on the role of the cyst in the life cycle and survival of free-living protozoa. trans. amer. micros. soc. 93: 578-593, 1974. delmonte corrado mu, chessa mg, pelli p. ultrastructural survey of mucocysts throughout the life cycle of colpoda cucullus (ciliophora, colpodea). 82 acta protozool. 35: 125-129, 1996. foissner w. colpodea (ciliophora). gustav fischer verlag, stuttgart, 1993. frenkel ma. the cyst wall formation in tillina magna (ciliophora, colpodidae). arch. protistenkd. 144: 17-29, 1994. janisch r. a freeze-etch study of the ultrastructure of colpoda cucullus protective cysts. acta protozool. 19: 239-246, 1980. kawakami h, yagiu r. an electron microscopical study of the change of fine structures in the ciliate, colpoda cucullus, during its life cycle. ii. from the preencystment stage to the early stage of the formation of the first layer of resting cyst membrane. zool. mag. 72: 146-151, 1963a. kawakami h, yagiu r. the electron microscopical study of the change of fine structure in the ciliate, colpoda cucullus, during its life cycle. iii. from the stage of completion of the first layer of resting cyst membrane to the completion of the resting cyst. zool. mag. 72: 224-229, 1963b. martín-gonzález a, benítez l, gutiérrez jc. cortical and nuclear events during cell division and resting cyst formation in colpoda inflata. j. protozool. 38: 338-344, 1991. martín-gonzález a, benítez l, gutiérrez jc. ultrastructural analysis of resting cysts and encystment in colpoda inflata. 2. encystment process and a review of ciliate resting cyst classification. cytobios 72: 93-106, 1992. martín-gonzález a, palacios g, gutiérrez jc. cyst wall precursors of colpoda inflata: a comparative ultrastructural study and a review of ciliate cyst wall precursors. cytobios 77: 215-223, 1994. ruthmann a, kuck a. formation of the cyst wall of the ciliate colpoda steinii. j. protozool. 32: 677-682, 1985. tibbs j. fine structure of colpoda steinii during encystment and excystment. j. protozool. 15: 725-732, 1968. watoh t, sekida s, yamamoto k, kida a, matsuoka t. morphological study on the encystment of the ciliated protozoan colpoda cucullus. j. protozool. res. 15: 20-28, 2005. 83 introduction materials and methods results discussion acknowledgement review 31 isj 15: 31-38, 2018 issn 1824-307x research report differential impact of pesticides and biopesticides on edaphic invertebrate communities in a citrus agroecosystem mz majeed1,2, m naveed2, ma riaz2,3, c-s ma1, m afzal2 1state key laboratory for biology of plant diseases and insect pests, institute of plant protection, chinese academy of agricultural sciences, beijing 100193, pr china 2department of entomology, college of agriculture, university of sargodha, 40100, sargodha, pakistan 3department of entomology, university of georgia, athens, ga 30602, usa accepted january 08, 2018 abstract edaphic invertebrate fauna is usually exposed directly or indirectly to a wide range of pesticides in agroecosystems worldwide. very few studies have assessed the negative effects of these pesticides on the diversity and population dynamics of soil invertebrates. in this study, the effect of most commonly used pesticides viz; bifenthrin (a synthetic pyrethroid), spinosad (a bio-insecticide), aliette (a synthetic fungicide) and trichoderma harzianum formulation (30x106 cells ml-1; a bio-fungicide) was assessed on soil invertebrate fauna in a citrus agroecosystem. secondary objective was to compare the impact of synthetic versus biological pesticides and insecticides versus fungicides. there was a significant effect of all pesticides on the population abundance of springtails (f4,14 = 16.53; p<0.001), mites (f4,14 = 12.07; p<0.001) and ants (f4,14 = 16.28; p<0.001). by and large, soil fauna got recovered after two to three weeks post-treatment. insecticides were more suppressive for soil invertebrates than fungicides. overall, biological pesticides i.e. spinosad and t. harzianum formulation were less disruptive to soil invertebrate fauna than synthetic conventional pesticides. hence, keeping in view the key role of soil invertebrates in soil sustainability and crop productivity, the utilization of biopesticides should be encouraged. key words: bio-pesticides; fungicides; insecticides; population abundance; soil invertebrates introduction citrus is an important fruit crop around the globe. however, its production is hampered by numerous species of insect pests including psyllids, leafminers, fruit flies and scales, and diseases including canker, greening and downy mildews (anjum and javaid, 2005; tahir et al., 2015). in order to control these pests and to protect their crop and yield, farmers indiscriminately and recurrently use a wide range of synthetic pesticides including insecticides and fungicides (monzo et al., 2014). although these pesticides provide the control of insect pests and diseases because of their rapid knockdown effect and save considerable yield loss by reducing the pest infestation, but concomitantly these agro-chemicals have many non-target effects ___________________________________________________________________________ corresponding author: muhammad zeeshan majeed state key laboratory for biology of plant diseases and insect pests institute of plant protection chinese academy of agricultural sciences beijing 100193, pr china e-mail: zeeshan.majeed@uos.edu.pk including disruption of beneficial organisms, insecticide resistance, pest resurgence and human health hazards (edwards, 2013; monzo et al., 2014). most of the pesticides being used by citrus growers in indo-pak region are broad spectrum and highly persistent in the environment diminishing many beneficial fauna along with the target pests (ashraf et al., 2014). regarding pesticide side effects, soil invertebrates are one of the non-target organisms which may be exposed to all pesticides either directly to spray splashes and drift or indirectly by contacting pesticidal residues on foliage, soil, litter and thatch (bünemann et al., 2006; larson et al., 2014). agricultural soils harbor a considerable diversity of invertebrates often classified as micro and mesofauna including collembolans, mites and tiny insects etc., and macro-fauna including earthworms, termites, ants, beetles and spiders etc. (lavelle et al., 1997; kocourek et al., 2013). these soil invertebrates play a crucial role in different ecological processes such as organic matter decomposition and nutrients cycling, and are indispensable for soil biological functioning and mailto:zeeshan.majeed@uos.edu.pk 32 table 1 list of treatments used in this study *water used in control plots was same as used for preparation of pesticides spray mixtures. sustained crop productivity in agro-ecosystems (lavelle et al., 1997; lardo et al., 2012; majeed, 2012; bagyaraj et al., 2016). soil fauna, particularly mesoand macro-fauna, improve soil physicochemical conditions through their feeding (ingestion, digestion and ejection) and foraging (tunneling, boring, mining, movement) activities (lee and pankhurst, 1992; edwards and bohlen, 1996; lavelle at al., 1997; jouquet et al., 2006; coleman and wall, 2015; bagyaraj et al., 2016). keeping in view the ecological importance of soil invertebrates and lack of information regarding the side effects of most commonly and widely used pesticides in indigenous citrus agroecosystems on the soil invertebrate fauna, this study sought to compare the impact of different type of pesticides (insecticides and fungicides) on the density (abundance) and diversity (community assemblage) of non-target soil invertebrates. our a priori hypothesis was that conventional pesticides would have more severe effects on soil fauna than biopesticides. to achieve these objectives, different pesticides were applied according their labelrecommended dose rates on the under canopy area of citrus plants and preand post-application data regarding soil invertebrate fauna was collected for different time intervals up to two months. materials and methods the study area is situated in the district sargodha of the province of punjab (pakistan) and is characterized by semi-arid sub-tropical climatic conditions with mean annual temperature and precipitation of 23 °c and 450 mm, respectively. citrus is the principal fruit crop of sargodha region. study was conducted in private citrus orchards of kinnow mandarin (cv. citrus reticulata) (32°05'n and 72°40'e) situated in the vicinity of the college of agriculture, university of sargodha. orchard age was approximately 12 years and was not treated with any pesticide for last nine weeks. fifteen healthy and equal sized citrus trees were selected and tagged at random leaving rows of plants on all sides of the orchard as buffer zone to avoid edge effect. five treatments as given in table 1 including four most commonly used pesticides by indigenous citrus growers and one control (sprayed only with tap water) were applied on 30 april, 2017 on the under-canopy soil area of the citrus trees according to randomized complete block (rcb) design with five replications for each treatment. pesticides were applied according to their label-recommended dose rates using a back-mounted knapsack pump sprayer from a height of 1.5 m to mimic the pesticide spray drift. physico-chemical characteristics of the study soil were also determined for pre-treatment soil samples (table 2). pesticides included bifenthrin (a synthetic pyrethroid insecticide), spinosad (a microbial bioinsecticide), aliette (an aluminium based (fosetyl-al) synthetic fungicide) and formulation of trichoderma harzianum (a bio-fungicide). there were two motives behind the selection of these pesticides. first was to have a comparative assessment of the impact of different pesticides (insecticides and fungicides) and different pesticide groups (i.e. synthetic and biological) on soil non-target fauna. table 2 physico-chemical characteristics of the soil of citrus orchards studied soil characteristic condition/content soil texture sandy loam ph 7.8 (0.03) ece ( µs cm-1) 2410.0 (221.4) soil organic matter (g kg-1) 8.1 (0.29) soil organic-c (g kg-1 soil) 4.7 (0.16) total soil n (mg kg-1) 408.6 (15.01) nahco3 extractable-p (mg kg -1 soil) 7.9 (0.40) extractable-k (mg kg-1 soil) 163.4 (5.65) values are means of five independent soil composite samples along with standard errors within parenthesis. treatment no. treatment application rate 1 bifenthrin (synthetic pyrethroid) 625 ml ha-1 2 spinosad (microbial bio-insecticide) 150 ml ha-1 3 aliette (fosetyl-al; synthetic fungicide) 7 kg ha-1 4 trichoderma harzianum (bio-fungicide) 30x106 conidia ml-1 5 control (water)* 33 table 3 diversity indices of different edaphic faunal (invertebrate) groups in citrus orchard soils treated with different types of pesticides dbt: days before treatment; dat = days after treatment second criterion was that these pesticides were the most commonly used by citrus famers in sargodha region as assessed from a preliminary survey of local pesticide dealers and citrus growers. three 0 15 cm deep soil samples were collected from each treatment 2 days before, 3, 15, 30 and 60 days after application of treatments. each sample (weighing about 1,750 g) was the composite of four sub-samples taken randomly from four sides of the treated plant using 10x10 cm metallic soil corer. these samples were brought to the laboratory of the department of entomology and data regarding soil invertebrate fauna was taken. macro-invertebrates were collected and enumerated from each sample manually, while mesoand micro-invertebrates were extracted from samples by installing them on tullgren-berlese funnel for 24 h. extracted invertebrates were preserved in 50% ethanol solution in transparent 20 ml plastic vials for their further identification and enumeration under light microscope up to higher taxonomic (order, genus or family) level. statistica® version 7.1 (statsoft®, france) was used for statistical interpretation of data. normality of data was checked and data were transformed by log10 (x+2) before further analyses where normality was not met. data regarding soil fauna was subjected to 2-way factorial analysis of variance with treatment and time interval as factors and twosample student’s t-tests were used to compare pesticides and/or their groups. for effect of each pesticide on soil invertebrates, one-way anova was applied at 95% confidence level followed by tukey’s highest significant difference (hsd) tests to compare treatment means. for faunal (invertebrates) community assemblage determination, shannon-wiener’s index, faunal group richness and evenness indices were calculated as described by ahmed et al. (2017) along with the graphical presentation (pie charts) of data. moreover, multivariate analysis of significance was also run to assess the impact of pesticides on soil invertebrate fauna. results impact of pesticides on the diversity of soil faunal groups results have demonstrated that shannonwiener diversity index, which estimates the relative richness and abundance of different groups or species of organisms collected and sampled from different locations (shannon-wiener, 1963), fluctuated among different time intervals for all treatments (table 3). maximum diversity index (1.90) was found for control treatment at 30dat (days after treatment) while minimum was recorded for bio-fungicide at 15dat (table 3) for all pesticide treatments, diversity of soil invertebrate faunal groups reduced for the first two weeks posttreatment as compared to the diversity index of control treatment which was least perturbed during the entire period of experiment. after 15 days posttreatment, shannon-wiener index increased to its maximum at 30 dat for all treatments but then reduced to normal level at 60dat. pesticide treatments had a differential impact on the evenness index of soil faunal groups. evenness index measures the relative abundance of species or organismal groups of an area. in case of insecticide (bifenthrin), maximum evenness (0.79) was observed just after three days of spray but then reduced slightly as compared to control (before spray) value. in case of bio-insecticide (spinosad), minimum evenness value (0.58) was observed at 30dat and then returned to normal (control) value after one month. in case of fungicides, both synthetic (aliette) and bio-fungicide (t. harzianum formulation) showed similar response regarding their effect on fauna group evenness with minimum values (0.55 and 0.53, respectively) at 15dat (table 3). similarly, soil faunal (invertebrate) groups’ richness was reduced up to 15dat, then increased to a maximum value of 11.00 at 30dat, and again decreased up to normal level at 60dat (table 3). among treatments, synthetic fungicide showed minimum values of richness of soil faunal groups as compared to others, while insecticide (bifenthrin) showed maximum fluctuation in faunal group richness index (table 3). effect of pesticides on community assemblages of invertebrate faunal groups gross higher-level taxonomic composition of treated soil samples have been presented in the form of pie charts (fig. 1). according to this graphical presentation of invertebrate fauna encountered in soil samples, collembolans and mites were the most abundant faunal groups found in all samples, followed by ants, spiders and rove beetles. the least dominant groups were oligochaeta (earthworms) and carabid beetles. the diversity indices shannon w iener's diversity index richness index evenness index treatments 1dbt 3dat 15dat 30dat 60dat 1dbt 3dat 15dat 30dat 60dat 1dbt 3dat 15dat 30dat 60dat insecticide 1.56 1.54 1.22 1.44 1.40 10.00 7.00 7.00 10.00 7.00 0.68 0.79 0.63 0.62 0.72 bio-insecticide 1.39 1.53 1.13 1.23 1.51 9.00 9.00 7.00 10.00 9.00 0.63 0.70 0.58 0.53 0.69 fungicide 1.44 1.35 1.20 1.59 1.51 9.00 8.00 9.00 9.00 10.00 0.65 0.65 0.55 0.72 0.66 bio-fungicide 1.44 1.44 1.04 1.70 1.46 10.00 9.00 7.00 11.00 8.00 0.63 0.66 0.53 0.71 0.70 control 1.47 1.55 1.74 1.90 1.68 9.00 9.00 9.00 11.00 10.00 0.67 0.71 0.79 0.79 0.73 34 fig. 1 pie-charts showing community assemblages of edaphic faunal (invertebrate) groups in a citrus agroecosystem at different time intervals in response to application of different types of pesticides. (dbt = days before treatment; dat = days after treatment). most prominent change occurred in the community assemblages of soils treated with insecticide (bifenthrin) and bio-insecticide (spinosad) till 15 days post-exposure. the most effected faunal groups were ants and collembolans (fig. 1). for the first month of experiment, the faunal community assemblages remained dominant by collembolans (springtails) in all samples while oribatid mites (cryptostigmata) outnumbered all other invertebrate faunal groups in the last observation at 60 days post treatment. impact of pesticides on population abundance of different soil faunal groups collembola (springtails), cryptostigmata (oribatid mites), formicidae (ants) and araneae (spiders) were the most abundant and dominant faunal groups in the study soils of citrus orchard. therefore, the response of only these faunal groups towards different pesticides was further analyzed statistically apart from its graphical representation (fig. 2). in case of micro-invertebrates (collembola and cryptostigmata), insecticide (bifenthrin) reduced three times the average population of springtails and oribatid mites at 3dat than their control population (i.e. 15.0 springtails and 13.3 mites sample-1). the maximum population of springtails and mites reached at 30dat (i.e. 35.0 springtails and 16.3 mites sample-1). however, in case of all treatments including control, population of springtails suddenly decreased at 60dat except mites (fig. 2). similarly, bio-insecticide (spinosad) had a little but significant effect while fungicide and bio-fungicide had no significant effect on population abundance of springtails and oribatid mites. similarly, regarding macro-invertebrates (formicidae and araneae), a similar trend had been observed for ants. while in case of spiders, there was no clear-cut trend regarding the impact of pesticides on their population dynamics. maximum population of ants and spiders (i.e. 8.67 ants and 3.67 spiders sample-1) were recorded at 30dat and 60dat respectively, while the minimum ones (i.e. 0.67 ants and 0.33 mites sample-1) at 15dat and 60dat, respectively. in general, population of all faunal groups in control soils, which were treated with water only, gradually increased till 30dat, and then decreased at 60dat for all groups except oribatid mites which continued to increase till 60dat. however, in control (water) treatment, spider population increased gradually from start to the end of experiment at 60dat (fig. 2). 35 fig. 2 impact of different types of pesticides on the population abundance of major edaphic invertebrate groups in a citrus agroecosystem. data points represent population means ± standard error (n = 4). for each faunal group, treatments bearing same superscripted letters are not statistically different from each other (factorial (two factor) anova; α = 0.05). (dbt = days before treatment; dat = days after treatment; bf = bio-fungicide; f = fungicide; bi = bio-insecticide; i = insecticide; c = control). according to two-way factorial analysis of variance, insecticide (bifenthrin) had the most drastic and significant effect on population abundance of all edaphic faunal groups followed by bio-insecticide (spinosad), while the least disturbing pesticide for soil invertebrate fauna was formulation t. harzianum (bio-fungicide) (fig. 2). fungicide (aliette) exhibited an intermediate response (fig. 2). overall, there was a significant effect of pesticides on the population abundance of springtails (f4,14 = 16.53; p<0.001), oribatid mites (f4,14 = 12.07; p<0.001), formicid ants (f4,14 = 16.28; p<0.001) but non-significant for the population abundance of spiders (f4,14 = 1.51; p=0.212). similarly, observation times had a significant effect on the population abundance of springtails (f4,14 = 4.46; p<0.01), oribatid mites (f4,14 = 3.01; p=0.027), formicid ants (f4,14 = 6.60; p<0.001) and spiders (f4,14 = 9.27; p<0.001). however, the interaction of treatment and time exhibited significant effect on the population of all faunal groups except spiders (araneae). nevertheless, according to multivariate analysis, all treatments (pesticides) and time intervals exhibited a significant (p < 0.05) effect on overall population abundance of soil invertebrate fauna encountered in soil samples of different treatments, except for time effect of bio-fungicide (p = 0.2455; table 4). discussion in sustainable agriculture, one of the contemporary ecological issues regarding extensive use of pesticides is their deleterious effects on nontarget organisms including soil invertebrate fauna (edwards, 2013; pisa et al., 2015). beneficial edaphic invertebrate fauna is usually exposed directly or indirectly to a wide range of pesticides (frampton and van den brink, 2006; adamski et al., 2009; larson et al., 2014). this study was carried out to determine the impact of different types of pesticides on edaphic soil fauna in a citrus orchard. two types of pesticides i.e. insecticides and fungicides with different origins i.e. synthetic and biological were evaluated under field conditions. most commonly used insecticides selected in this study were bifenthrin and spinosad, while fungicides were aliette and t. harzianum formulation containing 30x106 cells ml-1. soil fauna was sampled, identified up to group or order level and enumerated at regular time intervals for two months post treatment. the overall effect of these pesticides on diversity of soil invertebrate fauna was evaluated by calculating three diversity indices i.e. shannonwiener diversity index, faunal group evenness index and faunal group richness index, while the abundance of major soil faunal groups was compared 36 table 4 multivariate analysis of significance for the effect of different types of pesticides on the abundance of edaphic faunal (invertebrate) groups in citrus orchard soils treatments/effect wilk’s lambda value f-value hypothesis df error df p-value insecticide 0.000182 2061.87 8 3.00 0.0000 *** observation time 0.000026 6.51 32 12.66 0.0005 *** bio-insecticide 0.001722 331.34 7 4.00 0.0000 *** observation time 0.002005 2.60 28 15.84 0.0246 * fungicide 0.000939 608.02 7 4.00 0.0001 *** observation time 0.001874 2.66 28 15.84 0.0222 * bio-fungicide 0.001143 499.34 7 4.00 0.0000 *** observation time 0.011285 1.39 28 15.84 0.2455 *** p < 0.001; * p < 0.01; * p < 0.05; multivariate analysis (effective decomposition of the hypothesis) at α = 0.05 with control (water) treatment and with pretreatment data of soil fauna as well. study results revealed that the insecticide bifenthrin, a synthetic pyrethroid, exerted considerable reduction of soil invertebrate fauna particularly of springtails, mites, ants and earthworms. similar findings have been reported by frampton and van den brink (2007) and larson et al. (2014) that pyrethroid insecticides like cypermethrin and bifenthrin had more pronounced negative effects on soil-dwelling fauna as compared to organophosphate (chlorpyriphos) and neonicotinoids (clothianidin). similar effects of pyrethroid insecticides (deltamethrin and cypermethrin) on soil-inhabiting spiders have been described by pekár and beneš (2008). nevertheless, jänsch et al. (2006) also reported the most pronounced deleterious effects of pyrethroid and organophosphate insecticides to earthworms, spiders, mites and springtails. hence, the continuous use of such toxic insecticides may reduce the diversity and abundance of soil nontarget invertebrates (bünemann et al., 2006; frampton and van den brink, 2006; adamski et al., 2009). although spinosad, which is a bio-insecticide derived from metabolites of soil-borne actinomycete saccharopolyspora spinosa, reduced the diversity and abundance of soil fauna particularly of springtails, mites and ants, however, it exhibited relatively less effect on soil fauna as compared to bifenthrin. insecticides of biological origin such as spinosad are replacing conventional insecticides due to their higher target specificity, quick environmental biodegradation and more biorational and eco-friendly nature (williams et al., 2003; biondi et al., 2012). nevertheless, the combined effect of insecticide and bio-insecticide on soil non-target fauna was also significant. both type of chemicals suppressed population abundance and community assemblage of all soil faunal groups up to 15 days post-application. these observations are in accordance with those of pekár and beneš (2008) who demonstrated up to 100 % mortality of soilinhabiting spiders by their exposure to few hours to 20 days old residues of insecticides (chlorpyrifos, cypermethrin and deltamethrin). on the other hand, fungicide aliette (fosetyl aluminum) exerted very little reduction of some soil fauna only for three days post-application while t. harzianum formulation did not affect soil fauna as much as insecticides. al-assiuty et al. (2014) described similar findings that bio-fungicides have less severe effect on community structure and population size of oribatid mites than synthetic fungicides. however, possible reasons for the observed little or no significant effects on nontargets in case of fungicides might be due to the poor diversity of invertebrates in study soils as well as the application of treatments on limited soil patches close to citrus plants as manifested by babendreier et al. (2015). nevertheless, the effects of both fungicides were non-significant. this observation is in context with the findings of moreby et al. (1997) and jänsch et al. (2006) who found that fungicides have less negative effect on soil dwelling invertebrates than insecticides. however, prolonged exposure to fungicides may reduce the population abundance of soil fauna either by exerting immediate changes in soil food web system (jänsch et al., 2006) or by affecting the behavior and longterm survival of soil invertebrates (evans et al., 2010). regarding community assemblages of different soil faunal groups, the most prominent changes were observed in case of micro-invertebrates (springtails and mites) and ants in response to insecticides. in case of fungicides, community assemblages of soil fauna remained almost identical at all sampling dates. this trend corroborates the findings of moreby et al. (1997) and al-assiuty et al. (2014). nevertheless, diversity of different edaphic faunal groups collected and identified during the study was influenced by the application of pesticides. in general, all diversity indices i.e. shannon-wiener’ index, species (faunal group) evenness and richness indices were lowered in the first two weeks of application and then either 37 recovered to normal or increased from 15dat to 30dat levels. the same pattern of recovery was dominant in both micro-invertebrates (springtails and mites) and macro-invertebrates (ants, spiders, rove beetles and earthworms). soil invertebrates usually have high recovery rates following the pesticidal application but repeated use of pesticides may exacerbate the situation of soil biological functioning (desneux et al., 2007; iloba and ekrakene, 2008; evans et al., 2010). however, there was a sudden decline in the diversity and abundance as well for all the treatments including control one. this trend might be due to the increased temperature in the month of june. only mite population exhibited an increasing trend till 60dat, which may be due the fact that mites survive better in hot and dry environment than other invertebrates (behan et al., 2003; al-assiuty et al., 2014). nevertheless, some insecticides such as neonicotinoids have shown a positive effect on the population abundance of mites (pisa et al., 2014). apart from four major faunal groups, i.e. springtails (collembola), mites (cryptostigmata), ants (formicidae) and spiders (araneae), rove (staphylinidae) and ground (carabaiedae) beetles were also encountered in all soil samples. nevertheless, rove beetles showed the minimum impact of pesticides while ground beetles population showed a decreasing trend in all pesticidal treatments. these results are in line with those of huusela-veistola et al. (1996) who demonstrated that ground beetles are very sensitive to agrochemicals than other macro-invertebrates, particularly staphylinidae beetles (honěk et al., 2012). conclusion the principal finding of this study is that synthetic pesticides (insecticides and fungicides) have more adverse effects on soil biological components such as soil invertebrate fauna than pesticides of biological origin. therefore, keeping in view the key role of soil fauna in soil sustainability and crop productivity and the ecological implications of pesticides in citrus agroecosystems, farmers should opt for more eco-friendly plant protection options such as biopesticides. references adamski z, błoszyk j, piosik k, tomczak k. effects of diflubenzuron and mancozeb on soil microarthropods: a long-term study. biol. lett. 46: 3-13, 2009. al-assiuty ani, khalil ma, ismail awa, van straalen nm, ageba mf. effects of fungicides and biofungicides on population density and community structure of soil oribatid mites. sci. total environ. 466: 412-420, 2014. anjum t, javaid a. major diseases of citrus in pakistan: a review. int. j. biol. biotechnol. 2: 793-796, 2005. ashraf s, khan ga, ali s, iftikhar m, mehmood n. managing insect pests & diseases of citrus: on farm analysis from pakistan. pak. j. phytopathol. 26: 301-307, 2014. babendreier d, jeanneret p, pilz c, toepfer s. non‐target effects of insecticides, entomopathogenic fungi and nematodes applied against western corn rootworm larvae in maize. j. appl. entomol. 139: 457-467, 2015. bagyaraj dj, nethravathi cj, nitin ks. soil biodiversity and arthropods: role in soil fertility. in: chakravarthy ak, sridhara s. (eds) economic and ecological significance of arthropods in diversified ecosystems. springer, singapore: pp 17-51, 2016. biondi a, mommaerts v, smagghe g, viñuela e, zappalà l, desneux n. the non‐target impact of spinosyns on beneficial arthropods. pest manage. sci. 68: 1523-1536, 2012. bogya s, markó v. effect of pest management systems on ground-dwelling spider assemblages in an apple orchard in hungary. agric. ecosys. environ. 73: 7-18, 1999. bünemann ek, schwenke gd, van zwieten l. impact of agricultural inputs on soil organisms-a review. soil res. 44: 379-406, 2006. chelinho s, domene x, andrés p, natal-da-luz t, norte c, rufino c, et al. soil microarthropod community testing: a new approach to increase the ecological relevance of effect data for pesticide risk assessment. appl. soil ecol. 83: 200-209, 2014. coleman dc, wall dh. soil fauna: occurrence, biodiversity, and roles in ecosystem function. soil microbiol. ecol. biochem. 111-149, 2015. desneux n, decourtye a, delpuech jm. the sublethal effects of pesticides on beneficial arthropods. ann. rev. entomol. 52: 81-106, 2007. edwards, ca, ed. environmental pollution by pesticides. vol. 3. springer science & business media, 2013. el-naggar jb, zidan neha. field evaluation of imidacloprid and thiamethoxam against sucking insects and their side effects on soil fauna. j. plant prot. res. 53: 375-387, 2013. evans sc, shaw em, rypstra al. exposure to a glyphosate-based herbicide affects agrobiont predatory arthropod behaviour and long-term survival. ecotoxicology. 19: 1249-1257, 2010. everts jw, aukema b, hengeveld r, koeman jh. side-effects of pesticides on ground-dwelling predatory arthropods in arable ecosystems. environ. pollut. 59: 203-225, 1989. frampton gk, van den brink pj. collembola and macroarthropod community responses to carbamate, organophosphate and synthetic pyrethroid insecticides: direct and indirect effects. environ. pollut. 147: 14-25, 2007. francesena n, desneux n, de campos mr, schneider mi. side effects of spirotetramat on pupae and adults of a neotropical strain of eretmocerus mundus (hymenoptera: aphelinidae). effects on the life parameters and demography. environ. sci. pollut. res. 1-12, 2017. gill hk, garg h. pesticides: environmental impacts and management strategies. pesticides-toxic effects. in tech. rijeka, croatia, pp 187-230, 2014. 38 honěk a, kocian m, martinkova z. rove beetles (coleoptera: staphylinidae) in an apple orchard. plant protec. sci. 48: 116-122, 2012. huusela-veistola e. effects of pesticide use and cultivation techniques on ground beetles (col, carabidae) in cereal fields. ann. zool. fenn. 197-205, 1996. jänsch s, frampton gk, römbke j, van den brink pj, scott‐fordsmand jj. effects of pesticides on soil invertebrates in model ecosystem and field studies: a review and comparison with laboratory toxicity data. environ. toxicol. chem. 25: 2490-2501, 2006. kocourek f, saska p, řezáč m. diversity of carabid beetles (coleoptera: carabidae) under three different control strategies against european corn borer in maize. plant protec. sci. 49: 146-153, 2013. lardo e, coll p, le cadre e, palese am, villenave c, xiloyannis c, et al. electromagnetic induction (emi) measurements as a proxy of earthworm presence in southern french vineyards. appl. soil ecol. 61: 76-84, 2012. larson jl, redmond ct, potter da. impacts of a neonicotinoid, neonicotinoid-pyrethroid premix, and anthranilic diamide insecticide on four species of turf-inhabiting beneficial insects. ecotoxicology 23: 252-259, 2014. lee ke, pankhurst ce. soil organisms and sustainable productivity. soil res. 30: 855-892, 1992. majeed mz. nitrous oxide emissions by tropical soil macrofauna: impact of their feeding guilds and functional microbial communities involved in these emissions. [phd thesis.] universite montpellier ii, montpellier, france: pp 235, 2012. monzo c, qureshi ja, stansly pa. insecticide sprays, natural enemy assemblages and predation on asian citrus psyllid, diaphorina citri (hemiptera: psyllidae). bull. entomol. res. 104: 576-585, 2014. moreby sj, sotherton nw, jepson pc. the effects of pesticides on species of non-target heteroptera inhabiting cereal fields in southern england. pestic. sci. 51: 39-48, 1997. pekár s, beneš j. aged pesticide residues are detrimental to agrobiont spiders (araneae). j. appl. entomol. 132: 614-622, 2008. pisa lw, amaral-rogers v, belzunces lp, bonmatin jm, downs ca, goulson d, et al. effects of neonicotinoids and fipronil on nontarget invertebrates. environ. sci. pollut. res. 22: 68-102, 2015. stork ne, eggleton p. invertebrates as determinants and indicators of soil quality. am. j. alternative agr. 7: 38-47, 1992. tahir hm, nazarat i, naseem s, butt a, yaqoob r, mukhtar mk, et al. seasonal dynamics of spiders and insect pests in citrus orchards of district sargodha, pakistan. pak. j. zool. 47: 1673-1681, 2015. williams t, valle j, viñuela e. is the naturally derived insecticide spinosad® compatible with insect natural enemies? biocontrol sci. technol. 13: 459-475, 2003. chimerism a natural ability to tolerate kin, evolutionary traits connecting mammalian and protochordates isj 6: s9-s20, 2009 issn 1824-307x review chimerism a natural ability to tolerate kin, evolutionary traits connecting mammalian and protochordates a voskoboynik institute of stem cell biology and regenerative medicine, department of pathology, stanford university school of medicine, stanford, usa and department of developmental biology, stanford university hopkins marine station, pacific grove, usa accepted march 13, 2009 abstract in the middle of the 20th century, owen (1945, 1954) and billingham et al. (1953) immunological studies suggested that fetal exposure to foreign antigens during pregnancy induce immunologic tolerance in the fetus. recently, mold et al. found that a substantial number of maternal cells crosses the placenta to reside in fetal lymph nodes and induces the development of regulatory t cells (tregs) that suppress fetal anti-maternal immunity. these tregs cells persist till, at least, early adulthood. this result demonstrates how chimerism induces fetal tolerance to maternal antigens during mammalian pregnancy. natural chimerism is the coexistence of two or more genomic lineages within the same individual. it is a common phenomenon which can be detected in a wide variety of multi-cellular organisms. in mammals, natural chimerism can be established during pregnancy between the mother and the fetus or between fetuses in a multiple embryos pregnancy. restriction of natural chimerism mainly to kin is also observed in colonial marine protochordates. in protochordates, like botryllus schlosseri, natural chimerism can be established through fusion of vasculature, between a parent colony and its progeny or between siblings (adult distinct colonies).the ability to tolerate a partial allogeneic individual and to create a chimeric entity between these colonies is determined by a single, highly polymorphic, fusion/histocompatibility locus (fu/hc). colonies that share at least one allele in their fu/hc locus would fuse upon contact. a pair that does not share any fu/hc allele would not. in the chimera, cells transmigrate between partners and in some cases, replace the germline and/or the somatic tissues of the host. this genotype replacement is mediated by stem cells (termed somatic/germ cell parasitism). botryllus colonies propagate asexually through budding, therefore somatic stem cell parasitism in host colonies can induce the development of a partial allogeneic entity (buds) within the host colony. in this way, chimerism in protochordates serves as a state that enables the development of a “virtual embryo” within the host colony. in light of mold et al., study, which demonstrates a role to chimerism in tolerance induction during pregnancy, studying the immunological mediators for natural acceptance of partial allogeneic allograft in protochordates may reveal the evolutionary precursors to the tolerance state during mammalian pregnancy. key words: chimerism; immunologic tolerance; stem cells; tunicate; mammalian pregnancy; fu/hc; uterine nk chimerism in pregnancy an increasing number of studies have detected ___________________________________________________________________________ corresponding author: avelet voskoboynik institute of stem cell biology and regenerative medicine department of pathology stanford university school of medicine stanford, ca 94305, usa department of developmental biology stanford university hopkins marine station pacific grove, ca 93950 usa email: ayeletv@stanford.edu natural chimerism in a wide variety of multi-cellular organisms, including vertebrates. this suggests that chimerism is a common phenomenon in the wild (e.g. buss, 1982; van dijk et al., 1996; bianchi et al., 1996; marleau et al., 2003; pineda-krch and lehtila, 2004; rinkevich 2004a, b; kaplan and land, 2005; khosrotehraniet et al., 2005; loubiere et al., 2006; bianchi, 2007; mold et al., 2008). studies demonstrated that chimerism can be experimentally initiated by engraftment of a single stem cell (e.g. cao et al., 2004; laird et al., 2005). human chimerism which originated from twins was reported already in 1953. with the advent of blood typing, it was discovered that some people have blood cells s9 mailto:ayeletv@stanford.edu of more than one type (dunsford et al., 1953). until 1996, twins chimerism seems to be very rare in humans, only 40 cases of twins chimerism were reported (trippet, 1983). these cases were found during routine blood grouping, a procedure which detects a mixture of red blood cells only when the percentage of allogeneic cell in the blood is above 5 % of all blood cells (van dijk et al., 1996). van dijk et al. (1996) applied a more sensitive method and found that in humans, 8 % of non-identical twins and 21 % of triplets are chimeric (van dijk et al., 1996). twin chimerism with high levels of blood cells from the other twin yields tolerance to donor antigens. all chimeras twins which were identified in a routine blood grouping (frequency >5 % of chimeric cell population) had mutual tolerance to blood transfusion and skin grafts from their chimeric partner (trippet, 1983). chimeras with low frequencies of foreign cell population (like those identified by van dijk et al. (1996), with chimeric cell population frequency of ~0.1 %) were not tolerant to their twin foreign antigens and rejected blood grafts. fetal-maternal micro –chimerism is another natural chimerism that is developed during human pregnancy. cell trafficking, during normal human pregnancy, between the mother and the fetus, leads to the establishment of a chimeric state in both. fetal cells were identified in the maternal circulation (herzenberg, 1979; lo et al., 1989, 1996, 1998; petit et al., 1997; bianchi et al., 1997, 2002). maternal cells were found in the umbilical cord and in fetal blood samples (socie et al., 1994; hall et al., 1995; lo et al., 1996; petit et al., 1995; 1997; bauer et al., 2001). during human pregnancy, 20 %-50 % of the erythroblasts in maternal blood are originated from the fetal (sekizawa et al., 1996; von eggeline et al., 1997; oosterwijk et al., 1998; wachtel et al., 1998; troeger et al., 1999). these percentages indicate relatively high levels of chimerism between mother and fetal during pregnancy in the host mother. persistence of allogeneic fetal progenitor cells were detected in chimeric mothers (humans) as long as 27 years after birth (termed as microchimerism, mc; bianchi et al., 1996). fetal mesenchymal stem cells were detected in maternal bone marrow and in mother’s ribs even 35 years after delivery (1 fetal cell in ~105 maternal cells; o’donoghue et al., 2004a, b). long term persistence of maternal cells in progeny (maternal mc) was described too (maloney et al., 1999). loubiere et al. (2006) found that high percent of healthy adult women harbor maternal and fetal mc among their t and b lymphocytes, monocyte/macrophages and nk cells. 78 % (21/27) of healthy tested women carried fetal mc (tested women’s children average age 12.6 years), 39 % carried maternal chimerism (12/31 average age = 39; loubiere et al., 2006). maternal / fetal mc is not limited to blood cells, it has been identified in many organs including normal kidney, liver and heart of women with or without pregnancy history (chimerism in different organs was detected in 23 out of 75 tested women, ages 29-93 at death; koopmans et al., 2005). since stem cells are the only cells in the tissues with a self-renewing capability, the detection of maternal and fetal mc up to four decades following delivery indicates that healthy humans harbor long-surviving populations of maternal/fetal hematopoietic stem cells. the above studies suggest that during pregnancy fetal/maternal stem cells enter maternal/fetal blood engraft, differentiate in host tissues and remain presence throughout its entire life. tolerance in pregnancy acceptance of a fetus, which expresses paternally inherited alloantigens, by the mother during pregnancy is a unique example of the way immune system reshapes a destructive alloimmune response to a state of tolerance (guleria and sayegh, 2007). intolerance of the embryo, as an antigenetically foreign body growing within the female, can represent a significant obstacle to reproduction. in humans, it is estimated that 70 % of conceptions fail (hill, 1992). similar estimates of early fetal loss, ranging from 10 % to 60 % have been obtained for other mammalian species (baker and bellis, 1995). in human, approximately 45 % of the couples experiencing primary recurrent spontaneous abortion have been linked to immunological factors (clark, 1999). on the other hand, during successful mammalian pregnancy, the semiallogeneic fetus resides comfortably within the maternal uterus and is protected from response of maternal graft rejection. the maternal immune system is active during pregnancy, yet mothers tolerate and do not reject their genetically disparate fetuses (hunt et al., 2005; lightner et al., 2008). there is growing evidence that viviparous reproduction depends on a two-way interactions between fetal and maternal tissues that involve humoral, cellular, innate and adaptive immune responses (hill, 1995; guleria and sayegh 2007; mold et al., 2008). these include, expression of non-classical mhc molecules by trophoblast cells (king et al., 2000; ishitani et al., 2003; hunt et al., 2005; lightner et al., 2008), tryptophan catabolism by the enzyme ido (munn et al., 1998), t cell apoptosis (hunt et al., 1997), the complement system, regulatory t cells (tregs) and inhibitory costimulatory molecule programmed death ligand (pdl) 1 (guleria et al., 2005; lightner et al., 2008; mold et al., 2008). it was also suggested that dendritic cells have a potential role in reproductive immunology and chemokine decoy receptors (borroni et al., 2008; kammerer et al., 2008). the classic, highly polymorphic mhc loci has a minimal expression, immediately following implantation, but it is increased steadily in placental and extravillous cytotrophoblast tissues during pregnancy (kurpisz et al., 1995). restriction of polymorphic mhc gene expression to later stages of embryonic development is probably useful to limit maternal rejection of the fetus. the contribution of each mechanism and the potential interactions among the various pathways are just beginning to emerge (guleria and sayegh, 2007). liegeois (1983) hypothesized that chimerism has a role in tolerance induction and maintenance during placental pregnancy. this hypothesis, although never been tested directly, is consistent with historical and recent findings which connect s10 induction of donor specific tolerance in fetal with cell chimerism during pregnancy. in 1945, owen found that bovine fraternal twins shared for life the blood cells types of both calves. based on earlier observations, indicating that bovine twins share a single placenta and blood circulation during fetal phase (lillie, 1916), owen conjectured that blood cells and their precursors (which he called “embryonal cells ancestral to the erythrocytes”) move from one twin to the other in the uterus, allowing mixing of blood cells from both genotypes (owen, 1945). bovine fraternal twins frequently exhibit a complete lifelong mutual tolerance to each other’s leukocyte and a temporary (up to 15 months) tolerance to each other’s skin grafts (anderson et al., 1951; billingham et al., 1952; stone et al., 1965, 1971; emery and mccullagh 1980a, b). in 1954, owen et al. (1954), further detected another remarkable acquired tolerance phenomenon, connected to embryonic exposure. they reported that rhesus d negative pregnant mothers are less likely to produce antibodies against rhesus d positive child, in cases where the grandmother is rhesus d positive. this observation led to the discovery that a high percent of individuals tolerate non-inherited maternal hla antigens (nima), better than non-inherited paternal hla antigens nipa (claas et al., 1988, 1989). then, it was hypothesized that exposure of a child to nima during pregnancy may lead to nimaspecific tolerance later in life. chimerism has probably an important role in this acquired tolerance effect (owen et al., 1954; andrassy et al., 2003; van den boogaardt et al., 2006). billingham et al. (1953) were the first to induce specific tolerance to solid organs by exposing fetal and neonatal mice to the donor hematopoietic cell infusions. engrafted mice did not reject allogeneic material, accepting skin from mice of the leukocyte donor strain, but not from any other strain (6-8 weeks after birth; billingham et al., 1953; billingham and brent, 1956). tolerance to skin allograft was permanent or transient, depending on the mouse strain (billingham and brent, 1956). this acquired tolerance to donor tissues was associated with leukocyte chimerism, which was detected in the animals’ lymphoid organs (billingham et al., 1953; billingham and brent, 1956). since then, there have been numerous reports suggesting that transfer of foreign antigens from the mother to the fetus is common (review in adams and nelson, 2004). recently, mold et al. (2008) found that substantial numbers of maternal cells cross the placenta to reside in fetal lymph nodes, inducing the development of regulatory t cells (tregs) that suppress fetal anti-maternal immunity and persist at least until early adulthood (mold et al., 2008). the above study reveals a form of antigen-specific tolerance in humans induced in utero via chimerism and highlights the potential important role of cell chimerism for induction of fetal tolerance to maternal tissues. colonial protochordates may offer a unique evolutionary perspective on the maternal-fetal relationship during pregnancy. in these organisms, a genetically controlled allorecognition system, similar to the natural killer missing self model system in vertebrate, enables the creation of chimeric state (through vasculature fusion) with kin and prevents chimerism with non-related individuals (oka and watanabe, 1957; sabbadin, 1962; grosberg, 1988; buss, 1982, 1990; scofield et al., 1982). protochordates, are considered as the closest invertebrate relative of vertebrate (delsuc, 2006). studying the immunobiology of the tolerance to partial allogeneic allograft in these organisms may reveal the evolutionary precursors to the maternalfetal relationship during pregnancy. genetic basic for allograft acceptance or rejection in protochordate in colonial protochordate, like the botryllus schlosseri, homeostasis is defined by generation of all organ systems every week. colonies are initially formed by asexual reproduction of founder individuals, a product of sexual reproduction (review in manni and burighel, 2006; manni et al., 2007). the progeny clone members are united under a single gelatinous tunic by a network of anastomosed extracorporeal blood vessels. throughout adult life, the b. schlosseri generates its entire body every week. this cycle of development, includes the formation of all body organs including heart, respiration system, digestive system, and neural complex. ovary and testis are formed within each individual when sexual reproduction commences (burighel and cloney, 1997; manni and burighel, 2006; manni et al., 2007). in addition to their extraordinarily high developmental activity, allogeneic botryllus colonies can fuse and create a chimera. when co-specific botryllid colonies contact each other, several morphological and cellular allogeneic processes and reactions are developed, including: fusion, rejection, indifference (no reaction) and a temporary fusion followed by disconnection (see fig 1 for detailed fusion process; bancroft 1903; oka and watanabe 1957, 1960, 1967; sabbadin 1962, 1982; mukai 1967; tanaka and watanabe 1973; scofield et al., 1982; saito and watanabe 1982, 1984; scofield et al., 1983; hirose et al., 1988; 1990; boyd et al., 1990; sabbadin et al., 1992; rinkevich and weissman 1992a, b; rinkevich et al., 1994a, b, 1998; saito et al., 1994; chadwik-furman and weissman 1995; ballarin et al., 1995, 1998, 2002; cima et al., 2004, 2006; rinkevich, 2005). fusion or non fusion response between adjacent colonies is determined by a single, highly polymorphic, fusibility/histocompatibility (fu/hc) locus (oka and watanabe, 1957; sabbadin, 1962; scofield et al., 1982) which was isolated and characterized (de tomaso et al., 2005). colonies that share at least one allele in their fu/hc locus would fuse upon contact, and would become a chimera, while those that don’t share any allele would not (oka and watanabe, 1957). burnet (1971) suggested studying botryllus as a model for the evolution of self-recognition. he wrote, “although self recognition in ascidians is not analogous to the immunological processes of vertebrates, it presents a primitive type of ’self and not self‘ recognition from which adaptive immunity may have evolved” (burnet, s11 fig. 1 fusion process between kin. using time laps microscopy (imagexpress as described in voskoboynik et al., 2008), we followed and documented the fusion process between 6 different pairs of compatible b. schlosseri colonies. these observations revealed a more elaborate fusion process than the one described before. a) a mother colony and its embryo. b) larva seeks for a settlement site near its mother colony. c) initial contact is established between the tunics and ampullae of the mother colony and its offspring colony. d) following partial fusion of the tunics the offspring ampullae 1 and 2 penetrate into the tunic of the mother colony. e) the offsprings’ penetrating ampullae change the shape of their tips into a cone like shape and through dynamic cycles of extension and retraction further penetrate and explore the tunic of the mother colony. f) 64 hours later, these 2 navigating ampullae fuse with the marginal blood vessel of the mother colony. g) following partial fusion of the tunics, colony a ampulla number 3 penetrates into the tunic of its sibling colony b. h) penetrating ampulla, of colony a changes its tip shape into a cone like shape and through dynamic cycles of extension and retraction further penetrates and explores the tunic of its sibling colony. i) 16 hours later, ampulla 3 fuses with the marginal blood vessel of its sibling colony b. j) only a navigator ampulla fuses and creates a common blood vessel. once created, the number of common blood vessels in the chimera remains constant throughout the chimera life. k-l) 360 hours following ampullae penetration, colony b got resorbed by its sibling colony a. amp, ampulla; h, hours following ampullae contact; bv, blood vessel; mbv, marginal blood vessel; ampullae and common blood vessels are outlined by a dotted line. bar = 75 µm. s12 1971). an extensive study of the botryllid ascidians self-nonself recognition system in the last 25 years revealed that, while the colonial ascidian fu/hc and the mammalian major histocompatibility complex (mhc) share phenomenological features, including polymorphism and specificity, their molecular structure is different. similar to the mhc, the fu/hc is highly polymorphic (karakashian and milkman 1967; scofield et al., 1982; 1983; grosberg and quinn, 1986; grosberg 1987; rinkevich et al., 1995; paz et al., 2003; de tomaso et al., 2005; benshlomo et al., 2001, 2006, 2008). however, structural homologies were not found (weissman et al., 1990; rinkevich and weissman, 1992b; fagan and weissman, 1997; pancer et al., 1993, 1996a, b, c, 1997; muller et al., 1994; khalturin et al., 2003; de tomaso et al., 2005; nyholm et al., 2006). the botryllus fu/hc is not homologous to any molecules of the vertebrate mhc-based histocompatibility system. moreover, the whole botryllus fu/hc locus does not have a syntenic region in the ciona genome or in the genomes of vertebrates (de tomaso et al., 2005). genes involved in adaptive immunity, which include the polymorphic mhc class i and ii glycoproteins that present internal peptides to t cells, the clonally expressed t-cell receptors (tcrs), immunoglobulins (igs) and the recombination activating genes (rag1, rag2), have not been identified in protochordates (klein 1989, laird et al., 2000; dehal et al., 2002; kaufman, 2002; azumi et al., 2003; khalturin et al., 2003; de tomaso et al., 2005; litman et al., 2005, 2007). in both mice and humans, the mediators of the adaptive immune system are thought to be responsible for the rejection of a transplant. the adaptive immune t cell system rejects grafts even if one of two alleles is shared, as t cells make an immune reaction against non-self allele gene products. in contrast, allogeneic botryllus colonies tolerate each other and create a chimera even if only one of the two fu/hc alleles is shared. this is consistent with immune systems, like the nk system in vertebrates, wherein recognition of self prevents an immune reaction. recently, nk cells have been recognized as active participants in the acute and chronic rejection of solid tissue grafts (reviewed in kitchen et al., 2005). the importance of nk cell attributes has been first recognized in bone marrow transplantation, where nk cells are fully sufficient to reject hematopoietic cell transplants, even in the absence of t or b cell responses (lethally-irradiated or lack mhc class i expression recipients; hoglund et al., 1991; bix et al., 1991; manilay and sykes, 1998). recent studies suggest that nk contribute to organ rejection indirectly, by activating or helping effector cells, such as cytotoxic and helper t cells (reviewed in kitchen et al., 2005). studies also point to a potential involvement of nk in the induction of tolerance to solid graft in mix chimerism, a tolerance regimen that employs the generation of mixed hematopoietic chimerism through donor stem cell engraftment (zhao et al., 2003). another aspect that might connect mammalian nk and the botryllus fu/hc was recently raised by lighter et al. (2008). studying uterine nk, lighter et al., pointed to a unique phenotype that uterine nk and the botryllus fu/hc might share. uterine nk cells produce angiogenic growth factors and are potential regulators of decidual angiogenesis in early pregnancy (ashkar and croy, 2001; hanna et al., 2006; manaster and mandelboim, 2008). they suggested that, as botryllus fu/hc is probably involved in the generation of a common vascular system between two individuals, uterine nk may share the same evolutionary roots as the botryllus fu/hc. cell parasitism induces development of a foreign entity within host colony inspired by the genetic control for allograft acceptance and creation of chimerism within kin in botryllus, burnet (1971) hypothesized the emergence of intraspecific parasitism along the evolution. indeed, his hypothesis was later confirmed in botryllus chimeras, where the replacement of host germline by a donor genotype was demonstrated (sabbadin and zaniolo, 1979). in a remarkable set of experiments sabbadin and zaniolo (1979) demonstrated this kind of parasitism in b. schlosseri. years later pancer et al. (1995), stoner and weissman (1996) and stoner et al. (1999) confirmed these results and further showed that in a chimera, the blood, soma and germ cells, demonstrated the combine genotypes of both chimeric partners. moreover, in many cases the circulating pluripotent cells of one partner parasitized either the soma or the germ line of the other partner and replaced the whole mass of gonads or the soma (bud/zooid) of several individuals in the host colony (termed gonads or somatic cell parasitism; g/scp). in a few cases, a complete takeover of donor genotype occurred and the whole mass of gonads in the chimeric colony expressed solely the donor’s genotype (sabbadin and zaniolo 1979; pancer et al., 1995; stoner and weissman 1996; stoner et al., 1999). under invariant environmental conditions, both germline and somatic cell parasitism followed repeatable hierarchies of “winner strains” and “loser strains” (stoner et al., 1999). however, breeding experiments proved that only the hierarchical position of germ cell parasitism is sexually inherited (stoner et al., 1999). the hierarchy of somatic parasitism in botryllus chimeras is a plastic trait, as variations in the environmental conditions (such as seawater temperature) can be reversed; the winner – loser hierarchy at the somatic parasitism level (rinkevich and yankelevich, 2004). botryllus colonies propagate a-sexually through budding, therefore somatic stem cell parasitism in host colonies can induce the development of partial allogeneic entities (buds) within the host colony. as a result, chimerism in protochordate could serve as a state that enables the development of a “virtual embryo” within the host colony (voskoboynik et al., in press; fig. 2). beside cell parasitism chimerism may alter tolerance and intolerance state in the colonies. chimeras might fuse with colonies that they used to reject (on their non chimeric phase) or reject colonies that they used to fuse with (mukai 1967; sabbadin and astorri, 1988). moreover, in some cases, chimeric colonies will simultaneously fuse s13 fig. 2 virtual pregnancy: a development of a semi allogeneic entity in a host colony through asexual budding in a chimera of a mother colony and its offspring. botryllus colonies propagate asexually through budding, therefore, somatic stem cell parasitism in host colonies can induce the development of a partial allogeneic entities (buds) within the host colony. in this illustration, an offspring colony (red) is fused with its mother colony (blue). genetic analysis of the chimera’s buds and gonads can reveal several cell chimerism patterns. either both genotypes are detected or only one genotype is detected. bv, blood vessel; mbv, marginal blood vessel; red, offspring genotype; blue, mother genotype. and reject another colony (taneda, 1985; sabbadin and astorri, 1988). the presence, of a simultaneously fusion and rejection with a genotype that one of the chimera partner used to fuse with and the other partner used to reject, prove the persistence of both genotypes and suggest an uneven distribution of each genotype within the chimera. different fusibility patterns that the chimeric entity presents on different time points suggests genomes fluctuation and competitive interaction of the different genomes within the chimera (sabbadin and astorri, 1988). sabbadin and astorri observed changes in the tolerance state of the chimeric colonies and linked it to changes in the dynamic of the chimeric cells within the chimera. genetic analysis of the dynamic of chimeric cell revealed that chimeras exhibit either a sectorial pattern in which both genotypes are detected within some systems but not others, or a uniform pattern in which tissues throughout the entire chimera exhibit both genotypes (stoner and weissman, 1966). colonies which showed rejection and fusion on the same time probably expressed sectorial pattern and the others expressed uniform pattern. the dynamic of chimeric cells within the host is changing with time, as different patterns are observed during different time points (pancer et al., 1995; stoner and weissman, 1966; stoner et al., 1999). these studies show that the genetically controlled ability of botryllus colonies to tolerate or reject other colonies can be altered by chimerism. the temporal and spatial dynamic of the chimeric cells, patterns of host/donor cells competition, niche occupation and immunoregulatory mechanisms for routing, timing and frequencies of chimeric cells have probably important role in the induction of tolerance or intolerance. stem cells mediated chimerism the long-term ability of cells from one genotype to replace the germline and somatic cells of the host, led to hypothesize that cell parasitism in the chimeras is mediated by stem cells (sabbadin and zaniolo, 1979; rinkevich and weissman, 1987; pancer et al., 1995; stoner and weissman 1996; stoner et al., 1999). by transplanting a single cell, which expresses high enzymatic activity of aldehyde dehydrogenase and a set of serial engraftment assays, laird et al. (2005) revealed that multipotent stem cells are responsible for a stable long-term chimerism in adult b. schlosseri colonies. yet, the location of these cells remained unknown (laird et al., 2005). recently, by using cell engraftments, chimeric fusion techniques, and in vivo cell tracking by automated time lapse fluorescence microscopy, s14 we (voskoboynik et al., 2008) have demonstrated that the anterior ventral region of the endostyle (termed endostyle niche) harbors adult stem cells and exports them to developing and regenerating organs, wherein they participate in tissue formation. as few as 5-20 engrafted cells transplanted from the donor endostyle niche sufficed to generate a somatic chimerism in compatible hosts; however, no germline chimerism was demonstrated. the induction of somatic chimerism demonstrates a remarkable stemness capacity of the cells in the endostyle niche (voskoboynik et al., 2008). the endostyle produces thyroid hormones and serotonin and expresses several variety of factors that are involved in development and stem cell regulation like wnt, hox1, pax 2/5/8, pl10, cadherin, raldh and pcna (canestro et al., 2008; dunn, 1980; hiruta et al., 2005; nilsson et al., 1988; pennati et al., 2001; rosner et al., 2006; rosner et al., 2007; voskoboynik et al., 2008). the endostyle niche is the first somatic stem cell niche ever described in a protochordate and is one of the most accessible stem cell niches for in vivo studies. the discovery of a major stem cell niche in an organism with fu/hc controlled chimerism, pluripotent stem cell parasitism, and tolerance or intolerance induction via chimerism promotes the botryllus as an evolutionary model for studying molecular regulations of tolerance induction by cellular chimerism in the absence of an adaptive immune system. acknowledgements i thank a voskoboynik for critical review, il weissman and b rinkevich for discussion, p brown, r marinelli, r pesich l jerabek, kj ishizuka, and kj palmeri for support and helpful assistance. references adams km, nelson jl. microchimerism: an investigative frontier in autoimmunity and transplantation. jama 291: 1127-1131, 2004. anderson d, billingham re, lampkin gh, medawar pb. the use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. heredity 5: 379-397, 1951. andrassy j, kusaka s, jankowska-gan e, torrealba jr, haynes ld, marthaler br, et al. tolerance to noninherited maternal mhc antigens in mice. j. immunol. 171: 5554-5561, 2003. ashkar aa, croy ba. functions of uterine natural killer cells are mediated by interferon gamma production during murine pregnancy. semin. immunol. 13: 235-241, 2001. azumi k, takahashi h, miki y, fujie m, usami t, ishikawa h, et al. construction of a cdna microarray derived from the ascidian ciona intestinalis. zool. sci. 20: 1223-1229, 2003. baker rr, bellis ma. human sperm competition: copulation, masturbation and infidelity. chapman & hall, london, 1995. ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri. dev. comp. immunol. 22: 479-492, 1998. ballarin l, cima f, floreani m, sabbadin a. oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian botryllus schlosseri. comp. biochem. physiol. 133c: 411-418, 2002. bancroft fw. variation and fusion of colonies in compound ascidians. proc. calif. acad. sci. 3: 137-186, 1903. bauer m, orescovic i, schoell wm, bianchi dw, pertl b. detection of maternal dna in umbilical cord plasma by fluorescent pcr amplification of short tandem repeat sequences. ann. n.y. acad. sci. 945: 161-163, 2001. ben-shlomo r, douek j, rinkevich b. heterozygote deficiency and chimerism in remote populations of a colonial ascidian from new zealand. mar. ecol. prog. ser. 209: 109-117, 2001. ben-shlomo r, paz g, rinkevich b. postglacialperiod and recent invasions shape the population genetics of botryllid ascidians along european atlantic coasts. ecosystems 9: 11181127, 2006. ben-shlomo r, motro u, paz g, rinkevich b. pattern of settlement and natural chimerism in the colonial urochordate botryllus schlosseri. genetica 132: 51-58, 2008. bianchi dw, zickwolf gk, weil gj, sylvester s, demaria ma. male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. proc. natl. acad. sci. usa 93: 705-708, 1996. bianchi dw, williams jm, sullivan lm, hanson fw, klinger kw, shuber ap. pcr quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. am. j. hum. genet. 61: 822-829, 1997. bianchi dw, simpson jl, jackson lg, elias s, holzgreve w, evans mi, et al. fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of nifty i data. national institute of child health and development fetal cell isolation study. prenat. diagn. 22: 609-615, 2002. bianchi dw. fetomaternal cell trafficking: a story that begins with prenatal diagnosis and may end with stem cell therapy. j. pediatr. surg. 42:12-18, 2007. billingham re, lampkin gh, medawar pb, williams hl. tolerance to homografts, twin diagnosis and the freemartin condition in cattle. heredity 6: 200-212, 1952. billingham re, brent l, medawar pb. actively acquired tolerance of foreign cells. nature 172: 603-606, 1953. billingham re, brent l. acquired tolerance of foreign cells in newborn animals. proc. r. soc. lond. b biol. sci. 146: 78-90, 1956. bix m, liao ns, zijlstra m, loring j, jaenisch r, raulet d. rejection of class i mhc-deficient haemopoietic cells by irradiated mhc-matched mice. nature 349: 329-331, 1991. borroni em, bonecchi r, baracchi c, savino b, mantovani a, locati m. chemokine decoy s15 receptors: new players in reproductive immunology. immunol. invest. 37: 483-497, 2008. boyd hc, weissman il, saito y. morphologic and genetic verification that monterey botryllus and woods hole botryllus are the same species. biol. bull. 178: 239-250, 1990. burighel p, cloney ra. urochordata: ascidiacea. in: harrison fw, ruppert ee (eds), microscopic anatomy of invertebrates, wiley-liss, inc., ny, pp. 221-347, 1997. burnet fm. "self-recognition" in colonial marine forms and flowering plants in relation to the evolution of immunity. nature 232: 230-235, 1971. burnet fm. multiple polymorphism in relation to histocompatibility antigens. nature 245: 359361, 1973. buss lw. somatic cell parasitism and the evolution of somatic tissue compatibility. proc. natl. acad. sc.i usa 79: 5337-5341, 1982. buss lw, grosberg rk. morphogenetic basis for phenotypic differences in hydroid competitive behavior. nature 343: 63-66, 1990. canestro c, bassham s, postlethwait jh. evolution of the thyroid: anterior-posterior reorganization of the oikopleura endostyle revealed by otx, pax 2/5/8, and hox1 expression. dev dyn doi:10.1002/dvdy.21525, 2008 cao ya, wagers aj, beilhack a, dusich j, bachmann mh, negrin rs, et al. shifting foci of hematopoiesis during reconstitution from single stem cells. proc. natl. acad. sci. usa 101: 221-226, 2004. chadwick-furman ne, weissman il. life history plasticity in chimaeras of the colonial ascidian botryllus schlosseri. philos. trans. r. soc. lond. b biol. sci. 262: 157-162, 1995. cima f, sabbadin a, ballarin l. cellular aspects of allorecognition in the compound ascidian botryllus schlosseri. dev. comp. immunol. 28: 881-889, 2004. cima f, sabbadin a, zaniolo g, ballarin l (2006) colony specificity and chemotaxis in the compound ascidian botryllus schlosseri. comp. biochem. physiol. 145a: 376-382, 2006. claas fh, gijbels y, van der velden-de munck j, van rood jj. induction of b cell unresponsiveness to noninherited maternal hla antigens during fetal life. science 241: 1815-1817, 1988. claas fh, gijbels y, von veen a, de waal lp, d'amaro j, persijn gg, et al. selection of cross-match negative hla-a and/or -b mismatched donors for highly sensitized patients. transplant. proc. 21: 665-666, 1989. clark da. signaling at the fetomaternal interface. am. j. repros. immunol. 41: 169-173, 1999. de tomaso aw, nyholm sv, palmeri kj, ishizuka kj, ludington wb, mitchel k, et al. isolation and characterization of a protochordate histocompatibility locus. nature 438: 454-459, 2005. dehal p, satou y, campbell rk, chapman j, degnan b, de tomaso a, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157-2167, 2002. delsuc f, brinkmann h, chourrout d, philippe h. tunicates and not cephalochordates are the closest living relatives of vertebrate. nature 439: 965-968, 2006. dunn ad. properties of an iodinating enzyme in the ascidian endostyle. gen. comp. endocrinol. 40: 484-493, 1980. dunsford i, bowley cc, hutchison am, thompson js, sanger r, race rr. a human blood-group chimera. br. med. j. 2: 80-81, 1953. emery d, mccullagh p. immunological reactivity between chimeric cattle twins. i. homograft reaction. transplantation 29: 4-9, 1980a. emery d, mccullagh p. immunological reactivity between chimeric cattle twins. ii. normal lymphocyte transfer. transplantation 29: 10-16, 1980b. evans pc, lambert n, maloney s, furst de, moore jm, nelson jl. long-term fetal microchimerism in peripheral blood mononuclear cell subsets in healthy women and women with scleroderma. blood 93: 2033-2037, 1999. fagan mb, weissman il. hsp70 genes and historecognition in botryllus schlosseri: implications for mhc evolution. hereditas 127: 25-35, 1997. gaillard mc, ouvre e, liegeois a, lewin d. the concentration of fetal cells in maternal haematopoietic organs during pregnancy. an experimental study in mice (author's transl). j. gynecol. obstet. biol. reprod. (paris) 7: 10431050, 1978. grosberg rk. limited dispersal and proximity dependent mating success in botryllus schlosseri. evolution 41: 372-384, 1987. grosberg rk. the evolution of allorecognition specificity in colonial invertebrates. quart. rev. biol. 63: 377-412, 1988. grosberg rk, quinn jf. the genetic control and consequences of kin recognition by the larvae of a colonial marine invertebrate. nature 322: 456-459, 1986. guleria i, sayegh mh. maternal acceptance of the fetus: true human tolerance. j. immunol. 178: 3345-3351, 2007. hall jm, lingenfelter p, adams sl, lasser d, hansen ja, bean ma. detection of maternal cells in human umbilical cord blood using fluorescence in situ hybridization. blood 86: 2829-2832, 1995. hamada h, arinami t, kubo t, hamaguchi h, iwasaki h. fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age. hum. genet. 91: 427-432, 1993. hanna j, goldman-wohl d, hamani y, avraham i, greenfield c, natanson-yaron s, et al. decidual nk cells regulate key developmental processes at human fetal-maternal interface. nat. med. 12: 1065-1074, 2006. herzenberg la, bianchi dw, schroder j, cann hm, iverson gm. fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. proc. natl. acad. sci. usa 76: 1453-1455, 1979. s16 hill ja. immunological contribution to recurrent pregnancy loss. baillieres clin. obster. gynaecol. 6: 489-438, 1992. hirose e, saito y, watanabe h. anew type of the manifestation of the colony specificity in the compound ascidian, botrylloides violaceus oka. biol. bull. 175: 240-245, 1988. hirose e, saito y, watanabe h. allogeneic rejection induced by the cut surface contact in the compound ascidian, botrylloides simodensis. invertebr. dev. reprod. 17: 159-164, 1990. hiruta j, mazet f, yasui k, zhang p, ogasawara m. comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates. dev. dyn. 233: 1031-1037, 2005. höglund p, ohlén c, carbone e, franksson l, ljunggren hg, latour a, et al. recognition of beta-2microglobulin-negative (beta 2m-) t-cell blasts by natural killer cells from normal but not from beta 2m-mice: nonresponsiveness controlled by beta 2m-bone marrow ib chimeric mice. proc. natl. acad. sci. usa 88: 1033210336, 1991. hunt js, petroff mg, mcintire rh ober c. hla-g and immune tolerance in pregnancy. faseb j. 19: 682-693, 2005. ishitani a, sageshima n, lee n, dorofeeva n, hatake k, marquardt h, et al. protein expression and peptide binding suggest unique and interacting functional roles for hla-e, f, and g in maternalplacental immune recognition. j. immunol. 171: 1376-1384, 2003. kammerer u, kruse a, barrientos g, arck pc, blois sm. role of dendritic cells in the regulation of maternal immune responses to the fetus during mammalian gestation. immunol. invest. 37: 499533, 2008. kaplan j, land s. influence of maternal-fetal histocompatibility and mhc zygosity on maternal microchimerism. j. immunol. 174: 7123-7128, 2005. karakashian s, milkman r. colony fusion compatibility types in botryllus schlosseri. biol. bull. 133: 473-?, 1967. kaufman j. the origins of the adaptive immune system: whatever next? nat. immunol. 3: 11241125, 2002. khalturin k, becker m, rinkevich b, bosch tc. urochordates and the origin of natural killer cells: identification of a cd94/nkr-p1-related receptor in blood cells of botryllus. proc. natl. acad. sci. usa 100: 622-627, 2003. khosrotehrani k, johnson kl, guegan s, stroh h, bianchi dw. natural history of fetal cell microchimerism during and following murine pregnancy. j. reprod. immunol. 66: 1-12, 2005. king a, allan ds, bowen m, powis sj, joseph s, verma s, et al. hla-e is expressed on trophoblast and interacts with cd94/nkg2 receptors on decidual nk cells. eur. j. immunol. 30: 1623-1631, 2000. kitchens wh, uehara s, chase cm, colvin rb, russell ps, madsen jc. the changing role of natural killer cells in solid organ rejection and tolerance. transplantation 81: 811-817, 2006. klein j. are invertebrates capable of anticipatory immune responses?. scand. j. immunol. 29: 499-505, 1989. koopmans m, kremer hovinga ic, baelde hj, fernandes rj, de heer e, bruijn ja, et al. chimerism in kidneys, livers and hearts of normal women: implications for transplantation studies. am. j. transplant. 5: 1495-1502, 2005. kurpisz m, fernandez n, fiszer d. the expression of mhc genes in reproductive organs and their role in reproduction. in: kurspiz m, fernandez n (eds), immunology of human reproduction, bios scientific publishers, oxford, pp163-184, 1995. laird dj, de tomaso aw, cooper md, weissman il. 50 million years of chordate evolution: seeking the origins of adaptive immunity. proc. natl. acad. sci. usa 97: 6924-6926, 2000. laird dj, de tomaso aw, weissman il. stem cells are units of natural selection in a colonial ascidian. cell 123: 1351-1360, 2005. liegeois a. in: edelman p, sureau c (eds) immunologie de le reproduction humanie, edition boz., paris, pp 99-100, 1983. lightner a, schust dj, chen yba, barrier bf. the fetal allograft revisited: does the study of an ancient invertebrate species shed light on the role of natural killer cells at the maternal-fetal interface? clin. dev. immunol. 2008;2008:631920. lillie fr. the theory of the free-martin. science 43: 611-613, 1916. litman gw, cannon jp, dishaw lj. reconstructing immune phylogeny: new perspectives. nat. rev. immunol. 5: 866-879, 2005. litman gw, dishaw lj, cannon jp, haire rn, rast jp. alternative mechanisms of immune receptor diversity. curr. opin. immunol. 19: 526-534, 2007. lo es, lo ym, hjelm nm, thilaganathan b. transfer of nucleated maternal cells into fetal circulation during the second trimester of pregnancy. br. j. haematol. 100: 605-606, 1998. lo ym, patel p, wainscoat js, sampietro m, gillmer md, fleming ka. prenatal sex determination by dna amplification from maternal peripheral blood. lancet 2: 13631365, 1989. lo ym, lo es, watson n, noakes l, sargent il, thilaganathan b, et al. two-way cell traffic between mother and fetus: biologic and clinical implications. blood 88: 4390-4395, 1996. loubiere ls, lambert nc, flinn lj, erickson td, yan z, guthrie ka, et al. maternal microchimerism in healthy adults in lymphocytes, monocyte/macrophages and nk cells. lab. invest. 86: 1185-1192, 2006. maloney s, smith a, furst de, myerson d, rupert k, evans pc, et al. microchimerism of maternal origin persists into adult life. j. clin. invest. 104: 41-47, 1999. manastar i, mandelboim o. the unique properties of human nk cells in the uterine mucosa. placenta 22: s60-s66, 2008. s17 manilay jo, sykes m. natural killer cells and their role in graft rejection curr. opin. immunol. 10: 532-538, 1998. manni l, burighel p. common and divergent pathways in alternative developmental processes of ascians. bioessays 28: 902-912, 2006. manni l, zaniolo g, cima f, burighel p, ballarin l. botryllus schlosseri: a model ascidian for the study of asexual reproduction. dev. dyn. 236: 335-352, 2007. marleau am, greenwood jd, wei q, singh b, croy ba. chimerism of murine fetal bone marrow by maternal cells occurs in late gestation and persists into adulthood. lab. invest. 83: 673681, 2003. mold je, michaelsson j, burt td, muench mo, beckerman kp, busch mp, et al. maternal alloantigens promote the development of tolerogenic fetal regulatory t cells in utero. science 322: 1562-1565, 2008. mukai h. experimental alteration of fusibility in compound ascidians. sci. rep. tokyo kyoiku daigaku 13b: 51-73, 1987. mukai h, watanabe h. distribution of fusion incompatibility types in natural populations of the compound ascidian botryllus primigenus. proc. jpn. acad. 51: 44-47, 1975. muller we, pancer z, rinkevich b. molecular cloning and localization of a novel serine protease from the colonial tunicate botryllus schlosseri. mol. mar. biol. biotechnol. 3: 70-77, 1994. munn dh, zhou m, attwood jt, bondarev i, conway sj, marshall b, et al. prevention of allogeneic fetal rejection by tryptophan catabolism. science 281: 1191-1193, 1998. nilsson o, fredriksson g, ofverholm t, ericson le. electron-microscopic immunocytochemistry of 5-hydroxytryptamine in the ascidian endostyle. cell tissue res. 253: 137-143, 1988. nyholm sv, passegue e, ludington wb, voskoboynik a, mitchel k, weissman il, et al. fester, a candidate allorecognition receptor from a primitive chordate. immunity 25: 163173, 2006. o'donoghue k, chan j, de la fuente j, kennea n, sandison a, anderson jr, et al. microchimerism in female bone marrow and bone decades after fetal mesenchymal stemcell trafficking in pregnancy. lancet 364: 179182, 2004a. o'donoghue k, fisk nm. fetal stem cells. best. pract. res. clin. obstet. gynaecol. 18: 853875, 2004b. oka h, watanabe h. colony specificity in compound ascidians as tested by fusion experiments (a preliminary report). proc. jpn. acad. 33: 657-659, 1957. oka h, watanabe h. problems of colony specificity in compound ascidians. bull. mar. biol. stat. asamushi. 10: 153-155, 1960. oka h, watanabe h. problems of colony specificity, with special reference to the fusibility of ascidians. kagaku 37: 307-313, 1967. oosterwijk jc, mesker we, ouwerkerk-van velzen mc, knepfle cf, wiesmeijer kc, beverstock gc, et al. fetal cell detection in maternal blood: a study in 236 samples using erythroblast morphology, dab and hbf staining, and fish analysis. cytometry 32: 178-185, 1998. owen rd. immunogenetic consequences of vascular anastomoses between bovin twins. science 102: 400-401, 1945. owen rd, wood hr, foord ag, sturgeon p, baldwin lg. evidence for actively tolerance to rh antigens. proc. natl. acad. sci. usa 40: 420-424, 1954. pancer z, gershon h, rinkevich b. cdna cloning of a putative protochordate fk506-binding protein. biochem. biophys. res. commun. 197: 973977, 1993. pancer z, gershon h, rinkevich b. coexistence and possible parasitism of somatic and germ cell lines in chimeras of the colonial urochordate botryllus schlosseri. biol. bull. 189: 106-112, 1995. pancer z, cooper el, muller we. a tunicate (botryllus schlosseri) cdna reveals similarity to vertebrate antigen receptors. immunogenetics 45: 69-72, 1996a. pancer z, leuck j, rinkevich b, steffen r, muller i, muller we. molecular cloning and sequence analysis of two cdnas coding for putative anionic trypsinogens from the colonial urochordate botryllus schlosseri (ascidiacea). mol. mar. biol. biotechnol. 5: 326-333, 1996b. pancer z, scheffer u, muller i, muller we. cloning of sponge (geodia cydonium) and tunicate (botryllus schlosseri) proteasome subunit epsilon (prce): implications about the vertebrate mhc-encoded homologue lmp7 (prcc). biochem. biophys. res. commun. 228: 406-410, 1996c. pancer z, diehl-seifert b, rinkevich b, muller we. a novel tunicate (botryllus schlosseri) putative c-type lectin features an immunoglobulin domain. dna cell biol. 16: 801-806, 1997. paz g, douek j, mo cq, goren m, rinkevich b. genetic structure of botryllus schlosseri (tunicata) populations from the mediterranean coast of israel. mar. ecol. prog. ser. 250: 163174, 2003. pennati r, groppelli s, sotgia c, candiani s, pestarino m, de bernardi f. serotonin localization in phallusia mammillata larvae and effects of 5-ht antagonists during larval development. develop. growth differ. 43: 647656, 2001. petit t, dommergues m, socie g, dumez y, gluckman e, brison o. detection of maternal cells in human fetal blood during the third trimester of pregnancy using allele-specific pcr amplification. br. j. haematol. 98: 767771, 1997. petit t, gluckman e, carosella e, brossard y, brison o, socie g. a highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood. exp. hematol. 23: 1601-1605, 1995. pineda-krch m, lehtila k. costs and benefits of genetic heterogeneity within organisms. j. evol. biol. 17: 1167-1177, 2004. s18 rinkevich b. immunological resorption in botryllus schlosseri (tunicata) chimeras is characterized by multilevel hierarchical organization of histocompatibility alleles. a speculative endeavor. biol. bull. 184: 342-345, 1993. rinkevich b. primitive immune systems: are your ways my ways? immunol. rev. 198: 25-35, 2004a. rinkevich b. will two walk together, except they have agreed? j. evol. biol. 17: 1167-1177, 2004b. rinkevich b. rejection patterns in botryllid ascidian immunity: the first tier of allorecognition. can. j. zool. 83: 101-121, 2005. rinkevich b, weissman il. chimeras in colonial invertebrates a synergidtic symbiosis or somatic and germ cell parasitism. symbiosis 4: 117-134, 1987. rinkevich b, weissman il. allogeneic resorption in colonial protochordates: consequences of nonself recognition. dev. comp. immunol. 16: 275-286, 1992a. rinkevich b, weissman il. incidents of rejection and indifference in fu/hc incompatible protochordate colonies. j. exp. zool. 263: 105111, 1992b. rinkevich b, lilker-levav t, goren m. allorecognition/xenorecognition responses in botrylloides subpopulations from the mediterranean coast of israel. j. exp. zool. 270: 302-313, 1994. rinkevich b, porat r, goren m. allorecognition elements on urochordate histocompatibility locus indicate unprecedented extensive polymorphism. proc. biol. sci. 259: 319-324, 1995. rinkevich b, tartakover s, gershon h. contribution of morula cells to allogeneic responses in the colonial urochordate botryllus schlosseri. mar. biol. 131: 227-236, 1998. rosner a, paz g, rinkevich b. divergent roles of the dead-box protein bs-pl10, the urochordate homologue of human ddx3 and ddx3y proteins, in colony astogeny and ontogeny. dev. dyn. 235: 1508-1521, 2006. rosner a, rabinowitz c, moiseeva e, voskoboynik a, rinkevich b. bs-cadherin in the colonial urochordate botryllus schlosseri: one protein, many functions. dev. biol. 304: 687-700, 2007. sabbadin a. le basi geneticha della capacita di fusion fra colonies in botryllus schlosseri (ascidiacea). rend. accad. naz. lincei ser. 32: 1031-1035, 1962. sabbadin a, zaniolo g. sexual differentiation and germ cell transfer in the colonial ascidian botryllus schlosseri. j. exp. zool. 207: 289-304, 1979. sabbadin a. formal genetics of ascidians. am. zool. 22: 765-777, 1982. sabbadin a, astorri c. chimeras and histocompatibility in the colonial ascidian botryllus schlosseri. dev. comp. immunol. 12: 737-747, 1988. sabbadin a, zaniolo g, ballarin l. genetic and cytological aspects of histocompatibility in ascidians. boll. zool. 59: 167-173, 1992. saito y, watanabe h. colony specificity in the compound ascidian botryllus scalaris. proc. japn. acad. 58b: 105-108, 1982. saito y, watanabe h. partial biochemical characterization of humoral factors involved in the nonfusion reaction of a botryllid ascidian, botrylloides simodensis. zool. sci. 1: 229-235, 1984. saito y, hirose e, watanabe h. allorecognition in compound ascidians. int. j. dev. biol. 38: 237247, 1994. scofield vl, schlumpberger jm, west la, weissman il protochordate allorecognition is controlled by a mhc-like gene system. nature 295: 499-502, 1982. scofield vl, schlumpberger jm, nagashima ls. morphology and genetics of rejection reactions between oozooids from the tunicate botryllus schlosseri. biol. bull. 165: 733-744, 1983. sekizawa a, kimura t, sasaki m, nakamura s, kobayashi r, sato t. prenatal diagnosis of duchenne muscular dystrophy using a single fetal nucleated erythrocyte in maternal blood. neurology 46: 1350-1353, 1996. socie g, gluckman e, carosella e, brossard y, lafon c, brison o. search for maternal cells in human umbilical cord blood by polymerase chain reaction amplification of two minisatellite sequences. blood 83: 340-344, 1994. stone wh, cragle rg, swanson ew, brown dg. skin grafts: delayed rejection between pairs of cattle twins showing erythrocyte chimerism. science 148: 1335-1336, 1965. stone wh, cragle rg, johnson df, bacon ja, bendel s, korda n. long-term observations of skin grafts between chimeric cattle twins. transplantation 12: 421-428, 1971. stoner ds, weissman il. somatic and germ cell parasitism in a colonial ascidian: possible role for a highly polymorphic allorecognition system. proc. natl. acad. sci. usa 93: 15254-15259, 1996. stoner ds, rinkevich b, weissman il. heritable germ and somatic cell lineage competitions in chimeric colonial protochordates. proc. natl. acad. sci. usa 96: 9148-9153, 1999. tanaka k, watanabe h. allogeneic inhibition in a compound ascidian, botryllus primigenus oka. i. processes and features of nonfusion reaction. cell. immunol. 7: 410-426, 1973. taneda y. simultaneous occurrence of fusion and nonfusion reaction in two colonies in contact of the compound ascidian botryllus priminegus. dev. comp. immunol. 9: 371-375, 1985. tippett p. blood group chimeras. a review. vox sang. 44: 333-359, 1983. van den boogaardt dem, van rood jj, roelen dl, claas fh. the influence of inherited and noninherited parental antigens on outcome after transplantation. transpl. int. 19: 360-371, 2006. van dijk ba, boomsma di, de man aj. blood group chimerism in human multiple births is not rare. am. j. med. genet. 61: 264-268, 1996. von eggeling f, michel s, gunther m, schimmel b, claussen u. determination of the origin of single nucleated cells in maternal circulation by means s19 of random pcr and a set of length polymorphisms. hum. genet. 99: 266-270, 1997. wachtel ss, sammons d, twitty g, utermohlen j, tolley e, phillips o, et al. charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy. prenat. diagn. 18: 455-463, 1998. voskoboynik a, weissman il, rinkevich b. stem cells, chimerism, and tolerance: lessons from mammals and ascidians. in: rinkevich b, matranga v (eds), stem cells in marine organisms, springer-verlag [in press]. weissman il, saito y, rinkevich b. allorecognition histocompatibility in a protochordate species: is the relationship to mhc somatic or structural?. immunol. rev. 113: 227-241, 1990. voskoboynik a, soen y, rinkevich y, ueno h, rosner a, reshef r, et al. identification of the endostyle as a stem cell niche in a colonial chordate. cell stem cell 3: 456-464, 2008. zhao y, ohdan h, manilay jo, sykes m. nk cell tolerance in mixed allogeneic chimeras. j. immunol. 170: 5398-5405, 2003. s20 antimicrobial peptides in echinoderms isj 7: 132-140, 2010 issn 1824-307x minireview antimicrobial peptides in echinoderms c li, t haug, k stensvåg norwegian college of fishery science, faculty of bioscience, fisheries and economics, university of tromsø, breivika, n-9037 tromsø, norway accepted may 5, 2010 abstract antimicrobial peptides (amps) are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. here we summarize the knowledge of such peptides in echinoderms. strongylocins are a novel family of cysteine-rich amps, recently identified in the sea urchins, strongylocentrotus droebachiensis and s. purpuratus. although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, dissimilar to other known amps. a family of heterodimeric amps, named centrocins, are also present in s. droebachiensis. lysozymes and fragments of larger proteins, such as beta-thymocins, actin, histone 2a and filamin a have also been shown to display antimicrobial activities in echinoderms. future studies on amps should be aimed in revealing how echinoderms use these amps in the immune response against microbial pathogens. key words: sea urchin; host defence peptides; celomocyte; innate immunity introduction antimicrobial peptides (amps) are evolutionarily conserved small molecular weight proteins of the innate immune response, with a broad spectrum of antimicrobial activities against bacteria, viruses, and fungi (reviewed by mookherjee and hancock, 2007). amps appear naturally throughout all three domains of life from unicellular to multicellular organisms (zasloff, 2002; riley and chavan, 2007; wang et al., 2009). by february 2010, more than 1,500 amps have been identified (http://aps.unmc.edu/ap/main.php). amps are characterized as having less than 100 amino acids and are usually cationic in nature. nearly all antimicrobial peptides form amphipathic structures which reflect the relative abundance and polarization of hydrophobic and hydrophilic domains within the peptides. the hydrophobicity enables the water-soluble antimicrobial peptides to interact with the hydrophobic lipid bilayer of the microbial membranes. amps are likely to be attracted by and attach to the negatively charged bacterial surfaces because of their cationic nature. the mechanism by which amps interact with the cell wall structures of gram ___________________________________________________________________________ corresponding author: klara stensvåg norwegian college of fishery science faculty of bioscience, fisheries and economics university of tromsø, breivika, n-9037 tromsø, norway e-mail: klara.stensvag@uit.no negative and gram-positive bacteria has rarely been addressed and is therefore not yet understood (brogden, 2005). however, once the peptides come into contact with the outer leaflet of the cell membrane and the peptide/lipid ratio increases, the peptides start forming multimers or self-associating on top of the membrane (yang et al., 2001). at sufficiently high concentrations of the peptides orientate perpendicularly and insert into the bilayer, thereby interfering with membrane integrity. however, new paradigms imply that pore-forming is not the only mechanism of antimicrobial activity. some peptides are also able to interact with intracellular targets without disrupting the membrane integrity. intracellular targets of antimicrobial peptides vary from nucleic acids to enzymatic proteins (brogden, 2005). amps are crucial immune effector molecules for invertebrates which lack a vertebrate-type adaptive immune system. they have been identified both in plasma and in various blood cells and their distribution in the host can be site-specific or systemic. they are either expressed constitutively or the expression is induced by exposure to pathogens. they do not only inactivate bacteria in vitro and in vivo , thereby protecting host organisms against a variety of infections, but they also modulate immunity ( hancock and diamond, 2000; zasloff, 2002; hancock et al., 2006). it is worth mentioning that a few amps play a role as anti-endotoxins (scott et al., 2002; bowdish et al., 2005) and chemokines in vertebrates (durr and peschel, 2002; 132 http://aps.unmc.edu/ap/main.php mailto:klara.stensvag@uit.no davidson et al., 2004) and might also induce production of cytokines and chemokines (bals and wilson, 2003). these immunomodulatory functions do not directly kill microbes, but recruitment and activation of immune cells and signalling molecules improves the host defence system. in this review we will present an overview of amps identified and characterized from echinoderms and present their characteristics. in particularly, we will describe their antimicrobial activities, their primary structure, and posttranslational modifications of these molecules. finally, we will address some important questions regarding future amp research on echinoderms. antimicrobial activities in echinoderms the phylum echinodermata is a large phylum of deuterostomes containing approximately 7,000 living species divided into the groups crinoidea (sea lilies), ophiuroidea (brittle stars), asteroidea (starfishes), echinoidea (sea urchins) and holothuroidea (sea cucumbers). the adult echinoderms have a few major organs such as mouth parts, intestine, nerve ring, and gonad. these organs are located in the cavity, named celomic cavity. the remaining space of the celomic cavity is filled with celomic fluid which contains various “blood cells”, the celomocytes. these cells are though to mediate the main immune functions in echinoderms, including antimicrobial activities. many studies have been conducted to examine the activity of celomocytes lysates and celomic fluid against bacteria, fungi and even tumour cells. celomic fluid from echinus esculentus possess bactericidal activity against marine pseudomonas sp (wardlaw and unkles, 1978). gerardi et al. (1990) found that the highest bacterial growth inhibition against several vibrio sp. is shown by phagocytes (called amoebocytes) and red spherule cells of paracentrotus lividus.. stabili et al. (1996) also discovered antibacterial activity against v. alginolyticus in celomocyte lysates and celomic fluid of p. lividus. recently, it was reported that celomocytes of p. lividus show a cytotoxic activity against rabbit erythrocytes and the k562 tumour cell line (arizza et al., 2007). antibacterial activity was detected in extracts of several tissues from the green sea urchin s. droebachiensis, the common sea star asterias rubens, and the sea cucumber cucumaria frondosa (haug et al., 2002). some of the activities detected (in extracts of celomocytes and body walls) were caused by compounds of protein nature. several drug discovery projects have screened echinoderms for antibiotic activities. an early study by rinehart et al (1981) showed that antimicrobial activity was present in 43 % of 83 unidentified species of echinoderms (collected from the west coast of baja california and the gulf of california) and 58 % of 36 unidentified caribbean species displayed antimicrobial activities. in the northern gulf of mexico, 80 % of 22 echinoderm species showed antimicrobial activity (bryan et al., 1994). body wall extracts of echinoderms displayed activity against marine bacteria, but did also inhibit settlement of marine larvae (bryan et al., 1996). several antimicrobial molecules (other than amps) have been isolated from echinoderms, including echinochrome a (kuwahara et al., 2009; service and wardlaw, 1984), steroidal glycosides (andersson et al., 1989; chludil et al., 2002; levina et al., 2009), and polyhydroxylated sterols (iorizzi et al., 1995). although these results indicate that echinoderms present remarkable activity against microbes, only a few amps have been reported. antimicrobial peptides in echinoderms lysozymes are widely distributed throughout the animal kingdom and catalyze the hydrolysis of peptidoglycans of bacterial cell walls of especially gram-positive bacteria, and acts as a nonspecific innate immunity molecules against the invasion of bacterial pathogens (jollès and jollès, 1984). they are characterized as basic proteins of low molecular weight (15-25 kda) with an isoelectric point of 10.5– 11.0, stable at acidic ph and even at high temperatures. there are several lysozymes or lysozyme-like proteins identified from celomic fluid, celomocytes and other tissues of echinoderms (jollès and jollès, 1975; canicatti and roch, 1989; canicatti, 1990; canicatti and d´ancona, 1990; gerardi et al., 1990; stabili et al., 1994; stabili and canicatti, 1994; stabili and pagliara, 1994, 2009; shimizu et al., 1999; bachali et al., 2004; cong et al., 2009) (table 1). a study by beauregard and co-workers indicated the presence of amps in the celomic fluid of the orange-footed sea cucumber, c. frondosa (beauregard et al., 2001). the celomic fluid extract was purified by molecular sieve chromatography and antibacterial activity was detected in separate fractions. a peptide of approximately 6 kda was identified, but no sequence was reported (table 1). in a celomocyte extract from the starfish a. rubens showing antimicrobial activity, several protein/peptides with molecular mass around 2 kda were isolated (maltseva et al., 2004; maltseva et al., 2007) (table 1). two peptides were identified as part of the histone molecule, h2a. two other peptides were identified as fragments of actin, while one peptide was a fragment of filamin a. in addition, four other amps were detected, but not characterized. it had been known for long time that histones have antibacterial properties (hirsch 1958). histone-derived fragments (h1, from h2a and h2b) have shown to possess antimicrobial activity (from et al., 1996; robinette et al., 1998; patrzykat et al., 2001; richards et al., 2001; fernandes et al., 2002). although maltseva et al. (2004, 2007) also identified fragments of actin and filamin a in the active extract, the antimicrobial activity of these fragments need to be further investigated. recently, schillaci et al. (2009) reported that a 5 kda peptide fraction from the celomocytes of p. lividus presents activity against both gram-positive and gram-negative bacteria and fungi (table 1). the peptide fraction also showed the ability to inhibit the formation of staphylococcus biofilms. the authors suggested that the antimicrobial and antistaphylococcal biofilm activity of this peptide 133 table 1 antimicrobial peptides and proteins characterized in echinoderms class and genus origin (tissue/cell type) compound mw (kda) reference asteroidea asterias forbesi cell-free celomic fluid complement-like leonard et al., 1990 a. rubens body wall peptides/proteins <20 haug et al., 2002 celomocytes fragments of actin, histone h2a, and filamin a 1.82.4 maltseva et al., 2007; maltseva et al., 2004 celomocytes peptides 2.0-4.7 maltseva et al., 2007; maltseva et al., 2004 lysozyme 15.5 jollès and jollès 1975; bachali et al., 2004 marthasterias glacialis eggs lysozyme-like stabili and pagliara, 1994, 2009 body wall mucus lysozyme-like canicatti and d´ancona, 1990 echinoidea holothuria scabra celomic fluid lectin >10 gowda et al., 2008 h. polii celomocytes lysozyme-like canicatti and roch, 1989 various tissues lysozyme-like canicatti, 1990 paracentrotus lividus celomocytes lysozyme-like gerardi et al., 1990 celomocytes protein ~ 60 stabili et al., 1996 celomocytes peptide ~ 5 schillaci et al., 2009 larvae lysozyme-like stabili et al., 1994 seminal plasma lysozyme-like stabili and canicatti, 1994 various tissues lysozyme-like canicatti, 1990 strongylocentrotus droebachiensis celomocytes stongylocins 5.65.8 li et al., 2008 celomocytes centrocins 4.4-4.5 li et al., 2010b s. intermedius celomic fluid lysozyme 14 shimizu et al., 1999 s. purpuratus celomocyte cdna spstrongylocinsa 5.6-6.1 li et al., 2010a holothuroidea cucumaria echinata whole body fragments of lectin cel-iii 2.04.2 hatakeyama et al., 2004; hisamatsu et al., 2008 c. frondosa celomic fluid peptides ~ 6 beauregard et al., 2001 various tissues peptides/proteins <20 haug et al., 2002 parastichopus califormicus celomic cavity lps-binding compound dybas and fankboner, 1986 stichopus japonicus intestine cdna lysozymea 14 cong et al., 2009 a recombinantly produced and tested for activity. fraction is associated with beta-thymosin like fragments. beta thymosins have been reported to induce the production of metalloproteinases, display chemotactic and anti-inflammatory activities (huff et al., 2001) and even function as amps released by human platelets (tang et al., 2002). several amps have been isolated and characterized from the green sea urchin, s. droebachiensis. one group is the cysteine rich peptides named strongylocins (li et al., 2008). homologous genes, named spstrongylocins, were also identified in the sister species s. purpuratus (li et al., 2010a). all these native peptides or recombinant equivalents show antibacterial activity against both gram positive and gram negative bacteria (table 2). in addition, there are several heterodimer structured peptides, named centrocins, identified from s. droebachiensis (li et al, 2010b). they show strong potent activity, not only against gram-positive and gram-negative bacteria, but the longest peptide chain of the dimers also show strong activity against fungi and yeasts. in silico analysis of strongylocins and comparison of the native peptide sequences and the ones deduced from cdna sequences, show that these peptides contain three regions: a signal peptide, a prosequence and a native sequence (fig. 1). analysis , using both the neutral network model and the hidden markov model (bendtsen et al., 2004; http://www.cbs.dtu.dk/services/signalp/), indicates that a signal peptide cleavage site is located between the amino acid number 22 and 23 for all these peptides. 134 http://www.cbs.dtu.dk/services/signalp/ table 2 susceptibility of bacterial strains to the antibacterial peptides, strongylocins and centrocins, isolated from s. droebachiensis celomocytes, and recombinant spstrongylocins (li et al., 2008; li et al. 2010a, b) minimal inhibitory concentration (μm) peptide listonella anguillarum escherichia coli corynebacterium glutamicum staphylococcus aureus strongylocin 1 a 2.5 5.0 2.5 2.5 strongylocin 2 a 1.3 5.0 2.5 2.5 recombinant spstrongylocin 1 b 15.0 7.5 7.5 15.0 recombinant spstrongylocin 2 b 15.0 7.5 3.8 15.0 centrocin 1 a 2.5 1.3 1.3 2.5 centrocin 2 a 2.5 2.5 1.3 5.0 a minimal inhibitory concentration (mic) was determined as the lowest concentration of peptide causing an optical density less than 50 % of the growth control without any peptide present. b minimal inhibitory concentration (mic) was determined as the lowest concentration of peptide causing 100 % of the growth inhibition of the test organism compared to the growth control. it is not surprising that strongylocin 1 and spstrongylocin 1 contain an identical signal peptide sequence since they share high amino acid sequence similarity. however, spstrongylocin 2 does not share an identical signal peptide with strongylocin 2, but with strongylocin 1. although no experimental data show which mechanism assists the migration of these precursors of strongylocins and spstrongylocins within the cells, the diversity of the signal peptides suggests that strongylocin 2 likely employs a divergent intracellular trafficking pathway than the other sea urchin amps. analysis of strongylocins and spstrongylocins show that their prosequences are located at the nterminal region, following the signal peptide. it is common that prosequences are located at the nterminal or the c-terminal region, or even in between parts of the mature sequences. the prosequence is considered to act as an intracellular steric chaperone during the folding process (inouye, 1991), and it has been shown that prosequencesupported protein folding is crucial for processing of the proper protein having antifungal activity (reichhart and achstetter, 1990). in addition, it has been shown that the prosequences in some proteolytic enzyme precursors inhibit the activity of the mature proteins (neurath, 1989). the prosequences of strongylocins and spstrongylocins are negatively charged sequences which may neutralize the positive charges of the mature sequences (li et al., 2008; li et al., 2010a, b). therefore, the precursor molecules are likely less toxic to the host cells than the mature peptides. the peptides will obtain their activity during their maturation once the prosequences are removed. strongylocins contain six cysteine residues, which form three disulfide bridges (li et al., 2008; li et al., 2010a). cysteine-rich amps are found in various phyla, such as the defensin families in both vertebrates and invertebrates, tachystatins in horse shoe crabs and circulin a in plants (daly et al., 1999; wang et al., 2009). table 3 shows that strongylocins and spstrongylocins display a novel cysteine location pattern compared to other known cysteine-rich peptides containing 6 cysteines. this indicates that these amps may not form the same disulfide bridge linkages as the other peptides containing six cysteines, since proteins that have the same cysteine residue pattern tend to have identical disulfide connections (bania et al., 1999; bontems et al., 1991; pallaghy et al., 1994). disulfide bridges are crucial for the antimicrobial activity of most cysteine-containing amps (daher et al., 1986; mandal and nagaraj, 2002), and they may also stabilize the conformation of the molecules (selsted and ouellette, 2005). therefore, the disulfide linkages may aid strongylocins and spstrongylocins to resist proteolysis within the celomocytes, and/or after their release into protease-containing environments. strongylocin 2 contains a tryptophan in the mature sequence which is likely brominated (li et al., 2008). several amps with bromotryptophan have also been isolated from other marine organisms, for example styelin d from the tunicate, styela clavata (taylor et al., 2000), cathelicidin from the hagfish, myxine glutinosa (shinnar et al., 2003), and hedistin from the annelid, nereis diversicolor (tasiemski et 135 fig. 1 alignment of strongylocins from s. droebachiensis and spstrongylocins from s. purpuratus. the predicted cleavage site between the signal peptide and the proregion is shown (▼). the first amino acid in active strongylocin 2 and spstrongylocin 2 are likely a modified tryptophan (♦). identical amino acids are shown in boxes, similar amino acids are shaded in grey, and cysteines are highlighted in black. al., 2007). brominated amino acids may protect the peptides from proteolysis, and/or increase the biological activity of peptides (bittner et al., 2007). although spstrongylocin 2 also contains a tryptophan residue in the same position as strongylocin 2, the recombinantly produced spstrongylocins, having a non-brominated trp residue, are still antimicrobial active (li et al., 2010a). therefore, we speculate that the brominated tryptophan affects the peptides by enhancing their stability, but does not affect their antimicrobial activity (li et al., 2010a). this suggestion is supported by results showing no difference in the antimicrobial activity between the heavy chain of the dimeric peptide centrocin 1, with or without the brominated modification (stensvåg, unpublished). future studies on amps in echinoderms amps are important immune defence molecules for echinoderms that lack a vertebratetype adaptive immune system (reviewed by smith et al., 2010). it is therefore interesting to know whether these amps are secreted into the celomic fluid or stored in the granula of certain cell types. it has been documented that neutrophilic granules packed with human cathelicidin and defensins are fused with phagocytic vacuoles, where they contain certain concentrations of amps to eliminate phagocytosed pathogens (reviewed by ganz, 2003; lehrer, 2004; brogden, 2005). for penaeidins in shrimp, it seems that haemocytes migrate towards the infection sites and then release the amps presumably by lysis of the cells (munoz et al., 2002). echinoderms have a large celomic cavity filled with circulating fluid which could dilute amps if they were secreted or released by the cells. therefore, further investigation on the distribution of amps in echinoderms might uncover whether the peptides are involved in 1) intracellular elimination of pathogens; 2) local release of amps at the infection sites; or 3) massive release of amps to regulate other immune activities. although several studies have documented mammalian amps as immune regulatory molecules (reviewed by hancock et al., 2006 and diamond et al., 2009), unfortunately, there are not many investigations on invertebrate amps. it has been reported that tachypleus tridentatus haemocyte granules can release tachyplesin after 136 table 3 comparison of cysteine location patterns in amps containing six cysteines peptide family cysteine location patterns 1 animals strongylocins c – c – c – cc – c s. droebachiensis spstrongylocins c – c – c – cc – c s. purpuratus βeta-defensins c – c – c – c – cc bos taurus alpha-defensins c – c – c – c – cc homo sapiens tachystatins c – c – cc – c – c t. tridentatus knottin-type amps c – c – cc – c – c phytolacca americana thionins type iii and iv amps cc – c – c – c – c sorghum bicolor insect defensins c – c – c – c – c – c rhodnius prolixus 1 adjacent double cysteine residues are highlighted in yellow. information regarding cysteine arrangements in the different peptides was obtained from the antimicrobial peptide database (wang et al., 2009). encountering microbial endotoxins and form a complex (iwanaga et al., 1998; hirakura et al., 2002). this complex will then block the activation of factor c, which is crucial for haemolymph coagulation (nakamura et al., 1988). pancer et al. (1999) identified an nf-κb homologue, transcription factor in s. purpuratus celomocytes according to analysis of the immune related gene repertoire of s. purpuratus (hibino et al., 2006; rast et al., 2006), toll-like receptor (tlr) genes, interleukin (il)-17 genes, il receptors, and tumor necrosis factor (tnf) family members are present in the sea urchin genome. it is possible that amps are involved in the endotoxin-induced signalling pathway from the tlr to nf-κb, and/or suppress inflammation. therefore, it is important to keep in mind that amps may play multiple immune roles in the echinoderm´s immunity as in other species (hancock and diamond, 2000; lehrer, 2004; durr et al., 2006; hancock et al., 2006; mookherjee and hancock, 2007; cuthbertson et al., 2008; smith et al., 2008). the discovery of amps in echinoderms has attracted attention from both pharmaceutical and academic sectors. however, there should be more focus on searching for new amps in crinoidea and ophiuroidea by newly developed high-throughput screening systems or other proteomic techniques since there is limited information about antimicrobial compounds from these echinoderm classes. in summary, amps are important host defence molecules in invertebrates. investigations of antimicrobial peptides/proteins in echinoderms have revealed two novel families of amps in strongylocentrotus, lysozymes in various species and fragments of larger proteins having antibacterial activity. these amps do not only attract interest as potent drugs or drug leads, but may also become useful in studying the echinoderm immune system. acknowledgements writing this review was supported by grants from the university of tromsø, the marine biotechnology in tromsø (mabit) research program (no. bs0034) and the norwegian research council (nos. 178214/s40 and 184688/s40). oleg sokolov is appreciated for translating the russian papers. references andersson l, bohlin l, iorizzi m, riccio r, minale l, moreno-lópez w. biological activity of some saponins and saponin-like compounds from starfish and brittle-stars. toxicon 27: 179-188, 1989. arizza v, giaramita ft, parrinello d, carnmarata m, parrinello n. cell cooperation in coelomocyte cytotoxic activity of paracentrotus lividus, coelomocytes. comp. biochem. physiol. 147a: 389-394, 2007. bachali s, bailly x, jolles j, jolles p, deutsch js. the lysozyme of the starfish asterias rubens. eur. j. biochem. 271: 237-242, 2004. bals r, wilson jm, cathelicidins--a family of multifunctional antimicrobial peptides. cell. mol. life sci. 60: 711-720, 2003. bania j, stachowiak d, polanowski a. primary structure and properties of the cathepsin g/chymotrypsin inhibitor from the larval hemolymph of apis mellifera. eur. j. biochem. 262: 680-687, 1999. beauregard ka, truong nt, zhang h, lin w, beck g. the detection and isolation of a novel antimicrobial peptide from the echinoderm, cucumaria frondosa. adv. exp. med. biol. 484: 55-62, 2001. bendtsen jd, nielsen h, von heijne g, brunak s. improved prediction of signal peptides: signalp 3.0. j. mol. biol. 340: 783-795, 2004. bittner s, scherzer r, harlev e. the five bromotryptophans. amino acids 33: 19-42, 2007. bontems f, roumestand c, gilquin b, menez a, toma f. refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. science 254: 1521-523, 1991. bowdish dm, davidson dj, lau ye, lee k, scott mg, hancock re. impact of ll-37 on antiinfective immunity. j. leukoc. biol. 77: 451-459, 2005. brogden ka. antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? nat. rev. microbiol. 3: 238-250, 2005. 137 bryan pj, mcclintock jb, watts sa, marion kr, hopkins ts. antimicrobial activity of ethanolic extracts of echinoderms from the northern gulf of mexico. in: b david, a guille, j-p feral, m roux (eds), echinoderms through time, balkema, rotterdam pp 17-23, 1994. bryan pj, rittschof d, mcclintock jb. bioactivity of echinoderm ethanolic body-wall extracts: an assessment of marine bacterial attachment and macroinvertebrate larval settlement. j. exp. mar. biol. ecol. 196: 79-96, 1996. canicatti c, distribution d´une activite lysozymiale dans un echinoderme holothuroide, holothuria polii, et dans les oeufs et les larves d´un echinoderme echinoide, paracentrotus lividus. eur. arch. biol. (bruxelles) 101: 309-318, 1990. canicatti c, d´ancona g. biological protective substances in marthasterias glacialis (asteroidea) epidermal secretion. j. zool. (lond.) 222: 445-454, 1990. canicatti c, roch p, studies on holothuria polii (echinodermata) antibacterial proteins. i. evidence for and activity of a coelomocyte lysozyme. experientia 45: 756-759, 1989. chludil hd, seldes am, maier ms. antifungal steroidal glycosides from the patagonian starfish anasterias minuta: structure-activity correlations. j. nat. prod. 65: 153-157, 2002. cong l, yang x, wang x, tada m, lu m, liu h, et al. characterization of an i-type lysozyme gene from the sea cucumber stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein. j. biosci. bioeng. 107: 583-588, 2009. cuthbertson bj, deterding lj, williams jg, tomer kb, etienne k, blackshear pj, et al, diversity in penaeidin antimicrobial peptide form and function. dev. comp. immunol. 32:167-181, 2008. daher ka, selsted me, lehrer ri. direct inactivation of viruses by human granulocyte defensins. j. virol. 60: 1068-1074, 1986. daly n l, koltay a, gustafson kr, boyd mr, casas-finet jr, craik dj. solution structure by nmr of circulin a: a macrocyclic knotted peptide having anti-hiv activity. j. mol. biol. 285: 333-345, 1999. davidson dj, currie aj, reid gs, bowdish dm, macdonald kl, ma rc, et al. the cationic antimicrobial peptide ll-37 modulates dendritic cell differentiation and dendritic cell-induced t cell polarization. j. immunol. 172: 1146-1156, 2004. diamond g, beckloff n, weinberg a, kisich ko. the roles of antimicrobial peptides in innate host defense. curr. pharm. des. 15: 2377-2392, 2009. durr m, peschel a. chemokines meet defensins: the merging concepts of chemoattractants and antimicrobial peptides in host defense. infect. immun. 70: 6515-6517, 2002. durr uh, sudheendra us, ramamoorthy a. ll-37, the only human member of the cathelicidin family of antimicrobial peptides. biochim. biophys. acta 1758: 1408-1425, 2006. dybas l, fankboner pv. holothurian survival strategies: mechanisms for the maintenance of a bacteriostatic environment in the coelomic cavity of the sea cucumber. dev. comp. immunol. 10: 311-330, 1986. fernandes jmo, kemp gd, molle g, smith jv. novel antimicrobial properties of histone h2a from skin secretions of rainbow trout, oncorhynchus mykiss. biochem. j. 368: 611620, 2002. from m, gunne h, bergman ac, agerberth b, bergman t, boman a, et al. biochemical and antibacterial analysis of human wound and blister fluid. eur. j. biochem. 237: 86-92, 1996. ganz t. defensins: antimicrobial peptides of innate immunity. nat. rev. immunol. 3: 710-720, 2003. gerardi p, lassegues m, canicatti c. cellular distribution of sea urchin antibacterial activity. biol. cell 70: 153-157, 1990. gowda nm, goswami u, khan mi. t-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (holothuria scabra): possible involvement in differential recognition of bacteria. j. invert. pathol. 99: 141-145, 2008. hancock re, brown kl, mookherjee n. host defence peptides from invertebrates--emerging antimicrobial strategies. immunobiology 211: 315-322, 2006. hancock r e, diamond g, the role of cationic antimicrobial peptides in innate host defences. trends microbiol. 8: 402-410, 2000. hatakeyama t, suenaga t, eto s, niidome t, aoyagi h. antibacterial activity of peptides derived from the c-terminal region of a hemolytic lectin, cel-iii, from the marine invertebrate cucumaria echinata. j. biochem. 135: 65-70, 2004. haug t, kjuul ak, styrvold ob, sandsdalen e, olsen mo, stensvåg k. antibacterial activity in strongylocentrotus droebachiensis (echinoidea), cucumaria frondosa (holothuroidea), and asterias rubens (asteroidea). j. invert. pathol. 81: 94-102, 2002. hibino t, loza-coll m, messier c, majeske aj, cohen ah, terwilliger dp, et al, the immune gene repertoire encoded in the purple sea urchin genome. dev. biol. 300: 349-365, 2006. hirakura y, kobayashi s, matsuzaki m specific interactions of the antimicrobial peptide cyclic b-sheet tachyplesin i with lipopolysaccharides. biochim. biophys. acta 1562: 32-36, 2002. hirsch jg. bactericidal action of histone, j. exp. med. 108: 925-944, 1958. hisamatsu k, tsuda n, goda s, hatakeyama t. characterization of the alpha-helix region in domain 3 of the haemolytic lectin cel-iii: implications for self-oligomerization and haemolytic processes. j. biochem. 143: 79-86, 2008. huff t, muller cs, otto am, netzker r, hannappel e. beta-thymosins, small acidic peptides with multiple functions. int. j. biochem. cell biol. 33: 205-220, 2001. inouye m. intramolecular chaperone: the role of the pro-peptide in protein folding. enzyme 45: 314321, 1991. iorizzi m, bryan p, mcclintock j, minale l, palagiano e, maurelli s, et al. chemical and 138 biological investigation of the polar constituents of the starfish luidia clathrata, collected in the gulf of mexico. j. nat. prod. 58: 653-671, 1995. iwanaga s, kawabata s. evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab. front. biosci. 3, d973-984, 1998. jollès j, jollès p. the lysozyme from asterias rubens. eur. j. biochem. 54: 19-23, 1975. jollès p, jollès j. what's new in lysozyme research? always a model system, today as yesterday, mol. cell. biochem. 63: 165-189, 1984. kuwahara r, hatate h, yuki t, murata h, tanaka r, hama y. antioxidant property of polyhydroxylated naphthoquinone pigments from shells of purple sea urchin anthocidaris crassispina. lwt-food sci. technol. 42: 12961300, 2009. lehrer ri. primate defensins. nat. rev. microbiol. 2: 727-38, 2004. leonard la, strandberg jd, winkelstein ja. complement-like activity in the sea star, asterias forbesi. dev. comp. immunol. 14: 1930, 1990. levina ev, kalinovsky ai, dmitrenok pv. steroid compounds from two pacific starfish of the genus evasterias. russian j. bioorg. chem. 35: 123-130, 2009. li c, haug t, styrvold ob, jørgensen tø, stensvåg k. strongylocins, novel antimicrobial peptides from the green sea urchin, strongylocentrotus droebachiensis. dev. comp. immunol. 32:14301440, 2008. li c, blencke hm, smith lc, karp mt, stensvåg k. two recombinant peptides, spstrongylocins 1 and 2, from strongylocentrotus purpuratus, show antimicrobial activity against grampositive and gram-negative bacteria. dev. comp. immunol. 34: 286-292, 2010a. li c, haug t, moe km, styrvold ob, stensvåg k. centrocins: isolation and characterization of novel dimeric antimicrobial peptides from the green sea urchin, strongylocentrotus droebachiensis. dev. comp. immunol. doi: 10.1016/j.dci.2010.04.004, 2010b [in press]. maltseva al, aleshina gm, kokryakov vn, krasnodembsky eg. diversity of antimicrobial peptides in acidic extracts from coelomocytes of starfish asterias rubens l. vestnik sanktpeterburgskogo universiteta, seriya 3, biologiya, 1: 85-94, 2007 (in russian). maltseva al, aleshina gm, kokryakov vn, krasnodembsky eg, ovchinnikova tv. new antimicrobial peptides from coelomocytes of sea star asterias rubens l. vestnik sanktpeterburgskogo universiteta, seriya 3, biologiya 4: 101-108, 2004 (in russian). mandal m, nagaraj r. antibacterial activities and conformations of synthetic alpha-defensin hnp1 and analogs with one, two and three disulfide bridges. j. pept. res. 59: 95-104, 2002. mookherjee n, hancock re. cationic host defence peptides: innate immune regulatory peptides as a novel approach for treating infections. cell. mol. life sci. 64: 922-933, 2007. munoz m, vandenbulcke f, saulnier d, bachere e. expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp. eur. j. biochem. 269: 2678-2689, 2002. nakamura t, furunaka h, miyata t, tokunaga f, muta t, iwanaga s. tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (tachypleus tridentatus). j. biol. chem. 263: 16709-16713, 1988. neurath h, proteolytic processing and physiological regulation. trends biochem. sci. 14: 268-271, 1989. pallaghy pk, nielsen kj, craik dj, norton rs. a common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides. protein sci. 3: 1833-839, 1994. pancer z, rast jp, davidson eh. origins of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes. immunogenetics 49: 773-786, 1999. patrzykat a, zhang l, mendoza v, iwama g, hancock r. synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin. antimicrob. agents chemother. 45: 1337-1342, 2001. rast jp, smith lc, loza-coll m, hibino t, litman gw. genomic insights into the immune system of the sea urchin. science 314: 952-956, 2006. reichhart jm, achstetter t. expression and secretion of insect immune peptides in yeast. res. immunol. 141: 943-946, 1990. richards rc, o'neil db, thibault p, ewart kv. histone h1: an antimicrobial protein of atlantic salmon (salmo salar). biochem. biophys. res. commun. 284: 549-555, 2001. riley mar, chavan ma. bacteriocins: ecology and evolution. chapter 5, peptide and protein antibiotics from the domain archaea: halocins and sulfolobicins, pp 93-109, 2007. rinehart kl, shaw pd, shield ls, gloer jb, harbour gc, koker mes, et al. marine naturalproducts as sources of anti-viral, anti-microbial, and anti-neoplastic agents. pure appl. chem. 53: 795-817, 1981. robinette d, wada s, arroll t, levy mg, miller wl, noga ej. antimicrobial activity in the skin of the channel catfish ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins. cell. mol. life sci. 54: 467-475, 1998. schillaci d, arizza v, parrinello n, di stefano v, fanara s, muccilli v, et al. antimicrobial and antistaphylococcal biofilm activity from the sea urchin paracentrotus lividus. j. appl. microbiol. 108: 17-24, 2009. scott m g, davidson dj, gold mr, bowdish d, hancock re. the human antimicrobial peptide ll-37 is a multifunctional modulator of innate immune responses. j. immunol. 169: 38833891, 2002. selsted me, ouellette aj. mammalian defensins in the antimicrobial immune response. nat. immunol. 6: 551-557, 2005. service m, wardlaw ac. echinochrome a as a bactericidal substance in the coelomic fluid of 139 echinus esculentus (l.). comp. biochem. physiol. 79b: 161-165, 1984. shimizu m, kohno s, kagawa h, ichise n. lytic activity and biochemical properties of lysozyme in the coelomic fluid of the sea urchin strongylocentrotus intermedius. j. invert. pathol. 73: 214-222,1999. shinnar ae, butler kl, park hj. cathelicidin family of antimicrobial peptides: proteolytic processing and protease resistance. bioorg. chem. 31: 425-436, 2003. smith lc, ghosh j, buckley mk, clow al, dheilly mn, haug t, et al. echinoderm immunity. in: soderhall k (ed), invertebrate immunology, landes bioscience, inc. 2010 [in press]. smith vj, fernandes jm, kemp gd, hauton c. crustins: enigmatic wap domain-containing antibacterial proteins from crustaceans. dev. comp. immunol. 32: 758-772, 2008. stabili l, canicatti c. antibacterial activity of the seminal plasma of paracentrotus lividus. can. j. zool. 72: 1211-1216, 1994. stabili l, licciano m, pagliara p. evidence of antibacterial and lysozyme-like activity in different planktonic larval stages of paracentrotus lividus. mar. biol. 119: 501-505, 1994. stabili l, pagliara p. antibacterial protection in marthasterias glacialis eggs characterization of lysozyme-like activity. comp. biochem. physiol. 109b: 709-713, 1994. stabili l, pagliara p. effect of zinc on lysozyme-like activity of the seastar marthasterias glacialis (echinodermata, asteroidea) mucus. j. invert. pathol. 100: 189-192, 2009. stabili l, pagliara p, roch p, antibacterial activity in the coelomocytes of the sea urchin paracentrotus lividus. comp. biochem. physiol. 113b: 639-644, 1996. tang yq, yeaman mr, selsted me. antimicrobial peptides from human platelets. infect. immun. 70: 6524-6533, 2002. tasiemski a, schikorski d, le marrec-croq f, pontoire-van camp c, boidin-wichlacz c, sautiere pe. hedistin: a novel antimicrobial peptide containing bromotryptophan constitutively expressed in the nk cells-like of the marine annelid, nereis diversicolor. dev. comp. immunol. 31: 749-762, 2007. taylor sw, craig ag, fischer wh, park m, lehrer ri, styelin d. an extensively modified antimicrobial peptide from ascidian hemocytes. j. biol. chem. 275: 38417-38426, 2000. villasin j, pomory cm. antibacterial activity of extracts from the body wall of parastichopus parvimensis (echinodermata: holothuroidea). fish shellfish immunol. 10: 465-467, 2000. wang g, li x, wang z. apd2: the updated antimicrobial peptide database and its application in peptide design. nucleic acids res. 37: d933-937, 2009. wardlaw ac, unkles se. bactericidal activity of celomic fluid from sea-urchin echinus esculentus. j. invert. pathol. 32: 25-34, 1978. yang l, harroun ta, weiss tm, ding l, huang hw. barrel-stave model or toroidal model? a case study on melittin pores. biophys. j. 81: 14751485, 2001. zasloff m. antimicrobial peptides of multicellular organisms. nature 415: 389-395, 2002. 140 effects of ocean acidification and warming on the covering behavior of sea urchins glyptocidaris crenularis and strongylocentrotus intermedius 14 isj 15: 14-18, 2018 issn 1824-307x short communication effects of water temperature and ph value on covering behavior of the sea urchin glyptocidaris crenularis mf yang#, sb luo#, jn sun, dt shi, jy ding, yq chang, c zhao # these authors contributed equally to this work key laboratory of north mariculture and stock enhancement, ministry of agriculture, dalian ocean university, dalian, china, 116023 accepted november 28, 2017 abstract as marine calcifying organisms, sea urchins are sensitive in the changing ocean. more information is needed about the effects of ph value and water temperature on behaviors of sea urchins. here, we reported that ph value and water temperature significantly affected the covering behavior of sea urchins glyptocidaris crenularis. lower ph value (ph = 7.4) significantly reduced the time to first covering (p = 0.026), while significantly decreased number of covered sea urchins (p = 0.029) and number of shells used for covering (p = 0.007) in g. crenularis. water temperature did not significantly affect the time to first covering (p = 0.180) or number of covered sea urchins (p = 0.157), though significantly affected number of shells used for covering (p = 0.042). the present study provides preliminary information on behavioral ecology of sea urchins. notably, the effects of co2 induced acidification should be further investigated in future. key words: sea urchin; covering behavior; ph value; water temperature introduction over the past 250 years, atmospheric carbon dioxide (co2) levels increased by approximately 40 % (solomon et al., 2007), one third of which has been taken up by the oceans (sabine et al., 2004). due to absorbing increasing amounts of co2, ocean warming and acidification have been identified as the greatest anthropogenic threat to marine ecosystems (halpern et al., 2008; de madron et al., 2011). this highlights the risks to marine calcifying organisms in future (orr et al., 2005). as marine calcifying organisms, sea urchins are sensitive in the changing ocean (dupont et al., 2013). ocean warming and acidification have showed greatly impacts on reproduction (reuter et al., 2011), gamete health (morgan and galione, 2007), larval development (brennand et al., 2010; stumpp et al., 2011a, b) calcification (byrne et al., 2011) and gene expression (o'donnell et al., 2010) of sea urchins. effects of ph value and water temperature on ___________________________________________________________________________ corresponding author: chong zhao key laboratory of north mariculture and stock enhancement ministry of agriculture dalian ocean university email: chongzhao@dlou.edu.cn behaviors of sea urchins remain largely unknown, although the fragility has showed in various of behaviors of marine fish (munday et al., 2009; cripps et al., 2011; simpson et al., 2011) and bivalves (chan et al., 2011). covering behavior refers to echinoids using their tube feet and spines to manipulate environmental objects, such as shells, stones and algae fragments, to put onto their dorsal surface (verling et al., 2002). covering behavior has been well proposed to have multiple functions of protection from lights (verling et al., 2002), predation (agatsuma, 2001), floating sand (richner and milinski, 2000) and wave surge (dumont et al., 2007). recently, effects of elevated temperature and acidification have been investigated separately (challener and mcclintock, 2013; zhao et al., 2014; brothers and mcclintock, 2015; zhang et al., 2017). the effects of water temperature and ph value on covering behavior, however, were not simultaneously investigated. acidification can be experimentally imitated by co2 (for example, dupont et al., 2013) or hcl (for example, yamada and ikeda, 1999) in laboratory. a comparative study indicates that hcl-induced acidification showed lower acute toxicity to aquatic animals than that induced by co2 (zhang et al., 2011). mailto:chongzhao@dlou.edu.cn 15 table 1 characteristics of sea urchins and covering materials water temperature 15°c 25°c ph value 7.4 8.2 7.4 8.2 glyptocidaris crenularis test diameter (mm) 16.55 ± 1.98 16.44 ± 1.76 16.58 ± 2.04 16.50 ± 1.89 test height (mm) 7.87 ± 1.14 8.20 ± 0.98 8.09 ± 0.98 8.16 ± 1.23 body weight (g) 2.04 ± 0.73 2.10 ± 0.71 2.10 ± 0.74 2.06 ± 0.74 mytilus galloprovincialis shell length (mm) 11.82 ± 0.81 11.91 ± 0.85 11.74 ± 0.84 11.59 ± 0.76 shell height (mm) 19.84 ± 2.61 20.31 ± 1.45 19.96 ± 1.39 19.45 ± 2.02 shell weight (g) 0.12 ± 0.02 0.13 ± 0.02 0.12 ± 0.02 0.12 ± 0.02 (mean ± sd, n = 36 for glyptocidaris crenularis, n = 40 for mytilus galloprovincialis) thus, hcl-induced acidification can be used to provide preliminary information on behavioral response of sea urchins to ocean acidification. the aim of the present study is to investigate the effects of water temperature and ph value on the covering behavior of the sea urchin glyptocidaris crenularis. materials and methods sea urchins glyptocidaris crenularis were originally reared at dalian haibao seafood company in lvshun of dalian, china. individuals from one cohort were transported to key laboratory of north mariculture and stock enhancement, dalian ocean university, china. all sea urchins were acclimated for 7 days at 19 21 °c of water temperature, 30 31 ‰ of salinity and 8.2 of ph before the behavioral experiment. there was no significant difference of test diameter, test height and body weight among experimental groups in g. crenularis (p > 0.05, table 1). experimental design geochemical models indicates that ocean acidification will be over 1.4 ph units in the next 300 years (caldeira and wickett, 2003). consequently, two ph values (7.4 and 8.2) were comparatively studied in the coral oculina patagonica (fine and tchernov, 2007). we accordingly set ph = 7.4 and 8.2 in the present study. the low ph value (ph = 7.4) was simply produced by adding hydrochloric acid to the natural seawater (ph = 8.2). to maintain water temperatures, the behavioral experiments were carried out in buckets (diameter of the bucket bottom = 22 cm) bathed in temperature-controlled tanks, where two water temperatures (15 °c and 25 °c) were set. forty shells of mytilus galloprovincialis were randomly selected and put into each bucket according to our previous study (zhao et al., 2014). there were no significant differences in shell length, shell width and body weight of m. galloprovincialis among experimental groups (table 1, p > 0.05). we then placed 144 sea urchins into the 12 buckets (12 individuals per bucket, 4 treatments, 3 replicates). because the experimental duration was relatively short, ph values were well maintained. time to first covering refers to the time of covering by the first sea urchin that covered, indicating the behavioral reaction. the number of covered sea urchins and number of shells used for covering were measured every 10 min during 90 min, indicating the behavioral capability. fig. 1 time to first covering of glyptocidaris crenularis at different water temperatures and ph values (n = 3, mean ± sd). 16 fig. 2 number of covered glyptocidaris crenularis at different water temperatures and ph values (n = 3, mean ± sd). statistical analysis all original data were tested for normal distribution and homogeneity of variance. repeated measured anova was used to detect the differences of number of covered sea urchins and number of shells used for covering among experimental groups. time to first covering was analyzed using mann-whiney u test because the data showed heterogeneity of variance. all analysis was carried out using spss 13.0. a probability level of p < 0.05 was considered statistically significant. results time to first covering time to first covering was significantly lower in sea urchins exposed to lower ph (p = 0.026, fig. 1). however, water temperature did not significantly affect time to first covering, although g. crenularis showed obviously quicker reaction at 25 °c than those at 15 °c (p = 0.180, fig. 1). number of covered sea urchins and number of shells used for covering lower ph significantly decreased number of covered sea urchins (p = 0.029, fig. 2) and number of shells used for covering (p = 0.007, fig. 3). water temperature did not significantly affect number of covered sea urchins (p = 0.157, fig. 2), though significantly affected number of shells used for covering (p = 0.042, fig. 3). interaction between water temperature and ph value showed no significant effect on number of covered sea urchins (p = 0.752) or number of shells used for covering (p = 0.989). observational duration significantly affected both number of covered sea urchins (p < 0.001) and number of shells used for covering (p = 0.01). both number of covered sea urchins and number of shells used for covering increased in the first 20 or 30 min. and then obviously decreased in lower ph groups (figs 2, 3). discussion water temperature and ph value significantly affected calcification (byrne et al., 2011), growth (albright et al., 2012) and behavior (cripps et al., 2011) of marine organisms, ultimately impacting the entire ecosystem (strandberg et al., 2012). considering their roles in structuring marine benthic communities, it is critical to understand ecological response of sea urchins to water temperature and ph value. the present study investigated the behavioral response of sea urchins to water temperatures and ph values. it provides preliminary information on behavioral ecology of sea urchins. in the present study, we found that lower ph value (ph = 7.4) significantly affected covering behavior of g. crenularis. this result is in agreement with previous reports on the behavioral response of marine organisms to ph values. in reef fish pseudochromis fuscus, for example, ocean acidification significantly disturbed its prey detection (cripps et al., 2011). lower ph value significantly decreased the number of covered sea urchins and number of shells used for covering. this result is not in agreement with the finding of challener and mcclintock (2013) that ocean acidification did not significantly impact covering behavior of juvenile sea 17 fig. 3 number of shells used for covering of glyptocidaris crenularis at different water temperatures and ph values (n = 3, mean ± sd). urchins lytechinus variegatus. the disagreement is probably due to the difference of behavioral response to ocean acidification between the two species. interestingly, we found that lower ph value significantly increased the speed of covering reaction. a reasonable explanation is that the acute stress increased the covering reaction. consistently, brothers and mcclintock (2015) found that acute exposure to elevated temperature significantly increased covering behavior of l. variegatus, compared to chronical exposure. water temperature did not significantly affect the time to first covering and number of covered sea urchins. crook (2003) found that paracentrotus lividus covered more quickly during summer time and obviously less in winter time. these results confirm the difference of the covering behavior among different species (verling et al., 2004). interestingly, we found number of shells used for covering was significantly different between sea urchins at 15 °c and 25 °c, although the p value was very close to 0.05 (p = 0.042). this result is not consistent with our previous study, in which we found no significant difference of number of shells used for covering between g. crenularis in 15 °c and 25 °c (zhao et al., 2014). this disagreement might be due to limited sample size in both studies and strongly highlights the individual variation of covering behavior of sea urchins. considering the multiple functions of covering behavior, our finding indicates that lower ph value could probably trigger a series of indirect ecological impacts on sea urchins (at least on g. crenularis). notably, the present study used hcl rather than co2 to set the ph value. the effects of co2 induced acidification should be further investigated. acknowledgements this work was supported by the national natural science foundation of china (41506177) and the chinese national 863 project (2012aa10a412). we thank h zhou, x tian and w feng for their assistance. all authors have no conflict of interest. references agatsuma y. effect of the covering behavior of the juvenile sea urchin strongylocentrotus intermedius on predation by the spider crab pugettia quadridens. fish. sci. 67: 1181-1183, 2001. albright r, bland c, gillette p, serafy je, langdon c, capo tr. juvenile growth of the tropical sea urchin lytechinus variegatus exposed to near-future ocean acidification scenarios. j. exp. mar. biol. ecol. 426: 12-17, 2012. brennand hs, soars n, dworjanyn sa, davis ar, byrne m. impact of ocean warming and ocean acidification on larval development and calcification in the sea urchin tripneustes gratilla. plos one 5(6): e11372, 2010. brothers cj, mcclintock jb. the effects of climate-induced elevated seawater temperature on the covering behavior, righting response, and aristotle's lantern reflex of the sea urchin lytechinus variegatus. j. exp. mar. biol. ecol., http://apps.webofknowledge.com/oneclicksearch.do?product=wos&search_mode=oneclicksearch&colname=wos&sid=3fgak9b6h7k4bjahi3b&field=au&value=soars,%20n http://apps.webofknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&colname=wos&sid=3fgak9b6h7k4bjahi3b&author_name=byrne,%20m&dais_id=1963402 18 467: 33-38, 2015. byrne m, ho m, wong e, soars na, selvakumaraswamy p, shepard-brennand h, et al. unshelled abalone and corrupted urchins: development of marine calcifiers in a changing ocean. proc. r. soc. lond. b biol. sci. 278: 2376-2383, 2011. caldeira k, wickett me. anthropogenic carbon and ocean ph. nature 425: 365, 2003. chan kyk, grunbaum d, o'donnell mj. effects of ocean acidification on swimming performance in larval sand dollars and oysters. integr. comp. biol. 51: e22, 2011. challener rc, mcclintock jb. exposure to extreme hypercapnia under laboratory conditions does not impact righting and covering behavior of juveniles of the common sea urchin lytechinus variegatus. mar. freshw. behav. physiol. 46: 191-199, 2013. cripps il, munday pl, mccormick mi. ocean acidification affects prey detection by a predatory reef fish. plos one 6(7): e22736 (1-7), 2011. crook ac. individual variation in the covering behaviour of the shallow water sea urchin paracentrotus lividus. mar. ecol. 24: 275-287, 2003. de madron x, durrieu, guieu c, sempere r, et al. marine ecosystems' responses to climatic and anthropogenic forcings in the mediterranean. prog. oceanogr. 91: 97-166, 2011. dumont cp, drolet d, deschênes i, himmelman jh. multiple factors explain the covering behaviour in the green sea urchin, strongylocentrotus droebachiensis. anim. behav. 73: 979-986, 2007. dupont s, dorey n, stumpp m, melzner f, thorndyke m. long-term and trans-life-cycle effects of exposure to ocean acidification in the green sea urchin strongylocentrotus droebachiensis. mar. biol. 160: 1835-1843, 2013. fine m, tchernov d. scleractinian coral species survive and recover from decalcification. science 315: 1811, 2007. halpern bs, walbridge s, selkoe ka, kappel cv, micheli f, d’agrosa c, et al. a global map of human impact on marine ecosystems. science 319: 948-952, 2008. morgan aj, galione a. fertilization and nicotinic acid adenine dinucleotide phosphate induce ph changes in acidic ca2+ stores in sea urchin eggs. j. bio. chem. 282: 37730-37737, 2007. munday pl, dixson dl, donelson jm, jones gp, pratchett ms, devitsina gv, et al. ocean acidification impairs olfactory discrimination and homing ability of a marine fish. proc. natl. acad. sci. usa 106: 1848-1852, 2009. o'donnell mj, todgham ae, sewell ma, hammond lm, ruggiero k, fangue na, et al. ocean acidification alters skeletogenesis and gene expression in larval sea urchins. mar. ecol-prog. ser. 398: 157-171, 2010. orr jc, fabry vj, aumont o, bopp l, doney sc, feely ra, et al. anthropogenic ocean acidification over the twenty-first century and its impact on organisms. nature 437: 681-686, 2005. reuter ke, lotterhos ke, crim rn, thompson ca, harley cdg. elevated pco2 increases sperm limitation and risk of polyspermy in the red sea urchin strongylocentrotus franciscanus. global change biol. 17: 163-171, 2011. richner h, milinski m. on the functional significance of masking behaviour in sea urchins-an experiment with paracentrotus lividus. mar. ecol. prog. ser. 205: 307-308, 2000. sabine cl, feely ra, gruber n, key rm, lee k, bullister jl, et al. the oceanic sink for anthropogenic co2. science 305: 367-371, 2004. simpson sd, munday pl, wittenrich ml, manassa r, dixson dl, gagliano m, et al. ocean acidification erodes crucial auditory behaviour in a marine fish. biol. lett-uk 7: 917-920, 2011. solomon s, qin d, manning m, chen z, marquis m. et al. climate change 2007: the physical science basis: contribution of working group i to the fourth assessment report of the intergovernmental panel on climate change. new york: cambridge univ. press, 2007. strandberg m, damgaard c, degn hj, bak j, nielsen ke. evidence for acidification-driven ecosystem collapse of danish erica tetralix wet heathland. ambio. 41: 393-401, 2012. stumpp m, dupont s, thorndyke mc, melzner f. co2 induced seawater acidification impacts sea urchin larval development ii: gene expression patterns in pluteus larvae. comp. biochem. physiol. 160a: 320-330, 2011a. stumpp m, wren j, melzner f thorndyke mc, dupont st. co2 induced seawater acidification impacts sea urchin larval development i: elevated metabolic rates decrease scope for growth and induce developmental delay. comp. biochem. physiol. 160a: 331-340, 2011b. verling e, crook a, barnes d. covering behaviour in paracentrotus lividus: is light important? mar. biol. 140: 391-396, 2002. yamada y, ikeda t. acute toxicity of lowered ph to some oceanic zooplankto n. plankton biol. ecol. 46: 62-67, 1999. zhang dj, li sj, wang gz, guo dh. comparative study on the acute toxicity of ocean acidification driven by co2 and hcl on several marine copepods. j. xiamen univ. 50: 631-636, 2011. zhang l, zhang l, shi d, wei j, chang y, zhao c. effects of long-term elevated temperature on covering, sheltering and righting behaviors of the sea urchin strongylocentrotus intermedius. peer j. 5:e3122, 2017. zhao c, luo s, zhou h, tian x, chang y. effect of temperature on covering behavior of the sea urchins glyptocidaris crenulars and strongylocentrotus intermedius. oceanologia et limnologia sinica 45: 522-528 (in chinese with an english abstract), 2014. http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=byrne,%20m&ut=1963402&pos=%7b2%7d http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=ho,%20m http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=ho,%20m http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=soars,%20na http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=selvakumaraswamy,%20p&ut=14338795&pos=%7b2%7d http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=shepard-brennand,%20h characterization of a natural hemagglutinin in the serum of a mud crab scylla serrata isj 7: 79-88, 2010 issn 1824-307x research report physico-chemical characterization of bacterial and hemagglutinins from the serum of the mud crab scylla serrata ss jayaraj1, r thiagarajan2, m arumugam2, s vincent1 1unit of environmental health and biotechnology, department of advanced zoology and biotechnology, loyola college, chennai 600 034, india 2unit of pathobiology, department of zoology, university of madras, guindy campus, chennai 600 025, india accepted february 15, 2010 abstract a naturally occurring hemagglutinin (ha), with activity against bacteria and yeast cells were detected in the serum of scylla serrata using mammalian erythrocytes (rbc), various bacteria and yeast as indicator cells. the serum gave highest ha titer with rabbit rbc, tripsinized yeast and vibrio fluvialis. an analysis of the physico-chemical properties of the ha showed it to be specifically dependent on the presence of ca2+ for its activity, stable between ph 7 to 9 and showed thermal stability between 10 to 30 °c. further studies demonstrated that the ha is precipitable by ammonium sulphate and tca. hainhibition assays performed with carbohydrates revealed that the serum ha was specific for non-reducing terminal glucose with 1-2 glucosidic linkages. thus this agglutinin appears to be unique among all the known crustacean agglutinins. key words: hemagglutinin; bacterial agglutinin; yeast agglutinin; serum; mud crab; scylla serrata introduction mud crab scylla serrata is an economically important decapod crustacean in aquaculture in india. understanding the defence system of s. serrata at humoral and cellular levels is indispensable for preventing disease occurrence. lectins are carbohydrate-binding proteins and in invertebrates, lectins are vital means for non-self recognition and clearance of invading microorganisms. in invertebrates, phagocytosis is considered to be the primary mechanism of innate defense against foreign invaders (ratcliffe and rowley, 1981; coombe et al., 1984; ratcliffe et al., 1985). in this process, an intimate interaction of humoral substances, particularly as recognition factors, has been implicated (richards and renwrantz, 1991; zelck and becker, 1992). a variety of humoral factors, naturally occurring and/or formed after antigenic stimulation, have been detected in the serum of invertebrates and they include agglutinins (cornick and stewart, 1973; renwrantz, 1986; nalini et al., 1994; murali et al., 1999; jayasree, 2001; jayaraj et al., 2008a), lysins (osada et al., 1993), antibacterial (xylander and neverman, 1990), and antifungal proteins (iijima et al., 1993), phenoloxidase system (söderhäll, 1982), lps binding protein (jomori and natori, 1992) and β ___________________________________________________________________________ corresponding author: ss jayaraj department of advanced zoology and biotechnology loyola college, chennai, india e-mail: jayarajss@gmail.com 1, 3 glucan binding protein (jayaraj et al., 2008b). due to the probable functional similarities between agglutinins and vertebrate antibodies and the indications that agglutinins serve a defensive function (ofek and sharon, 1988), invertebrate agglutinins have been extensively studied. agglutinins (= lectins) are dior multivalent carbohydrate-binding proteins with the ability to agglutinate cells with complementary carbohydrates on their surfaces (sharon and lis, 1972; barondes, 1988). they are known to specifically recognize the whole sugar (bretting and kabat, 1976), a specific site in a sugar (shimizu et al., 1977), a sequence of sugars (kobiler and mirelman, 1980), or their glycosidic linkages (koch et al., 1982). the agglutinating molecules are widely distributed in microorganisms (sasmal et al., 1992), plants and animals (gold and balding, 1975). the body fluid or hemolymph of almost all invertebrate species tested contains agglutinins (yeaton, 1981; ratcliffe et al., 1985; renwrantz, 1986; nalini et al., 1994; murali et al., 1999; jayasree, 2001; jayaraj et al., 2008a). the presence of agglutinins has also been detected in the mucus as well as in certain tissues of invertebrates (renwrantz, 1986; mullainadhan and renwrantz, 1989; suzuki and mori, 1991). however, its immunological role is best understood in the hemolymph, and recent studies have shown that purified, hemolymph-derived agglutinins served as opsonin in a few insects and molluscs (renwrantz and stahmer, 1983; pendland 79 mailto:jayarajss@gmail et al., 1988; fryer et al., 1989; richards and renwrantz, 1991; jayaraj et al., unpublished observations). although a number of studies have demonstrated the presence of humoral agglutinins in several crustacean species, it can be noted that the immunological roles of these agglutinins remain largely unknown and that the carbohydrate specificity of serum agglutinins from crustaceans have been elucidated only in a few species (vasta et al., 1983; smith and chisholm, 1992; nalini et al., 1994; kondo et al., 1998; jayasree, 2001). this study thus describes rbc and bacterial binding activities, physico-chemical properties and carbohydrate specificity of a naturally occurring agglutinin in the serum of the marine crab s. serrata. material and methods experimental animals and laboratory maintenance the marine crab scylla serrata weighing 150 to 200 g were obtained from muttukadu estuary, chennai. in the laboratory, these crabs were maintained in plastic tanks (90 x 45 x 60 cm) containing aerated natural seawater and the medium was changed every day. the crabs were fed with donax spp. during the period of acclimation (24 h) and only male crabs were used. preparation of serum hemolymph samples (1 to 2 ml) from individual crabs were collected from the cut end of the dactylus region of the walking leg. the samples were collected in clean polystyrene plastic tubes held on ice and allowed to clot at room temperature (rt: 28 ± 2 °c for 20 min). serum was separated by centrifugation (400xg, 10 min, rt) and the resulting clear supernatant (=serum) was used immediately. preparation of erythrocyte (rbc) suspension human and other mammalian blood samples were obtained by venous or cardiac puncture and collected in sterile alsever’s solution (garvey et al., 1979) containing 10 µg/ml of streptomycin. prior to use, the rbcs were washed thrice with 0.9% saline and once with tbs-i (50 mm tris-hcl, 115 mm nacl, 10 mm cacl2, 300 mosm) by centrifugation (400xg, 5 min, rt). unless specified, the rbc pellet was finally resuspended in tbs-i as 1.5% suspension (v/v). preparation of yeast cell suspension a hundred mg commercial grade baker’s yeast (saccharomyces cerevisiae) purchased from local market were suspended in 10 ml of 0.9 % saline, washed extensively with saline by centrifugation (400xg, 5 min, rt) and suspended in the same medium. the yeast cell suspension was heatinactivated by autoclaving the suspension for 15 min at 15 psi. after cooling the suspension to rt, the heat-inactivated yeast cells were washed extensively with 0.9 % saline and finally resuspended in tbs-i as 0.5 % (v/v) suspension. trypsinization of yeast cells five μl of washed yeast cells were suspended in 1 ml of tbs-i containing trypsin (0.5 %) to give a final concentration of 0.5 % yeast. this suspension was incubated for 1 h at 37 oc with occasional gentle shaking. after incubation, the trypsinized yeast cells were washed once with tbs-i by centrifugation (400xg, 5 min, rt) and finally resuspended in tbs-i as 0.5 % (v/v) suspension. hemagglutination (ha) assay ha assays were performed in v-bottom microtiter plates (greiner, nürtingen, germany) by serial two-fold dilution of a 25 µl serum sample with an equal volume of tbs-i. after dilution, 25 µl rbc suspension was added to each well and incubated for 45 min at rt. the ha titers were recorded as the reciprocal of the highest dilution of the sample causing complete agglutination of rbc (garvey et al., 1979). controls for all assays consisted of the substitution of the sample by tbs-i. all the ha assays were performed in duplicate. yeast agglutination assay the agglutinating activity of serum against yeast cells was performed in v-bottom microtiter plates by serial two-fold dilution of 25 µl serum with an equal volume of tbs-i. after dilution, 25 µl of 0.5 % native or trypsinized yeast cell suspension was added to each well and incubated for 45 min at 26 °c. control consisted of substitution of serum with tbs-i. the agglutination of yeast cells by serum was assessed under microscope (40 x) and the agglutination titers were recorded as the reciprocal of the highest dilution of the sample causing complete agglutination. the experiment was performed using duplicates. bacterial agglutinating activity frozen stock culture of bacteria were inoculated in tbs-i and incubated for 6 h. the broth cultures were then centrifuged (5,000xg, 10 min). the pellet was collected and washed 3 times by centrifugation with tbs-i. the final concentration was adjusted to 1x108 cells ml-1 in tbs-i before use. two-fold serial dilutions of serum samples (in duplicates) were made in tbs-i. then, 25 µl of each serum dilution was incubated with 25 µl bacterial suspension. the reaction mixture was incubated at 20 ± 2 °c for 1 h. the appearance of clumps of bacteria was then recorded by microscopic examination (40x). agglutination titer was defined as the reciprocal of the last dilution giving evidence of agglutination after incubation. the negative controls comprised mixed equal volumes of bacterial suspension and tbs-i. divalent cation dependency and edta sensitivity serum samples (each 300 µl in duplicates) were dialysed (mw exclusion limit <10,000) extensively at 20 °c against divalent cation-free tbs-ii (50 mm tris-hcl, 135 mm nacl, 300 mosm) to examine cation dependency or in tbs-iii containing 50 mm edta (50 mm tris-hcl, 72 mm nacl, 40 mm cacl2, 300 mosm) to test edta sensitivity of the agglutinating activity of serum. the samples dialysed against tbs-iii were subsequently re-equilibrated by dialysis in tbs-ii. all the resulting dialysates were centrifuged (400xg, 10 min, 20 °c). the supernatant was tested for hemagglutinating activity using rabbit rbc in the presence of tbs 80 that did or did not contain 10 mm cacl2, mgcl2 (or) mncl2. a serum sample (300 µl) concurrently dialysed against, tbs containing 10 mm cacl2 (tbs-i) was also tested for the hemagglutinating activity against rabbit rbc in tbs-i. ph and thermal stability the stability of serum ha activity (in duplicates) at different ph was examined by dialyzing (24 h, 4 °c) 300 μl serum samples against the following buffers at ph ranging from 3 to 12 (lillie, 1954; pearse, 1968): 0.2 m acetate buffer (ph 3 to 6), 0.2 m tris-hcl buffer (ph 7 to 9) and 0.1 m glycinenaoh buffer (ph 10 to 12). after dialysis, all the samples were finally re-equilibrated by dialysis against tbs-i and the ha titer was determined with rabbit rbc. in another experiment designed to study the thermal stability of ha, 300 μl serum samples were held for 30 min at temperatures ranging from 10 to 100 °c, centrifuged and tested for ha activity with rabbit rbc. precipitation by ammonium sulphate and trichloro acetic acid precipitation of ha activity from serum (in duplicates) was attempted using 25, 50 and 75 % ammonium sulphate [(nh4)2so4] solution as well as 10 % trichloroacetic acid (tca) as described previously (millar, 1987). the ha activity was finally measured using rabbit rbc. ha-inhibition assays several carbohydrates were tested for their ability to inhibit serum ha activity. they were dissolved in tbs-iii (50 mm tris-hcl, 115 mm nacl, 50 mm edta, 300 mosm) and if necessary, the ph was adjusted to 7.5 using concentrated naoh. serum samples were diluted with tbs-iv (50 mm trishcl, 5 mm nacl, 30 mm cacl2, 135 mosm) to a ha titer of 4 against rabbit rbc. the inhibitor to be tested (25 μl) was serially diluted two-fold with an equal volume of diluted sample in microtiter plates and incubated for 1 h at rt. rabbit rbc suspension (25 μl) was added to each well and kept for 3 h at rt. the minimal concentration of carbohydrate that completely inhibited ha activity was recorded. the experiments were performed in duplicates. protein determination total protein concentration was measured using bovine serum albumin (bsa) as a standard (lowry et al., 1951). result serum ha profile the serum of mud crab scylla serrata agglutinated a variety of mammalian rbc types. among the various rbc types tested, the highest titer of 128 was obtained with rabbit erythrocytes. the serum did not discriminate human a, b and o rbc types and agglutinated them to the same degree. sheep and goat rbc were agglutinated at relatively low titers (table 1). however serum did not agglutinate ox rbc and the serum showed highest agglutinating activity against tripsinized yeast cells when compare to native yeast cells (table 2). table 1 hemagglutinating (ha) activity of serum from the mud crab s. serrata against various mammalian erythrocyte (rbc) types rbc types tested ha titer* rabbit 128 mice 64 rat 32 human b 32 buffalo 16 human a 8 human o 8 horse 4 goat 2 sheep 2 ox 0 * based on 20 determinations for each rbc type bacterial agglutination the serum strongly agglutinated vibrio fluvialis (titer 8), weekly agglutinated vibrio parahemolyticus, vibrio mimicus, escherichia coli, pseudomonas spp. and aerobacter aerogenes. the results of bacterial agglutination was assessed using a phase-contrast microscope (table 3). divalent cation dependency an edta sensitivity the serum tested in tbs containing 10 mm cacl2 (tbs-i) gave a hemagglutianation titer of 128 against rabbit rbc. when the serum was dialysed against tbsi and then tested in the absence of divalent cation, the agglutination titer reduced to 16. but, this serum sample recovered it’s ha activity only upon addition of ca2+ to the reaction mixture. further, substitution of ca2+ with mg2+ showed a considerable improvement in ha titer, while mn2+ could not reverse the effect of edta treatment. the serum dialyzed against tbs-iii containing 50 mm edta and tested in the absence of divalent cation, considerably lost its agglutinating activity against rabbit rbc (table 4). further the addition of mg2+ or 81 mn2+ to this sample could not restore the original ha activity and addition of ca2+, rescued the activity to 64 (table 4). ph and thermal stability the serum hemagglutinating activity of the marine crab s serrata was tested in the ph range of 3-12. as shown in figure 1, the hemagglutinating activity against rabbit rbc was found to be relatively stable between ph 7 and 9 reduced at ph below or above this ph range and completely lost at ph 11 and 12. the activity of the serum against rabbit rbc was unaffected only up to 30 °c but it was reduced considerably at 40 °c and 50 °c and completely inactivated at 60 °c and above (fig. 2). precipitation of serum ha activity by ammonium sulphate and tca serum was incubated with ammonium sulphate (25, 50 and 75 %) for 3 h. the 50 % concentration of ammonium sulphate completely precipitated ha activity from the serum, whereas ammonium sulphate concentration at 25 and 75 %, moderately precipitated the serum ha activity. by contrast 10 % tca failed to precipitate serum ha activity (table 5). carbohydrate binding specificity among the 24 carbohydrates tested, as many as 15 carbohydrates were found to inhibit serum hemagglutinating activity at concentrations ranging from 50 to 100 mm. all the three acetylated hexosamines (glcnac, galnac and mannac), but not their hexoses and hexosamine counterparts, were inhibitory at 50 or 100 mm. but the few sialic acids examined in this study and 9 other carbohydrates were not inhibitory when tested up to concentrations from 20 to 200 mm (table 6). among the six different polysaccharides tested (table7), only mannan and laminarin inhibited the ha activity at 0.25 and 0.50 mg/ml, respectively. among all the inhibitory carbohydrates, mannan was found to be most potent. table 2 agglutinating activity of s. serrata serum against native and trypsinized yeast cells yeast cells tested ha titer* native 16 trypsinized 64 * based on 20 determinations for native and trypsinized yeast cells discussion the serum of the marine mud crab s. serrata was found to possess naturally occurring agglutinating activity which showed the highest reactivity with rabbit rbc among the rbc types tested. these results also suggest that the rbc types agglutinated by the serum of s. serrata probably share a common surface receptor but with a quantitative difference in its ha binding sites. the serum agglutinated a variety of bacteria including gram + and gramtypes and the species of vibrio tested are known to be the most frequent opportunistic pathogens of aquatic crustaceans (equidius, 1987; vargas-albores et al., 1993) and the plasma showed highest agglutinating activity against trypsinized yeast cells (jayaraj et al., 2008b). the ability of the serum of s. serrata to agglutinate bacteria, particularly the potential pathogens, implicates a possible involvement of the humoral agglutinins in host defense response. table 3 agglutinating activity of s. serrata serum against various bacterial species bacterial species tested bacterial agglutination* (o.d: 0.8) vibrio fluvialis 16 vibrio alginolyticus 8 vibrio vulnificus 8 vibrio anguillarum 4 vibrio parahemolyticus 4 vibrio mimicus 2 escherichia coli 2 pseudomonas sp 2 bacillus subtilis 4 aerobacter aerogenes 2 * the assay was repeated six times for each bacterial species with identical results using samples from different preparations 82 table 4 effect of divalent cations and edta on the hemagglutinating (ha) activity of serum of s. serrata serum sample tested cation (10 mm) in sample diluting and rbc suspension ha titer* before dialysis cacl2 128 after dialysis against divalent cation free tbs (tbs-ii). none cacl2 mgcl2 mncl2 16 128 64 16 after dialysis against tbs+10 mm cacl2 (tbs-i) cacl2 128 after dialysis against tbs+50 mm edta (tbs-iii) followed by dialysis against tbsii none cacl2 mgcl2 mncl2 8 64 4 4 * determination using rabbit rbc and the results based on six determinations the serum agglutinin was heat-labile and susceptible to ph extremes. its ph stability was comparable to that found in one earlier report (nalini et al., 1994) and the proteinaceous nature of agglutinin has been well demonstrated (mckay and jenkin, 1969; acton et al., 1969; miller et al., 1972). the serum lost most of it’s ha activity after dialysis against cation-free tbs and when tested in the absence of cations. however, the activity in this sample completely regained only upon addition of ca2+ and the ha titer of serum did not change after dialysis against tbs containing ca2+. these observations demonstrated that the serum agglutinin of s. serrata specifically requires ca2+ for it’s ha activity. furthermore, the activity was sensitive to edta treatment, since dialysis of serum against tbs containing edta resulted in a significant reduction in the ha activity. none of the cations tested could restore the ha activity, albeit ca2+ moderately rescued the activity in these samples, thereby indicating that the ha of s. serrata appears to be irreversibly sensitive to edta which is in contrast with other crustacean agglutinins (hall and rowlands, 1974a; ravindranath et al., 1985; kamiya et al., 1987). crustacean serum agglutinins were shown to be specific for fucose (amirante and basso, 1984), glucose (umetsu et al., 1991), galactose (kamiya et al., 1987; umetsu et al., 1991), galnac (amirante and basso, 1984; vargas-albores et al., 1993), or sialic acids such as neuac (hall and rowlands, 1974b; vasta et al., 1983; vasta and cohen, 1984; cassels et al., 1986; ratanapo et al., 1990), 4and 9-0-acetyl neuac (ravindranath et al., 1985), 9-0acetyl neuac (vazquez et al., 1993) and neugc (mercy and ravindranath, 1993). the hemagglutination-inhibition test performed in this study using different carbohydrates, encompassing several diverse, unrelated monosaccharides and their derivatives as well as diand oligo-saccharides inhibited the serum agglutinating activity. furthermore, all the three acetylated hexosamines tested consistently inhibited the ha activities of crab table 5 ammonium sulphate and tca precipitation of haemagglutination (ha) activity against rabbit rbc from the serum of s. serrata ammonium sulphate and tca (%) saturation ha titer* (rabbit rbc) untreated sample 128 25 % 16 50 % 128 75 % 32 tca 10 % 0 * determination using rabbit rbc and the results based on six determinations 83 table 6 inhibition of agglutinating activity (titer = 4) of serum from the mud crab s. serrata by various carbohydrates carbohydrates tested maximum concentration tested (mm) minimum inhibitory concentration (mm)* monosaccharides simple sugars d-mannose 200 100 l-sorbose 100 100 d-fucose 100 100 l-fucose 100 50 deoxy sugars l-rhamnose 200 100 n-acetyl sugars n-acetyl-d-glucosamine (glcnac) 200 100 n-acetyl-d-galactosamine (galnac) 200 100 n-acetyl-d-mannosamine (mannac) 200 50 disaccharides trehalose (glcα1 → 1 glc) 200 50 cellobiose (glcβ1 → 4 glc) 200 100 β-gentiobiose (glcβ1 → 6 glc) 200 50 sucrose 200 100 palatinose (glcα1 → 6 fruc) 200 50 melibiose (galα1 → 6 glc) 200 100 lactose (galβ1 → 4 glc) 200 100 the following carbohydrates also did not inhibit the agglutinating activity and unless otherwise stated, all carbohydrate was tested at concentrations upto 200 mm: d-glucose, d-galactose, β-allose, d-fructose, dglucosamine (glcn), d-galactosamine (galn), mannosamine (mann), maltose (glcα1 → 4 glc), turanose (glcα1 → 3 fruc). * the assay was repeated five times for each carbohydrate with identical results table 7 agglutination–inhibition of s. serrata serum (agglutination titer = 4) by polysaccharides against rabbit rbc polysaccharides tested structural linkages maximum concentration tested (mg. ml-1) minimum inhibitory concentration (mg. ml-1)* mannan (α 1-6 homopolymer of mannose) 1 0.25 laminarin (β 1-3 homopolymer of glucose) 1 0.50 dextran t70 (α 1-6,3,2 homopolymer of glucose) 2 ni dextran t500 (α 1-6,3,2 homopolymer of glucose) 2 ni inulin (α 2-6 homopolymer of fructose) 5 ni colominic acid (α 2-8 homopolymer of neu5ac) 5 ni * the assay was repeated three times for each polysaccharide with identical results using samples from different preparations ni: no inhibition 84 fig. 1 ph stability of hemagglutinating activity of serum of s. serrata against rabbit rbc serum and this has been earlier demonstrated (amirante and basso, 1984; vargas-albores et al., 1993; mercy and ravindranath,1993). the serum ha activity of s. serrata was not inhibited by the amino sugar tested. but it was inhibited by the simple hexoses namely mannose, l-sorbose, dfucose and l-fucose. this finding is supported by previous studies wherein simple hexoses were shown to inhibit agglutinating activity, including fucose (amirante and basso, 1984), glucose (umetsu et al., 1991). the c-l position of these hexoses is essential for interaction with the agglutinin. the amino derivatives (glcn, galn and mann) did not inhibit the ha activity. however, their n-acetyl derivatives (glcnac, galnac and mannac) were able to inhibit the serum ha activity. recently, alpuche et al. (2005) have shown the inhibition of purified shrimp lectin by nacetylated sugars and this lends support to our study. the disaccharides d-maltose and turanose failed to inhibit the ha activity but all other disaccharides were inhibitory. fig. 2 thermal stability of hemagglutinating activity of serum of s. serrata against rabbit rbc 85 all these observations clearly demonstrate that the presence of acetyl group at c-2 position of hexosamines does not favour the interaction with agglutinin whereas this position with a free hydroxyl group or its substitution with amino group is essential for the interaction. ha inhibition tests employing polysaccharides indicated that only laminarin and mannan inhibited the serum agglutinating activity. this indicates that the agglutinin molecules in crab serum tend to exhibit affinity for extended structures particularly for polysaccharides with β-linked hexoses. this is significant and shows the ability of the serum agglutinating activity to recognize a wide range of carbohydrates that will potentially help the animal to recognize a variety of pathogens based on their surface molecules (zhang et al., 2009). thus, all the results obtained from the inhibitory effects of various carbohydrates and glycoproteins taken together clearly indicate that the agglutinins present in the serum of s. serrata interact with a wide range of carbohydrates including acetylated hexosamines, acetylated or non-acetylated sialic acids and several other carbohydrates, though their preference for a specific carbohydrate structure could not be ascertained. however, these findings in turn strongly suggest the natural occurrence of multiple agglutinins in the serum of this crab. the agglutinins in several crustaceans have been characterized (cornick and stewart, 1968; mckay and jenkin, 1969, 1970; huang et al., 1981; vasta et al., 1983; ratanapo et al.,1990; adams, 1991; nalini et al., 1994; jayasree, 2001). this agglutinin appears to be unique among all the known crustacean agglutinins. thus, based on the hemagglutination and bacterial agglutinating activity of the serum agglutinin, it is possible that this component of the crab is probably involved in non-self recognition and eliciting immune response in the mud crab s. serrata, against invading pathogens. the identification of immune effectors like agglutinin and the understanding of their regulation in response to infection will open the way to the selection of pathogen resistant animals. this can be achieved through the characterization and purification of this novel agglutinin of s. serrata as a prerequisite to elucidate the immunological roles of crustacean agglutinins. references acton rt, bennet jc, evans ee, schrohenloher re. physical and chemical characterization of an oyster haemagglutinin. j. biol. chem. 244: 4128-4135, 1969. adams a. response of penaeid shrimp to exposure to vibrio species. fish shellfish immunol. 1: 5970, 1991. alpuche j, pereyra a, agundis c, rosas c, pascual c, slomianny m-c, vázquez l, zenteno e. purification and characterization of a lectin from the white shrimp litopenaeus setiferus (crustacea decapoda) hemolymph. biochim. biophy. acta 1724: 86-93, 2005. amirante ga, basso v. analytical study of lectins in squilla mantis l. (crustacea stomatopoda) using monoclonal antibodies. dev. comp. immunol. 8: 721-726, 1984. barondes sh. bifunctional properties of lectins: lectins redefined. trends biochem. sci. 13: 480-482, 1988. bretting h, kabat ea. purification and characterization of agglutinins from the sponge axinella polypoides and a study of their combining sites. j. biochem. 15: 3228-3236, 1976. cassels fj, marchalonis jj, vasta gr. heterogeneous humoral and hemocyteassociated lectins with n-acylamino-sugar specificities from the blue crab, callinectes sapidus rathbun. comp. biochem. physiol. 85b: 23-30, 1986. coombe dr, ey pl, jenkin cr. self/nonself recognition in invertebrates. quart. rev. biol. 59: 231-255, 1984. cornick jw, stewart je. interaction of the pathogen gaffkya homari with natural defence mechanisms of homarus americanus. j. fish res. board can. 25: 695-709, 1968. cornick jw, stewart je. partial characterization of a natural agglutinin in the hemolymph of the lobster, homarus americanus. j. inverteber. pathol. 21: 255-262, 1973. equidius e. vibriosis: pathogenicity and pathology. rev. aquacul. 67: 15-28, 1987. fryer se, hull cj, bayne cj. phagocytosis of yeast by biomphalaria glabrata: carbohydrate specificity of hemocyte receptors and a plasma opsonin. dev. comp. immunol. 13: 9-16, 1989. garvey js, cremer ne, sussdorf dh. methods in immunology, w.a. benjamin inc., reading, massachusetts, 1979. gold er, balding p. receptor-specific proteins, plant and animal lectins, excerpta medica, elsevier publishing co., amsterdam, 1975. hall jl, rowlands dt. heterogeneity of lobster agglutinins. i. purification and physicochemical characterization. j. biochem. 13: 821-827, 1974a. hall jl, rowlands dt. heterogeneity of lobster agglutinins. ii. specificity of agglutininerythrocyte binding. j. biochem. 13: 828-832, 1974b. huang mtf, eble af, hammen cs. immune response of the prawn macrobrachium rosenbergii, to bacterial infection. j. invertebr. pathol. 38: 213-219, 1981. iijima r, kurata s, natori s. purification, characterization and cdna cloning of an antifungal protein from the hemolymph of sarcophaga peregrina (flesh fly) larvae. j. biol. chem. 268: 12055-12061, 1993. jayasree s. biological properties of a natural agglutinin in the hemolymph of indian white prawn, penaeus indicus h. milne edwards. aquaculture 194: 245-252, 2001. jayaraj ss. thiagarajan r, vincent s, arumugam m. characterization of a natural haemagglutinin from the plasma of marine mussel perna viridis. bull. europ. assoc. fish pathol. 28: 76-84, 2008a. jayaraj ss, thiagarajan r, arumugam m, mullainadhan p. isolation, purification and characterization of β-1, 3-glucan binding protein from the plasma of marine mussel perna viridis. fish shellfish immunol. 24: 715-725, 2008b. 86 http://www.sciencedirect.com/science/journal/03044165 http://www.sciencedirect.com/science/journal/03044165 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22amirante%20ga%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22basso%20v%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); javascript:al_get(this,%20'jour',%20'j%20biochem%20(tokyo).'); http://www.sciencedirect.com/science/journal/00222011 http://www.sciencedirect.com/science/journal/00222011 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%236888%231973%23999789996%23544715%23flp%23&_cdi=6888&_pubtype=j&view=c&_auth=y&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=d35323e9f6b8eee0663a00411fa82786 javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); javascript:al_get(this,%20'jour',%20'j%20biochem%20(tokyo).'); javascript:al_get(this,%20'jour',%20'j%20biochem%20(tokyo).'); http://www.sciencedirect.com/science/journal/00222011 http://www.sciencedirect.com/science/journal/00222011 jomori t, natori s. function of the lipopolysaccharide-binding protein of periplaneta americana as an opsonin. febs lett. 296: 283-286, 1992. kamiya h, muramoto k, ooto r. isolation and characterization of agglutinins from the hemolymph of an acorn barnacle megabalanus volcano. dev. comp. immunol. 11: 297-307, 1987. kobiler d, mirelman d. lectin activity in entamoeba histolytica trophozoites. infect. immun. 29: 221225, 1980. koch am, lee ck, uhlenbruck g. cerianthin lectins: a new group of agglutinins from cerianthus membraneceus. immunobiology 163: 53-62, 1982. kondo m, itami t, takahashi y. lectin in the hemolymph of kuruma prawn. fish pathol. 33: 429-435, 1998. lillie rd. histopathologic technique and practical histochemistry, blackiston company inc., new york, 1954. lowry oh, rosenborough n, farr al, randall rj. protein measurement with the folin-phenol reagent. j. biol. chem. 193: 265-275, 1951. mckay d, jenkin cr. immunity in the invertebrates. ii adaptive immunity in the crayfish (parachaeraps bicarinatus). immunology 17: 127-137, 1969. mckay d, jenkin cr. immunity in the invertebrates. the role of serum factors in phagocytosis of erythrocytes by haemocytes of the freshwater crayfish (parachaeraps bicarinatus). aust. j. exp. biol. med. sci. 48: 139-150, 1970. mercy srpd, ravindranath mh. purification and characterization of n-glycolyneuraminic-acidspecific lectin from scylla serrata. eur. j. biochem. 215: 697-704, 1993. millar vh, ballaback rs, pauley gb, krassnis sm. a preliminary physico-chemical characterization of agglutinin found in the hemolymph of the crayfish procambarus clarkii. j. inverterbr. pathol. 19: 83-93, 1972. millar da, ratcliffe na. activity and preliminary characterization of a haemagglutinin from the hemichordate saccoglossus ruber. dev. comp. immunol. 11: 309-320, 1987. mullainadhan p, renwrantz l. comparative analysis of agglutinins from hemolymph and albumin gland of helix pomatia. j. comp. physiol. 159b: 443-452, 1989. murali s, mullainadhan p, arumugam m. purification and characterization of a natural agglutinin from the serum of the hermit crab diogenes affinis. biochim. biophys. acta 1472: 13-24, 1999. nalini m, mullainadhan p, arumugam m. characterization of a natural haemagglutinin in the serum of a freshwater crab parathelphusa hydrodromus (herbst). arch. inter. physiol. bioch. biophys. 102: 259-264, 1994. ofek i, sharon n. lectin phagocytosis: a molecular mechanism of recognition between cell surface sugars and lectins in the phagocytosis of bacteria. infect. immun. 56: 539-547, 1988. osada m, ito t, matsutani t, mori k. partial purification and characterization of hemolysin from the coelomic fluid of strongylocentrotus nudus. comp. biochem. physiol. 105b: 43-49, 1993. pearse age. histochemistry: theoretical and applied, vol. i, churchill livingston, london, 759 pp, 1968. pendland jc, health ma, boucias dg. function of a galactose-binding lectin from spodoptera exigua larval hemolymph: opsonization of blastospores from entomogenous hypomycetes. j. insect physiol. 34: 533-540, 1988. ratanapo s, chulavatnatol m, monodin m. a new sialic acid-specific lectin from black tiger prawn (penaeus monodon). comp. biochem. physiol. 97b: 515-520, 1990. ratcliffe na, rowley af. invertebrate blood cells, vol. 2, academic press, new york, 1981. ratcliffe na, rowley af, fitzgerald sw, rhodes cp. invertebrate immunity: basic concepts and recent advances. inter. rev. cytol. 97: 183-350, 1985. ravindranath mh, higa hh, cooper el, paulson jc. purification and characterization of an oacetylsialic acid-specific lectin from a marine crab cancer antennarius. j. biol. chem. 260: 8850-8856, 1985. renwrantz l. lectins in molluscs and arthropods: their occurrence, origin and roles in immunity. in: immune mechanisms in invertebrate vectors, am lackie (ed), symp. zool. soc. lond. 56: 81-93, clarendon press, oxford, 1986. renwrantz l, stahmer a. opsonizing properties of an isolated hemolymph agglutinin and demonstration of lectin-like recognition molecules at the surface of hemocytes from mytilus edulis. j. comp. physiol. 149b: 535546, 1983. richards eh, renwrantz lr. two lectins on the surface of helix pomatia haemocytes: ca2+dependent, galnac-specific lectin and a ca2+independent, mannose 6-phosphate-specific lectin which recognises activated homologus opsonins. j. comp. physiol. 16b: 43-54, 1991. sasmal d, guhathakurta b, ghosh an, pal cr, datta a. n-acetyl-d-glucosamine specific lectin purified from vibrio cholerae 01. fems microbiol. lett. 98: 217-224, 1992. sharon n, lis h. lectins: cell-agglutinating and sugar-specific protein. sci. 177: 949-959. 1972. shimizu s, ito m, niwa m. lectins in the hemolymph of japanese horseshoe crab, tachypleus tridentatus. biochim. biophys. acta 500: 71-79, 1977. smith vj, chisholm rs. non-cellular immunity in crustaceans. fish shellfish. immunol. 2: 1-31, 1992. söderhäll k. prophenoloxidase activating system and melanization-a recognition mechanism of arthropod. dev. comp. immunol. 6: 601-611, 1982. suzuki t, mori k. immunolocalization and in vitro secretion of hemolymph lectin of the pearl oyster, pinctada fucata martensii. zool. sci. 8: 23-29, 1991. umetsu k, yamashita k, suzuki t. purification and carbohydrate-binding specificities of a blood 87 javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); http://www.sciencedirect.com/science/journal/00222011 http://www.sciencedirect.com/science/journal/00222011 javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); http://www.ncbi.nlm.nih.gov/pubmed/10572921?ordinalpos=19&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/10572921?ordinalpos=19&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/10572921?ordinalpos=19&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22umetsu%20k%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22yamashita%20k%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22suzuki%20t%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplus type b binding lectin from hemolymph of a crab charybdis japonica. j. biochem. 109: 718-721, 1991. vargas-albores f, guzman ma, ochoa jl. a lipopolysaccharide-binding agglutinin isolated from brown shrimp penaeus californiensis holmes haemolymph. comp. biochem. physiol. 104b: 407-413, 1993. vasta gr, cohen e. carbohydrate specificities of birgus latro (coconut crab) serum lectins. dev. comp. immunol. 8: 197-202, 1984. vasta gr, warr gw, marchalonis jj. serological characterization of humoral lectins from the freshwater prawn macrobrachium rosenbergii. dev. comp. immunol. 7: 13-20, 1983. vázquez l, massó f, rosas p, montaño l, centeno e. purification and characterization of a lectin from macrobrachium rosenbergii. comp. biochem. physiol. 105b: 617-623, 1993. xylander wer, neverman l. antibacterial activity in the hemolymph of myriapods (arthropoda). j. invertebr. pathol. 56: 206-214, 1990. yeaton rw. invertebrate lectins: i. occurrence. dev. comp. immunol. 5: 391-402, 1981. zelck u, becker w. biomphalaria glabrata: influence of calcium, lectins, and plasma factors on in vitro phagocytic behavior of hemocytes of noninfected or schistosoma mansoni-infected snails. exp. parasitol. 75: 126-136, 1992. zhang x-w, xu w-t, wang x-w, mu y, zhao x-f, yu x-q, et al. a novel c-type lectin with two crd domains from chinese shrimp fenneropenaeus chinensis functions as a pattern recognition protein. mol. immunol. 46:1626-1637, 2009. 88 javascript:al_get(this,%20'jour',%20'j%20biochem%20(tokyo).'); javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); http://www.sciencedirect.com/science/journal/00222011 http://www.sciencedirect.com/science/journal/00222011 javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); http://www.sciencedirect.com/science/journal/01615890 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235121%232009%23999539991%231045056%23fla%23&_cdi=5121&_pubtype=j&view=c&_auth=y&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=0e26e5290e78608baf0c21c302be43e2 monosaccharides disaccharides isj109.pdf 152 isj 2: 152-158, 2005 issn 1824-307x review insights into brown spider and loxoscelism mh appel1, r bertoni da silveira1,3, w gremski1,2, ss veiga1 1department of cell biology, federal university of paraná, jardim das américas, curitiba, paraná , brazil 2catholic university of paraná, health and biological sciences institute, curitiba, paraná brazil 3department of biochemistry, federal university of são paulo, são paulo, brazil accepted december 27, 2005 abstract loxosceles is a genus of cosmopolitan spiders comprising several species, and popularly known as brown spiders or brown recluses. brown spider bites can cause dermonecrotic lesions and systemic reactions known as loxoscelism. systemic effects are less common but may be severe or even fatal in some patients. systemic manifestations include intravascular hemolysis, disseminated intravascular coagulation and acute renal failure. a rapid diagnosis and an understanding of the venom’s molecular activity are crucial for satisfactory treatment. mechanisms by which venoms exert their deleterious effects are under investigation, and searches are underway for diagnostic envenomation assays. molecular biology is being used to produce quantities of several of the most important venom molecules and has contributed to the study and understanding of their mechanisms of action. key words: brown spider; loxoscelism; venom; recombinant toxins; dermonecrosis introduction more than 40,000 spider species exist, with probably 100,000 to be described, but only 3 taxa are recognized as dangerous, namely theridiidae, loxoscelidae and ctenidae. moreover, only the genera atrax, lactrodectus and loxosceles are associated with human deaths (escoubas et al., 2000; rash and hodgson, 2002). early european tales during the middle ages linked injuries or illness to spider bites (schienle et al., 2005). for example the tarantula bite was associated with a disease (tarantism) for which the cure was a frenetic dancing for 3-4 days. this energetic dance, called tarantella, is now a typical italian dance (isbister, 2004). today, as a consequence of mistaken diagnoses of spider bites, scientists are looking for methods to characterize and identify spider bites and their manifestations as well as to better understand the biological and molecular corresponding author: silvio s. veiga department of cell biology, federal university of paraná, jardim das américas,81531-990, curitiba, paraná, brazil e-mail: veigass@ufpr.br mechanisms of envenomation. the genus loxosceles (variously known as the brown spider, brown recluse, fiddleback, or gaucho spiders) is important in these studies because of its commonness in and around human dwellings. their bite is characterized by dermonecrosis and systemic effects known as loxoscelism (hogan et al., 2004). the first case of documented loxoscelism occurred in 1879 in tennessee. however, consistent data traced back about 50 years ago and were collected in chile, then other observations were made in brazil followed by the united states. these reports linked brown spider bite with necrotic skin lesions (macchiavello, 1947; atkin et al., 1958; sams et al., 2001). spiders’ habits have caused a close association with humans, and the number of bites is increasing and has become a public health problem in brazil, chile and the united states (da silva et al., 2004). most bites occur during sleep or dressing, and women are bitten more often than men. thighs, trunk, hands and arms are more often bitten (hogan et al., 2004). loxosceles spiders loxosceles spiders are known as violin (fiddleback) spiders due to a characteristic violin 153 shape on their cephalothorax (futrell, 1992). they are also known as brown spiders because their colour varies from a pale (l. laeta) to a dark brown (l. gaucho). loxosceles body length ranges from 8 to 15 mm with legs measuring from 8 to 30 mm (da silva et al., 2004). they are sedentary and nocturnal (andrade et al., 1999) with a lifetime of 3 – 7 years (andrade et al., 2000). brown spiders have three pairs of eyes (an important characteristic useful to identify the genus) (vetter and visscher, 1998). they build irregular, cottony webs (futrell, 1992) and normally prefer dead scavenged prey rather than live preys (sandidge, 2003). they can survive months without food or water and withstand temperatures ranging from 8 °c to 43 °c. they are not aggressive and prefer dark dry places (futrell, 1992; málaque et al., 2002; vetter and barger, 2002; da silva et al., 2004). the sexes produce venom with differences in volume, toxicity and compounds proportion (oliveira et. al., 1999). comparative analysis of sex and species in l. laeta and l. intermedia venom showed some biological activities (complement-dependent hemolysis and dermonecrosis) more prominent in venom from female spiders, especially from l. laeta (oliveira et al., 2005). epidemiology loxosceles spiders can be found distributed all over the world. in north america, the most important species are l. reclusa, l. deserta, l arizona, l. rufences (united states and mexico) and l. laeta (canada) (sams et al., 2001; vetter and bush, 2002a). europe, africa, middle east, some parts of asia, israel, and australia are hosts to some loxosceles species (futrell, 1992; borkkan et al., 1995; young and pincus, 2001; nicholson and graudins, 2003). in brazil, seven species have been described but three are the most implicated in human bites l. intermedia, l. gaucho and l. laeta (sezerino et al., 1998). from 1990 to 1993, the brazilian ministry of health received 17.781 reports of spiders’ bites, of which 36 % were due to loxosceles (sezerino et al., 1998). in the metropolitan area of curitiba, in the state of parana (southern brazil) about 3.000 brown spider bites are reported annually (málaque et al., 2002). in a retrospective study in florianopolis, in the state of santa catarina, brazil, 487 suspected cases of brown spider bites were found, 267 of which fulfilled the criteria for inclusion in the study (sezerino et al., 1998). in 359 cases of loxoscelism between january 1985 and december 1996 at butantan intitute, são paulo, brazil, 14 % of patients captured the spiders so that 28 were classified as l. gaucho, 5 as l. laeta and 18 as non-classified loxosceles (málaque et al., 2002). more bites occur in warmer months (schenone, 1996). in curitiba, from 1998 to 2001 the incidence of loxosceles bites was 1.4 cases per 1,000 habitants. of these, 23 % were in the thigh, 16.7 % in the trunk, 14 % in the arm and 13 % in the lower leg. only 1 % of cases were severe (health secretary, curitiba, parana, brazil, 2002). pathophysiology of loxoscelism dermonecrosis is the hallmark of loxoscelism (fig. 1). histopathology and clinical data are obtained from biopsies of human patients after brown spider bites. rabbit skin artificially injected with loxosceles venom is used for more controlled investigation since this animal model reproduces human skin lesions that follow envenomation (ospedal et al., 2002). systemic effects, such as renal failure, are less common and are usually reproduced in mouse (luciano et al., 2004). observation of human skin biopsies showed an inflammatory infiltrate, thrombosis, hemorrhage, dermatitis, erythema, induration of affected area and liquefactive necrosis of the epidermis and dermis consistent with pyoderma grangrenosum (futrell, 1992; yannias and winkelmann, 1992). symptoms in an experimental study in rabbits showed that after 4 h oedema, hemorrhage, degeneration of blood vessel walls, plasma exudation, thrombosis, neutrophil accumulation in and around blood vessels with an intensive diapedesis, a diffuse collection of inflammatory cells (polymorphonuclear leucocytes) in the dermis, and subcutaneous muscular oedema all occur. over the following hours and up to 5 days after envenomation, the changes progressed to a massive neutrophil infiltration into the dermis and even into subcutaneous muscle tissue, destruction of blood vessels, thrombosis, hemorrhage, myonecrosis, and coagulative necrosis on the 5th day (ospedal et al., 2002). neutrophil participation and the inflammatory response seem to be dependent on an endothelial cell agonist effect triggered by the venom that leads to an indirect and dysregulated neutrophil activation involved in dermonecrosis (patel, 1994). envenomation of rabbit skin with l. reclusa venom after 14 days results in a mixed inflammatory cell infiltrate, coagulative tissue necrosis, vasculitis and a dense band of neutrophils bordering the zone of necrosis (elston et al., 2000). l. intermedia venom damaged vessel endothelia, as shown by vessel instability, endothelium cell vacuolization in biopsies of rabbit skin (veiga et al., 2001a; zanetti et.al., 2002). in vitro experiments on rabbit aorta endothelium cell cultures showed cytotoxicity of l. intermedia venom associated with loss of cell adhesion to the culture substrate and the shedding of proteoglycans from the extracellular matrix and cell surface into the medium (veiga et al., 2001a). in human umbilical vein endothelial cell (huvec) cultures treated with l. reclusa venom, agonist activity ensued, inducing endothelial cell expression of e-selectin and the release of interleukin (il)-8 and granulocyte macrophage colony-stimulating factor, resulting in dysregulated inflammatory response (patel et al., 1994). huvec exposed to l. deserta venom produced il-8, growth-related oncogene-α and monocyte chemoattractant protein-1 via an nf-κbdependent pathway (desai et al., 1999; gomez et al., 1999). l. deserta venom induces the expression of vascular endothelial growth factor (vegf) in human keratinocytes, suggesting that keratinocyte-derived vegf may contribute to vasodilatation, oedema and erythema in brown spider envenomation (desai et al., 2000). primary cultures of keratinocytes exposed to 100 ng/ml of l. gaucho venom release tumour necrosis factor (tnf)-α into the medium after 6 h (málaque et al., 1999). mice injected with l. reclusa venom developed local hemorrhage after 6 h accompanied by blistering of the ear skin (sunderkötter et al., 2001). 154 fig. 1 cellular and molecular aspects of brown spider and loxoscelism. (a) loxosceles intermedia (brown spider) male. (b) l. intermedia (brown spider) female. (c) sds-page 3-20 % venom profile stained by coomassie blue dye. (d) dermonecrotic lesion on rabbit skin after 24 h post-l. intermedia venom (10 µg) exposure. arrowhead indicates the site of venom injection with characteristic black and white eschar named marble plate. black arrow points an erythema surrounding the lesion and white arrow shows the gravitational spreading of lesion (a hallmark of dermonecrotic loxoscelism). (e) microscopical view of dermonecrotic lesion showing inflammatory leukocytes accumulated in the connective tissue (arrowhead) and disorganization of collagen fiber and oedema (black arrow) (magnification 400x). the inset shows inflammatory cells of the infiltrate represented by neutrophils (white arrow) (magnification 1.000x). histopathology showed a vasculitis reaction 2 h after exposure. the microscopical analysis of some mouse organs injected with different doses of l. intermedia venom revealed remarkable kidney alterations. acute tubular necrosis accompanied by deposition of eosinophilic material inside the proximal and distal renal tubules was seen in several nephrons (tambourgi et.al., 1998). mouse kidneys, treated with l. intermedia venom showed hyalinisation and erythrocytes in the bowman’s space, glomerular collapse, tubular epithelial cell cytotoxicity and deposition of eosinophilic material within the tubular lumen (luciano et al., 2004). confocal microscopy observations of double staining immunofluorescence against type iv collagen or laminin and l. intermedia venom showed that toxins deposit and bind along the tubular and glomerular basement membrane of mice kidneys. ultrastructural observations showed glomerular epithelial and endothelial cell cytotoxicity, the collapse and destruction of glomerular basement membrane and tubular epithelial cell degeneration. the basement membrane is a target for brown spider venom, as shown administrating l. intermedia venom to murine tumor engelbreth-holm-swarm (ehs), which is rich in basement membrane molecules. l. intermedia venom degraded and fragmented the basement membrane (veiga et.al., 2000a). venom 29 44 12 116 205 kd a male female a b c d e 1cm 1cm 1cm 155 displays hydrolytic activity for entactin and heparan sulphate proteoglycan, two important constituents of basement membranes, while having no apparent activity on purified type iv collagen and laminin (veiga et al., 2000a, 2001a,b). in the bone marrow and peripheral blood cells, l. intermedia initially causes a decrease in the number of nucleated red cells, bone-marrow depression of megakariocytes with thrombocytopenia in peripheral blood and decrease of platelet count (da silva et al., 2003). neutropenia in peripheral blood and low neutrophil counts were observed as consequence of bone-marrow depletion, which may reflect an extensive neutrophil influx to the tissues. eosinophils are apparently unaffected. brown spider venom toxins l. intermedia and l. laeta have different protein patterns of glycosylation and the same is between sexes of the same species (oliveira et al., 2005). hemolytic and dermonecrotic activities have been described for l. similes venom. sphingomyelinase d molecules, with molecular mass ranging from 30 to 35 kda and having hemolytic, necrotic and platelet aggregation activity were found in l. reclusa, l. rufescens, l. gaucho, l. laeta and l. intermedia venoms (futrell 1992; barbaro et al., 1994; mota and barbaro, 1995; tambourgi et al., 1995; barbaro et al., 1996a,b, 1997). three sphingomyelinase d isoforms were purified from l. boneti venom (lb1, lb2 and lb3). only lb1 and lb2 had dermonecrotic activity (ramos-cerrillo et al., 2004). an alkaline phosphatase was described in l. reclusa venom (futrell, 1992). hyaluronidase (32.5 kda) was found in l. refescens and l. reclusa (futrell, 1992; young and pincus, 2001). l. deserta, l. gaucho, l. intermedia, l. laeta and l. reclusa venoms contained an enzyme of similar molecular size (44 kda), which digested hyaluronic acid (barbaro et al., 2005). a 5’ribonucleotide phosphohydrolase was found in l. reclusa venom (futrell 1992). loxnecrogin a (31.4 kda) and loxnecrogin b (31.6 kda) with necrotic activity on rabbit skin were found in l. gaucho venom (cunha et al., 2003). l. intermedia has a range of proteases described in its venom: loxolysin a (20-28 kda) with fibronectinolytic and fibrinogenolytic activity; loxolysin b (32-35 kda) with gelatinolytic activity (feitosa et al., 1998); a serin protease (85 kda) with gelatinolytic activity (veiga et.al., 2000b) and proteases able to hydrolyse entactin, heparan sulphate proteoglican and basement membrane (veiga et al., 2000b, 2001a,b). l. rufescens also has a broad molecular range of caseinolytic, gelatinolytic and fibrogenolytic metalloproteases (young and pincus, 2001). to test whether proteases in l. intermedia venom were due to natural constitution and not a digest fluid contamination, da silveira et al., (2002) compared the proteolytic activity of the venom obtained directly from venom glands with that obtained by electroshock. both protein profiles showed very similar electrophoretic and enzymatic characteristics. at present, a new generation of molecules developed through cloning techniques is under study. l. intermedia lid1 recombinant protein (31.4 kda) is a sphingomyelinase d family molecule without dermonecrotic activity but with antigenic activity (kalapothakis et al., 2002). l. laeta recombinant protein (33 kda) is a sphingomyelinase isoform able to degrade sphingomyelin (pedrosa et al., 2002). this recombinant protein induced complement susceptibility, release of glycophorins and had dermonecrotic activity. l. intermedia recombinant protein (lirecdt, 34 kda) has dermonecrotic activity and was able to directly induce nephrotoxicity in mice (chaim et al., 2005). l. laeta recombinant phospholipase d generated lysophosphatidic acid and was hemolytic (lee and lynch, 2005). clinical features, diagnosis and treatment of brown spider bites diagnosis of loxoscelism is rarely based on spider identification and therefore clinical features, epidemiological and historical findings must be well known (wright et al., 1997; vetter, 1999; málaque et al., 2002). lesion recovery improves once the patient is treated. however, brown recluse bites have been misdiagnosed in north america because they occurred in regions of non-endemicity (vetter, 1999; nishioka, 2001; vetter and barger, 2002; vetter and bush, 2002a,b; vetter et al., 2003). a typical necrotic skin lesion begins soon after the spider bites the victim, followed by gravitational spreading (da silva et al., 2004). the bite is painless, hence the patient is often unaware that he has been bitten (futrell, 1992), and the delay between the bite and when the victim pursues help makes the treatment less effective. from mild to severe pain begins 2-8 h after the bite. at the bite a small puncture wound may appear, associated with transient erythema with itching and swelling and mild to severe tenderness (futrell, 1992; da silva et al., 2004). blebs or blisters appear (12-24 h), may become hemorrhagic, and surrounded by a halo of ischemic tissue. in the following days, necrotized lesions become a dull blue-violet, the area of the gravitational spread turns blue, and the size of the blue area increases. within three to seven days an eschar may form, after which the lesion hardens. the eschar may drop off leaving an ulcer that may require a skin graft (schenone, 1996; sezerino et al., 1998; málaque et al., 2002; da silva et al., 2004). success of therapy depends upon a correct and rapid diagnosis, the volume of the venom injected, and the patient susceptibility to the venom (futrell, 1992; da silva et al., 2004). phentolamine, heparin, topical nitro-glycerine, cyproheptadine and hyperbaric oxygenation have been used for therapy, but the efficacy of these therapies is inconclusive and their use is not recommended (futrell, 1992; wendell, 2003; da silva et al., 2004). the established therapy is dapsone, acetylsalicylic acid (aspirin), antibiotics (erythromycin and cephalosporin), ice and elevation, avoidance of strenuous activity and heat and, when necessary, surgery. early surgical excision has not been shown to be effective and often delays healing (futrell, 1992; merigian and blaho, 1996; goddard, 1998, monteiro et al., 2002; da silva et al., 2004). serum anti-loxosceles venom is used only in severe cases and effectiveness is doubtful especially against local manifestation. systemic envenomation studies in 156 animals and humans have demonstrated that antivenom neutralizes the deleterious effects of the venom and reduces paediatric mortality (isbister et al., 2003). effectiveness of antivenom to prevent dermonecrotic lesions seems to be time dependent and usually patient looks for medical help 4 h after the bite when lesions is already established (ospedal et al., 2002; nicholson and graudins, 2003). some local and systemic noxious activities of the venom are attributed to proteolytic toxins that degrade fibrinogen, fibronectin, entactin and heparan sulphate proteoglycan and disrupt basement membrane structures, thereby causing local hemorrhage, gravitational spreading, disseminated intravascular coagulation and renal failure (feitosa, et al., 1998; veiga et al., 1999, 2000b, 2001a,b; luciano et al., 2004; chaim et al., 2005). biotechnological products from brown spider venom recently developed technologies are being used to produce biotechnological products from loxosceles venom. arachnase (hemostasis diagnosis international co., denver, co, usa), normal plasma containing l. reclusa venom, mimics a lupus anticoagulant and may provide a positive control for anticoagulant testing (mcglasson et al., 1993). an antiserum against venoms of l. gaucho, phoneutria nigriventer, tityus serrulatus, and tityus bahiensis that reacts with l. intermedia and l. laeta toxins is produced by the butantan institute, são paulo, brazil. the ccpi (production center of immunobiologic products, parana, brazil) has also produced antiserum using l. intermedia venom that is able to neutralize some activities of loxosceles venom. l. laeta antiserum is produced by the national institute of health (peru) (da silva et al., 2004). these antisera have all been used as bioproducts for serum therapy (roodt et al., 2002; barbaro et al., 1994; 1996a; health secretary, curitiba, parana, brazil). guilherme et al. (2001) produced monoclonal antibodies recognising l. gaucho venom toxins, which were able to neutralize the dermonecrotic effect and lethal activities of this species venom but not those of heterologous venoms. monoclonal and polyclonal antibodies are not only powerful tools for neutralizing the effects of venom; they are also useful for research. they can be used to purify toxins from venom by affinity chromatography. they can be used on location of specific toxins on cell and tissue treated with venom toxins. immunofluorescence techniques such as confocal microscopy and flow cytometry are modern techniques based on antibody specific binding. in contrast to the collection of snake venom, spiders provide very little venom, which limits the ability to study spider venom toxins. protein cloning techniques are helping to solve this problem. after cloning it is possible to have milligrams of the same protein thereby improving the quality of research work and allowing more controlled experimental studies. today several spider venom recombinant proteins are under investigation most of which are in the sphingomyelinase protein family (kalapothakis et al., 2002; pedrosa et al., 2002; chaim et al., 2005). future perspectives toxins from loxosceles spiders are a group of proteins with a great range of different activities. each toxin may be used to investigate molecular and cellular effects of venom. also each of these proteins is a putative molecular model for drug design and to develop knowledge on some effects not yet fully understood such as the inflammatory reaction of dermonecrosis and platelet aggregation. acknowledgements this work was supported by cnpq, capes, fundação araucária and secretaria de estado de ciência, tecnologia e ensino superior do parana, brazil. references andrade rmg, oliveira kc, giusti al, silva wd, tambourgi dv. ontogenetic development of loxosceles intermedia spider venom. toxicon 37: 627-632, 1999. andrade rmg, lourenço wr, tambourgi dv. comparison of the fertility between loxosceles intermedia and loxosceles laeta spiders. j. arachnol. 28: 245-247, 2000. atkin ja, wingo cw, sodeman wa, flynn je. necrotic arachnidism. am. j. trop. med. 7: 165-184, 1958. barbaro kc, eickstedt vrd, mota i. antigenic cross-reactivity of venoms from medically important loxosceles (araneae) species in brazil. toxicon 32: 113-120, 1994. barbaro kc, ferreira ml, cardoso df, eickstedt vrd, mota i. identification and neutralization of biological activities in venoms of loxosceles spiders. braz. j. med. biol. res. 29: 1491-1497, 1996a. barbaro kc, souza mv, morhy l, eickstedt vrd, mota i. compared chemical properties of dermonecrotic and lethal toxins from spiders of genus loxosceles (araneae). j. protein chem. 15: 337-343, 1996b. barbaro kc, ferreira ml, cardoso df, souza mv, morhy l, eickstedt vrd, mota i. study of loxosceles spider venom. j. ven. anim. toxins 3: 232, 1997. barbaro kc, knysak i, martins r, hogan c, winkel k. enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five loxosceles spider venoms of medical importance in the americas. toxicon 45: 489-499, 2005. borkkan j, gross e, lubin y, oryan i. an outbreak of venomous spider bites in a citrus grove. am. j. trop. med. hyg. 52: 228-230, 1995. chaim om, sade yb, da silveira rb, toma l, kalapothakis e, cháves-olórtegui c, mangili oc, gremski w, dietrich cp, nader hb, veiga ss. brown spider dermonecrotic toxin directly induces nephrotoxicity. toxicol. app. pharmacol. 2006 (in press). cohen n, sarafian da, alon i, gorelik o, zaidenstein r simantov r, blatt a, litinsky i, modai d, golik a. dermonecrotic loxoscelism in the mediterranean region. j. toxicol. cut. ocul. toxicol. 18: 75-83, 1999. cunha rb, barbaro kc, muramatsu d, portaro fcv, fontes w, sousa mv. purification and characterization of loxnecrogin, a dermonecrotic toxin from loxosceles gaucho brown spider venom. j. prot. chem. 22: 135146, 2003. da silva ph, hashimoto y, santos fa, mangili oc, gremski w, veiga ss. hematological cell findings in bone marrow and peripheral blood of rabbits after experimental acute exposure to loxosceles intermedia (brown spider) venom. toxicon 42: 155-161, 2003. da silva ph, da silveira rb, appel mh, mangili oc, gremski w, veiga ss. brown spider and loxoscelism. toxicon 44: 693-709, 2004. 157 da silveira rb, filho jfs, mangili oc, veiga ss, gremski w, nader hb, dietrich cp. identification of proteases in the extract of venom glands from brown spiders. toxicon 40: 815-822, 2002. desai a, miller,mj, gomez hf, warren js, loxosceles deserta spider venom induces nf-κb-dependent chemokine production by endothelial cells. clin. toxicol. 37: 447-456, 1999. desai a, lankford ha, warren js. loxosceles deserta spider venom induces the expression of vascular endothelial growth factor (vegf) in keratinocytes. inflammation 24: 1-9, 2000. elston dm, eggers js, schmidt we, storrow ab, doe rh, mcglasson d, fischer jr. histological findings after brown recluse spider envenomation. am. j. dermatopathol. 22: 242-246, 2000. escoubas p, diochot s, corzo g. structure and pharmacology of spider venom neurotoxins. biochimie 82: 893-907, 2000. feitosa l, gremski w, veiga ss, elias mcqb, graner e, mangili oc, brentani rr. detection and characterization of metalloproteinases with gelatinolytic, fibronectinolytic and fibrinogenolytic activities in brown spider (loxosceles intermedia) venom. toxicon 36: 1039-1051, 1998. futrell j. loxocelism. am. j. med. sci. 304: 261-267, 1992. goddard j. loxoscelism. n. engl. j. med. 339: 1944-1945, 1998. gomez hf, miller mj, desai a, warren js. loxosceles spider venom induces the production of alpha and beta chemokines: implications for the pathogenesis of dermonecrotic arachnidism. inflammation 23: 207-2015, 1999. guilherme p, fernandes i, barbaro kc. neutralization of dermonecrotic and lethal activities and differences among 32-35kda toxins of medically important loxosceles spider venoms in brazil revealed by monoclonal antibodies. toxicon 39: 1333-1342, 2001. hogan c, barbaro kc, winkel k. loxoscelism: old obstacles, new directions. ann. emerg. med. 44: 608-624, 2004. isbister gk, graudins a, white j, warrell d. antivenom treatment in arachnidism. j. toxicol. clin. toxicol. 41: 291-300, 2003. isbister gk. necrotic arachnidism: the mythology of a modern plague. lancet 364: 549-553, 2004. isbister gk, vetter rs. loxoscelism and necrotic arachnidism: more myths and minor corrections. ann. emerg. med. 46: 205-206, 2005. kalapothakis e, araujo sc, castro cs, mendes tm, gomez mv, mangili oc, gubert ic, chavez-olórtegui c. molecular cloning, expression and immunological properties of lid1, a protein from the dermonecrotic family of loxosceles intermedia spider venom. toxicon 40: 1691-1699, 2002 lee s, lynch kr. brown recluse spider (loxosceles reclusa ) venom phospholipase d (pld) generates lysophosphatidic acid (lpa). biochem. j. 391: 317-323, 2005. luciano mn, silva ph, chaim om, santos vl, franco crc, soares mfs, zanata sm, mangili oc, gremski w, veiga ss. experimental evidence for a direct cytotoxicity of loxosceles intermedia (brown spider) venom on renal tissue. j. histochem. cytochem. 52: 455-467, 2004. macchiavello a. cutaneous arachnidism or gangrenous spot of chile. pub. health trop. med. 22: 425-466, 1947. málaque cms, ori m, santos sa, andrade dr. production of tnf-α by primary cultures of human keratinocytes challenged with loxosceles gaucho venom. rev. inst. med. trop. s. paulo, 41, 1999. málaque cms, castro-valencia je, cardoso jlc, frança fos, bárbaro kc, fan hw. clinical and epidemiological features of definitive and presumed loxoscelism in são paulo, brazil. rev. inst. med. trop. s. paulo, 44, 2002. mcglasson dl, babcock jl, berg l, triplett da. arachnase. an evaluation of a positive control for platelet neutralization procedure testing with seven commercial activated partial thromboplastin time reagents. am. j. pathol. 100: 576-578, 1993. merigian ks, blaho r. envenomation from brown recluse spider: review of mechanism and treatment options. am. j. ther. 10: 724-734, 1996. monteiro clb, rubel r, cogo ll, mangili oc, gremski w veiga ss. isolation and identification of clostridium perfringens in the venom and fangs of loxosceles intermedia (brown spider): enhancement of the dermonecrotic lesion in loxoscelism. toxicon 40: 409418, 2002. mota i, barbaro kc. biological and biochemical properties of venom from medically important loxosceles (araneae) species in brazil. j. toxicol. tox. rev. 14: 401-421, 1995. nicholson gm, graudins a. antivenoms for treatment of spider envenomation. j. toxicol. tox. rev. 22: 35-59, 2003. nishioka sa. misdiagnosis of brown recluse spider bite. w est. j. med. 174: 240, 2001. oliveira kc, andrade rmg, giusti al, silva wd, tambourgi dv. sex-linked variation of loxosceles intermedia spider venoms. toxicon 37: 217-221, 1999. oliveira kc, andrade rmg, piazza rmf, ferreira jmc, van den berg cw, tambourgi dv. variations in loxosceles spider venom composition and toxicity contribute to the severity of envenomation. toxicon 45: 421-429, 2005. ospedal kz, appel mh, neto jf, mangili oc, veiga ss, gremski w. histopathological findings in rabbits after experimental acute exposure to the loxosceles intermedia (brown spider) venom. int. j. exp. pathol. 84: 287-294, 2002. patel kd, modur v, zimmerman ga. the necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation: differential induction of gm-csf, il-8, and e-selectin expression. j. clin. invest. 94: 631-642, 1994. pedrosa mff, azevedo ilmj, andrade rmg, berg cw, ramos crr, ho pl, tambourgi dv. molecular cloning and expression of functional dermonecrotic and haemolytic factor from loxosceles laeta venom. biochem. bioph. res. commun. 298: 638-645, 2002. ramos-cerrillo b, olvera a, odell gv, zamudio f, paniaguasolis j, alagón a, stock rp. genetic and enzymatic characterization of sphingomyelinase d isoforms from the north american fiddleback spiders loxosceles boneti and loxosceles recluse. toxicon 44: 507-514, 2004. roodt ar, salomon, od, lloveras sc, orduna ta. poisoning by spiders of loxosceles genus. medicina 62: 83-94, 2002. rash ld, hodgson wc. pharmacology and biochemistry of spider venoms. toxicon 40: 225-254, 2002. sams hh, dunnick ca, smith ml, king le. necrotic arachnidism. j. am. acad. dermatol. 44: 561-537, 2001. sandidge js. arachnology: scavenging by brown recluse spiders. nature 426: 30, 2003 schenone h. diagnosis in 1348 patients which consulted for a probable spider bite or insect sting. bol. chil. parasitol. 51: 20-27, 1996. schienle a, schäfer a walter b, stark r, vaitl d. brain activation of spider phobics towards disorder-relevant, generally disgustand fear-inducing pictures. neurosci. lett. 388: 1-6, 2005. sezerino um, zannin m, coelho lk, gonçalves j, grando m, mattosinho sg, cardoso jlc, eickstedt vr, frança fos, bárbaro kc, fan hw. a clinical and epidemiological study of loxosceles spider envenoming in santa catarina, brazil. trans. r. soc. trop. med. hyg. 92: 546-548, 1998. sunderkötter c, seeliger s, schönlau f, roth j, hallmann r, luger ta, sorg c, kolde g. different pathway leading to 158 cutaneous leukocytoclastic vasculitis in mice. exp. dermatol. 10: 391-404, 2001. tambourgi dv, magnoli fc, eickstedt vrd, benedetti zc, petricevich vl, silva wd. incorporation of 35kda purified protein from loxosceles intermedia spider venom transforms human erythrocytes into activators of autologous complement alternative pathway. j. immunol. 155: 4459-4466, 1995. tambourgi dv, petricevich vl, magnoli fc, assaf slmr, mancar s, silva wd. endotoxemic-like shock induced by loxosceles spider venoms: pathological changes and putative mediators. toxicon 36: 391-403, 1998. veiga ss gremski w, santos vlp, feitosa l, mangili oc, nader hb, dietrich cp, brentani rr. oligosaccharide residues of loxosceles intermedia (brown spider) venom proteins: dependence on glycosylation for dermonecrotic activity. toxicon 37: 587-607 1999. veiga ss, feitosa l. santos vlp, souza ga, ribeiro as, mangili oc, porcionatto ma, nader hb, dietrich cp, brentani rr, gremski w. effect of brown spider venom on basement membrane structures. histochem. j. 32: 397-408, 2000a. veiga ss, da silveira rb, dreyfuss jl, haoach j, pereira am, mangili oc, gremski w. identification of high molecular weight serine-proteases in loxosceles intermedia (brown spider) venom. toxicon 38: 825-839, 2000b. veiga ss, zanetti vc, franco crc, trindade es, porcionatto ma, mangili oc, gremski w, dietrich cp, nader hb. in vivo and in vitro cytotoxicity of brown spider venom for blood vessel endothelial cells. throm. res. 102: 229-237, 2001a. veiga ss, zaneti vc, braz a, mangili oc, gremski w. extracellular matrix molecules as target for brown spider venom toxins. braz. j. med. biol. res. 34: 843-850, 2001b. vetter rs. identifying and misidentifying the brown recluse spider. dermatol. online j. 5: 7, 1999. vetter rs, barger dk. an infestation of 2,055 brown recluse spiders (araneae:sicariidae) and no envenomation in a kansas home: implications for bite diagnoses in nonendemic areas. j. med. entomol. 39: 948-951, 2002. vetter rs, bush sp. reports of presumptive brown recluse spider reinforce improbable diagnosis in region of north america where the spider is not endemic. clin. inf. dis. 35: 442-445, 2002a. vetter rs, bush sp. the diagnosis of brown recluse spider bite is overused for dermonecrotic wounds of uncertain etiology. ann. emerg. med. 39: 544-546, 2002b. vetter rs, cushing pe, crawford rl, royce la. diagnoses of brown recluse spider bites (loxoscelism) greatly outnumber actual verifications of spider in four western american states. toxicon 42: 413-418, 2003. vetter rs, visscher pk. bites and stings of medically important venomous arthopods. int. j. dermatol. 37: 481-496, 1998. w endell rp. brown recluse spiders: a review to help guide physicians in nonendemic areas. south. med. j. 96: 486-490, 2003. wright sw, wrenn kd, murray l, seger d. clinical presentation and outcome of brown spider bite. ann. emerg. med. 30: 28-32, 1997. yannias ja, winkelmann rk. persistent painful plaque due to a brown recluse spider bite. cutis 50: 273-275, 1992. young ar, pincus sj. comparison of enzymatic activity from three species of necrotising arachnids in australia: loxosceles rufescens, badumma insignis and lampona cylindrata. toxicon 39: 391-400, 2001. zanetti vc, da silveira rb, dreyfuss jl, haoach j, mangili oc, veiga ss, gremski w. morphological and biochemical evidence of blood vessel damage and fibrinogenolysis triggered by brown spider venom. blood coag. fibrin. 13: 135-148, 2002. antimicrobial peptides in caenorhabdites elegans isj 7: 45-52, 2010 issn 1824-307x review antimicrobial peptides in caenorhabditis elegans a bogaerts, i beets, l schoofs, p verleyen research group of functional genomics and proteomics, k.u.leuven, leuven, belgium accepted january 19, 2010 abstract the nematode caenorhabditis elegans is one of the most successful model species for experimental research because of its sequenced genome, the versatile genetic toolkit and the straightforward breeding among others. in natural conditions however, this tiny worm is constantly surrounded by micro-organisms, simultaneously a source of indispensable nutrition and inevitable pathogens. lacking an adaptive immune system, the worm solely relies on its innate immune defence to cope with its challenging life style. hence c. elegans is an excellent model to gain more insight in innate immunity, which is remarkably preserved between invertebrate and vertebrate animals. the innate defence consists of receptors to detect potential pathogens, a complex network of signalling pathways and last but not least, effector molecules to abolish harmful microbes. in this review, we focus on the antimicrobial peptides, a vital subgroup of effector molecules. we summarise the current knowledge of the different families of c. elegans antimicrobial peptides, comprising nlps, caenacins, abfs, caenopores, and a recently discovered group with antifungal activity among which thaumatinlike proteins. key words: caenacins; abfs; caenopores; insulin signalling; immunity; host-pathogen interaction introduction as a free living soil nematode, caenorhabditis elegans forms an extremely interesting model to study the interaction with bacteria, its main food source and substrate. as the distribution of bacteria is mixed in natural conditions, worms should continuously search for regions in the soil where the benefit of energy-rich and benign bacteria exceeds the possible presence of harmful bacteria, be it slow or fast killers (shtonda and avery, 2006; abada et al., 2009). it is clear that discerning different types of bacteria and employing an effective battery of antibacterial molecules are crucial for worms. c. elegans has a tremendous variety of chemosensory receptors to detect both interesting and harmful bacteria, the latter provoking pathogen avoiding behaviour (zhang et al., 2005; pradel et al., 2007; schulenburg and ewbank, 2007). in contact with harmful bacteria, recognition molecules activate specific signalling pathways which ultimately induce the release of immune molecules. in this review we specifically focus on the variety of antimicrobial ___________________________________________________________________________ corresponding author: annelies bogaerts research group of functional genomics and proteomics k.u.leuven, zoological institute naamsestraat 59, 3000 leuven, belgium e-mail: annelies.bogaerts@bio.kuleuven.be peptides (amps), produced by c. elegans as part of its defence system. amps are defined as relatively short molecules with a low molecular weight (below 5 kda), often containing 10 up to 150 amino acids (bulet et al., 1999; jenssen et al., 2006). their expression can be either constitutive or inducible at the time of infection (kato et al., 2002; alegado and tan, 2008). amps posses a natural antimicrobial activity often thanks to their cationic and amphipathic structure which facilitates the disruption of anionic cell walls and phospholipids membranes of microbes, although other microbicidal mechanisms have also been proposed (bulet et al., 2004; brogden, 2005). worms have an innate immune system which constitutively expresses certain amps whereas complex mixtures of amps are induced upon encounter with different pathogens. note that by unfolding a specific mixture of ‘antibiotics’ the worm can prevent a straightforward development of resistant pathogenic strains. therefore, studying amps in an experimentally favourable immunological model such as c. elegans, forms a lead for the development of new strategies to deal with pathogenic infections in the future. we highlight the diverse amp families in c. elegans and summarise the evidence for their biological function, specificity, expression and the pathways involved as far as known. 45 mailto:annelies.bogaerts@bio.kuleuven.be neuropeptide like proteins the c. elegans genome encodes at least 42 neuropeptide like proteins (nlps) which can be divided into minimal 11 subgroups according to their unique bioactive motifs (nathoo et al., 2001). although most of the nlp genes are translated into conventional neuropeptides, others may possess distinct or additional functions. the first indication of an antimicrobial role for nlp genes came from a study in 2002 by mallo et al. in which they observed the induced expression of nlp-29 upon infection with the gram-negative bacterium serratia marescens. later on in 2004, ewbank’s group demonstrated an antifungal activity for nlp-31 against drechmeria coniospora in vitro (couillault et al., 2004). in c. elegans, most of the infection-inducible nlp genes were named as such because of their limited sequence similarity with yggxamide neuropeptide genes, sharing yggwg and yggyg motifs (nathoo et al., 2001). they form a monophyletic group, found in a 12 kb region on the left arm of chromosome v, referred to as “the nlp-29 cluster” (pujol et al., 2008b). this cluster comprises nlp-27 to 31 and the adjacent gene nlp-34. two other caenorhabditis species c. briggsae and c. remanei possess orthologous genes. in c. brigssae, these genes are orientated in different clusters: cbrnlp-27 and cbr-nlp-34.1, cbr-nlp-34.2 and cbr-nlp34.3. phylogenetic analysis indicates that gene duplications driven by natural selection form the basis for the evolutionary diversification of these clusters. at the time of divergence of the different caenorhabditis lineages from a common ancestor, two genes were at the nlp locus: nlp-27 and nlp-34. via gene duplication, nlp-27 gave rise to 5 genes in c. elegans whilst nlp-34 gave rise to 3 genes in c. brigssae (pujol et al., 2008b). expression of the genes belonging to the nlp-29 cluster is predominately limited to the epidermis and the intestine and is controlled by a diverse interplay of mechanisms. besides infection, physical injury of the epidermis as well seems to have an influence on expression of nlp-29 and nlp-31 (pujol et al., 2008a). in both conditions, the expression levels are controlled by distinct pathways that converge in a conserved signalling cascade: the p38 mitogen activated kinase (mapk) pathway. this pathway, involving mapk pmk-1, mapk kinase (mapkk) sek-1 and mapkk kinase (mapkkk) nsy-1, lies downstream of the tir (toll-interleukine 1 receptor) adaptor protein tir-1, an ortholog of the human protein sarm (selective androgen receptor modulator). involvement of tir-1 in the control of nlp expression upon fungal infection was established in 2004 by couillault et al. these researchers generated transgenic worms expressing gfp (green fluorescent protein) under control of the nlp-29 promotor and observed an increased fluorescent signal in the hypodermis upon infection with d. coniospora and s. marescens. rnai of tir-1 in the pnlp-29::gfp reporter lines diminished the constitutive and infection-induced expression of pnlp-29::gfp. moreover, tir-1 (rnai) worms are more susceptible to the deleterious effects of both fungi (d. coniospora) and bacteria (s. marescens). further studies have unravelled additional components of the immune signalling pathway. expression of nlp-29 is regulated by the upstream factor tpa-1, homologous to the mammalian protein kinase c (pkc) delta (ziegler et al., 2009). in the epidermal response to fungal infection, pkc delta is activated by a tribbles-like kinase nipi-3. however, upon wounding nipi-3 is not required for nlp-29 induction, supporting the existence of a pathogenspecific reaction, in addition to a non-specific protective response (pujol et al., 2008a). as mentioned above, also bacteria can trigger the nlp gene expression. in contrast to fungal infection, expression predominately occurs in the intestinal epithelium and is regulated by a c. elegans protein kinase d (dkf-2) which lies downstream of tpa-1, and acts in pmk-1 dependent and independent ways (ren et al., 2009). to make things even more complicated nlp-28 and nlp-29 expression is also induced as a consequence of osmotic stress and this by yet another transcriptional response which is pmk independent (pujol et al., 2008b). caenacins similarly to the neuropeptide like proteins, specific genes belonging to the caenacin family are induced upon infection with d. coniospora amongst other pathogens. cnc-1 up to cnc-5 and cnc-11 form a genomic cluster, also situated on the left arm of chromosome v, referred to as “the cnc-2 cluster”. mature peptides belonging to the nlp and cnc classes are rich in glycine and aromatic amino acids and most of them can be distinguished by the qwgyg motif present just c-terminal to the predicted signal sequence cleavage site. despite the fact that they are structurally and phylogenetically related to the nlps, these cnc genes are regulated in a very distinct way (zugasti et al., 2009). unlike the nlp genes, osmotic stress has only little effect on the expression of cnc-11 and no effect on the other members of the cnc-2 cluster. while induction of genes, belonging to the nlp-29 cluster, upon wounding or infection relies almost entirely on a p38 mapk signalling cascade, only physical injury and not infection seems to have a lowering effect on induction of cnc-2 cluster genes in pmk-1 mutants. this indicates that the expression of cnc genes in the epidermis upon fungal infection is dependent on a different immune pathway (zugasti et al., 2009). as the expression of cnc-2 was more strongly induced upon infection than upon wounding, researchers focused on this gene. they constructed transgenic strains expressing either gfp (pcnc2::gfp) or the ‘mcherry’ fluorescent protein (pcnc2::mcherry) under control of the cnc-2 promotor and reported that the cnc-2 gene was exclusively expressed in the epidermis. furthermore, induced expression of reporter genes was observed upon infection with d. coniospora but not upon bacterial infection with s. marescens and pseudomonas aeruginosa (zugasti et al., 2009). searching for the specific signalling pathway involved in cnc-2 gene expression they found that the transcription is controlled in a paracrine way by 46 the c. elegans transforming growth factor β ortholog dbl-1 as in dbl-1 mutant worms the induced expression of the cnc-2 reporter was much lower (zugasti et al., 2009). antibacterial factor (abf) peptides the c. elegans genome encodes six homologues (abf-1 to abf-6) of the ascaris suum antibacterial factor (asabf) peptides, which are microbicidal factors that were first discovered in the body fluid of the parasitic nematode a. suum (kato and komatsu, 1996). sequence identity and structural commonality reveals that these nematode abfs are genetically related: all known abf peptides share a cysteine-array consisting of eight conserved cysteine residues and a secretory signal sequence at the n-terminus. high homology appears in the region encompassing the eight cysteines with 25-95% similarity. in contrast, the region c-terminal to the last cysteine is divergent and varies in length (froy, 2005). most likely this part is cleaved off post-translationally, as was shown for asabf-α (kato and komatsu, 1996; zhang et al., 2000). apart from their sequence similarity, several other properties support a direct role for abfs in the innate immune response of c. elegans. recombinant abf-2 exhibits antimicrobial activity in vitro against a broad range of gram-positive, gramnegative and fungal pathogens. however, grampositive bacteria tend to be more sensitive and some gram-negative and fungal strains are resistant to abf-2 (kato et al., 2002). asabf-α possesses a similar antimicrobial specificity (zhang et al., 2000). the exact bactericidal mechanism of abf peptides remains to be elucidated, but based upon similarities in primary structure and antimicrobial effects, the killing of microbes could be caused by disruption of their cytoplasmic membrane (zhang et al., 2000; kato, 2007). abf peptides are constitutively expressed under normal growth conditions when c. elegans is cultivated on a lawn of the non-pathogenic strain e. coli op50, as was shown for abf-1, abf-2 and abf-3 (kato et al., 2002; alper et al., 2007; alegado and tan, 2008). the pharyngeal tissue represents the main production site for abf-1 and abf-2 (kato et al., 2002). together with abf-3, abf-1 is also produced in the intestine (alper et al., 2007). in this way, the constitutive expression of abfs may be part of a general defence mechanism in the worm that protects the digestive tract from microbial infection, an important threat since c. elegans mainly feeds on bacteria. expression of abf-2 and abf-3 was also observed in the excretory cells of the worm, presumably to protect the openings to the exterior (e.g., anus and excretory pores) that are continuously in contact with potential pathogens from the environment (kato et al., 2002; alper et al., 2007). recent evidence indicates that on top of the general defences against various microbes abfs also participate in a specific immune response induced upon infection (alper et al., 2007; alegado and tan, 2008; means et al., 2009). in a. suum a number of the asabf peptides were demonstrated to be induced after injection of heat-killed bacteria in the pseudocoelom (pillai et al., 2003; minabi et al., 2009). this induction was also proven for some of the asabf-type homologues in c. elegans. worms infected with the gram-negative pathogen salmonella typhimurium displayed an increase in abf-2 transcript levels by a hundred-fold. deficiencies in abf-2 after rnai treatment correlated with a significantly higher bacterial load in the intestine after exposure to s. typhimurium in comparison with control animals. these findings suggest that c. elegans induces the expression of abf-2 as part of an immune response to s. typhimurium infection that is essential for limiting bacterial growth in the worm’s digestive tract. however, transcript levels were initially indistinguishable within the first 24h of exposure which implies that the induced amp response may not be due to direct sensing of pathogenic microbes but due to later events such as the production of specific bacterial factors or damage to host tissues (alegado and tan, 2008). upregulation of abf-1 and abf-2 was also observed in wild type c. elegans after infection with the yeast cryptococcus neoformans (means et al., 2009) and exposure to the gram-positive bacteria staphylococcus aureus elicited a weak induction of abf-3 (alper et al., 2007). therefore, we can conclude that nematode abfs play a crucial role in the general and more specific induced immune response to pathogenic attack. the signalling pathways responsible for the upregulation of abf peptides upon infection of c. elegans have not yet been fully elucidated. tol-1, the single homologue of the toll-like receptor encoded in the worm’s genome, seems to be required for the correct expression of abf-2 since quantitative reverse transcription-pcr analysis indicates a decreased level of abf-2 transcripts in tol-1 mutants (tenor and aballay, 2008). moreover tol-1 mutants display a reduced lifespan on salmonella enterica due to a rapid invasion of the pharynx, which is the main expression site for the antibacterial abf-2 peptide (tenor and aballay, 2008). these results demonstrate that tlrmediated signalling probably contributes to the elicitation of a specific immune response in c. elegans on top of its established role in the pathogenic avoiding behaviour of the worm (pujol et al., 2001). in addition neurons expressing g proteincoupled receptors (gpcrs) also participate in the regulation of abf expression levels. a deficiency in the neural circuit involving npr-1, which encodes a gpcr related to the mammalian neuropeptide y receptors, reduces the expression level of abf-1 in response to infection by p. aeruginosa (styer et al., 2008). recently an evolutionary conserved pathway consisting of ced-1 and c03f11.3, orthologs of the mammalian scavenger receptors scarf1 and cd36, was shown to activate antimicrobial peptides including abf-1 and abf-2 upon yeast-infection (means et al., 2009). up till now, the phylogenetic relationship between nematode abfs and other metazoan amps remains unclear. asabf-type peptides contain a cysteine-stabilised α/β (csα/β) consensus motif consisting of an α-helix and two antiparallel β 47 strands stabilised by four internal disulfide bridges. recently, a novel member of asabf-type peptides was discovered in a. suum containing only six cysteines. so far this amp forms the single exception and probably arose after the divergence of ascaridida and rhabditida (minaba et al., 2009). structural similarities between nematode abfs and invertebrate defensins suggest a common ancestry in the evolution of these antimicrobial factors. first of all, the csα/β motif of asabf-type peptides is also found within invertebrate defensins that are further classified according to the number of cysteine residues contributing to the intramolecular disulfide bonds: the cysteine array of insect/arthropod defensins typically comprises six cysteine residues, mollusk defensins are characterized by eight cysteines (froy, 2005). secondly, the primary structure of asabf-type peptides includes an insect/arthropod consensus sequence (cys1-[…]-cys2-xaa-xaa-xaa-cys3-[…]gly-xaa-cys4-[…]-cys5-xaa-cys6) with six conserved cysteines and one glycine residue (kato and komatsu, 1996; kato et al., 2002). as a third argument zhang and kato (2003) showed that nematode abfs share even more characteristics with two mollusk defensins: myticin and an amp isolated from the mediterranean mussel mytilus galloprovincialis (mgd-1). asabf-type peptides and mollusk defensins both contain eight cysteine residues with an identical pairing and a similar precursor organization consisting of an n-terminal secretory signal sequence, followed by the mature polypeptide and a cleavable “pro-region “ at the cterminus. in classical csα/β type amps, such as insect defensins, this “pro-region” is located directly after at the n-terminus. in summary, nematode abfs and mollusk defensins share several structural properties and could therefore be generated from a common ancestor. however, the absence of highly reliable evidence such as a significant sequence similarity or a conserved genomic organization (exon-intron structure) cannot exclude that these two groups of amps developed through convergent evolution (froy, 2005). hopefully, the identification of new csα/β type amps in different phyla will clarify the evolutionary trajectory of nematode and invertebrate defensins (froy, 2005; rodriguez de le vega and possani, 2005). caenopores caenopores are the saposin-like proteins (spp) of c. elegans. saposins form a multifarious protein family characterized by an alpha helix bundle stabilized by 3 unique disulphide bonds and the ability to interact with phospholipid membranes (see for review bruhn, 2005). based on these hallmarks, patthy (1996) designated the putative protein product of gene t07c4.4 as the first c. elegans saposin-like protein. two years later, 5 additional spp genes were found in this nematode. all six predicted spps appeared similar to the amoebapores of entamoeba histolytica and granulysin from human cytotoxic t lymphocytes as they consist of a secretory signal peptide followed by a single saposin-like domain (banyai and patthy, 1998). note that amoebapore-like spps might have been the first antimicrobial peptides since this protein family emerged even before the advent of metazoans (leippe 1999). banyai and patthy (1998) recombinantly expressed t07c4.4 (spp-1) which allowed to demonstrate the characteristic helix bundle structure and an antibacterial effect on e. coli (jm-109). in addition, the three-dimensional structure of spp-5 was studied in great detail by mysliwy et al. (in press). they confirmed that spp-5 has five amphiphatic helices, connected by three disulfide bonds, arranged in a folded leaf typical for the saposin-like protein family. further examination of the worm’s genome lead to the discovery of 28 different spp genes coding for 33 saposin-like proteins which were named caenopores because of their structural and functional resemblance with amoebapores (roeder et al., 2010). indeed, like amoebopores, at least spp-5 was shown to display pore-forming activity capable of killing bacteria by permeabilizing their cytoplasmic membrane. a phylogenetic analysis of the 33 spps shows different clusters. in case of the cluster comprising spp-2 to spp-6, all corresponding genes are located in the same chromosomal region, consistent with a series of gene duplications. roeder et al. (2010) investigated the functional significance of spp-1, spp-3, spp-4, spp-5 and spp-6 by means of rnai-mediated gene silencing. one gene, spp-5, significantly affected the overall fitness of worms measured as the number of laid eggs and the deposition of fat tissue. silencing this same gene, contrary to the other genes tested, had a huge impact on the number of e. coli that could survive in the intestinal lumen (roeder et al., 2010). when grown on standard culture medium ngm, wild type worms consequently expressed spp-5 for all the bacteria/conditions tested. the spp-3 gene, on the other hand, was induced only when confronted with b. megaterium, m. luteus and starvation. further analyses of spp-5 learnt that it is as potent as spp-1 against the gram-positive b. megaterium and even more active against the gramnegative e. coli (roeder et al., 2010). the genome wide microarray analysis of wong et al. (2007) searched for pathogen specific signatures in the immune response of c. elegans. no expression change of any spp gene was observed against erwinia carotovora, whereas spp-18 was upregulated upon infection with photorhabdus luminescens as well as spp-5, spp-8, spp-14 and spp-21 in case of an enterococcus faecalis infection (wong et al. 2007). moreover, spp-1 was shown to be induced upon infection with s. typhimurium. it was found that salmonella strains lacking, among others, the virulence factor spi-2 had difficulties to persist in c. elegans intestine. interestingly, such persistence deficiencies could be rescued when spp-1 of the worm was reduced by rnai (alegado and tan, 2008). however, evans et al. (2008) showed that spp-1 is repressed upon infection with p. aeruginosa. next, they showed that downregulation of spp-1 expression, among others, is a key strategy to overcome the immune system of its host. this downregulation depends on the response regulator gaca and the quorum sensing 48 regulators lasr and rhlr; and interferes with the insulin-like signalling via the daf-2/daf-16 axis, which is important for the regulation of stress response, longevity and immune function (evans et al., 2008). this discovery correlates perfectly with the observation that spp-1 (and spp-12) expression is downregulated by loss of insulin signalling. decreasing the expression of either of these spp genes by rnai reduced the lifespan of c. elegans on e. coli (murphy et al., 2003). the exact mechanism of how the diverse spp genes are controlled is still not known. the only additional information currently available is that expression of spp-9 and spp-18, along with more than 80 defencerelated genes, appears to be regulated by the protein kinase d (ren et al., 2009). worth mentioning is that spp-5 is exclusively expressed in the gut (roeder et al., 2010). the same accounts for spp-1, but spp-7 is also expressed in the pharyngeal muscles and head neurons (alper et al., 2007). given that most caenopore genes (except spp-2, spp-12, spp-16 and spp-19) have the intestine specific transcription factor elt-2 binding domain in their putative promotor regions, it can be assumed that most caenopores will be expressed in the intestine. this finding prompted roeder et al. (2010) to state that “caenopores or spps are most likely the only candidates to tackle the diverse mixture of microbes c. elegans is confronted with in the natural environment”. in their opinion, other antimicrobial peptides described for c. elegans are either not numerous enough (abfs), or they are expressed at the wrong location (hypodermis: cncs and nlps). although roeder is perhaps correct when postulating that the spps are the most significant group of amps, this statement should be relativized as the importance of other types of amps, functioning alone or in cooperation with other defence molecules of the immune system of c. elegans, should not be underestimated. other potential amps besides the above mentioned members of the nlp, cnc, abf and spp families, which are induced upon infection with different types of pathogens (troemel et al., 2006; muir et al., 2008), pujol et al. (2008b) found other previously uncharacterised genes that seem to be specifically induced upon fungal challenge. hence they were named: fungus-induced proteins (fip), fip related proteins (fipr) and glycine-rich secreted protein 2 (grsp-2). a comparison to peptides with known antimicrobial activity (fjell et al., 2007) revealed that each of the fip, fipr and grps genes could potentially encode amps (pujol et al., 2008b). however, further biochemical and functional analysis will be required to confirm this statement. recently, homologs of thaumatins and other pathogenesis-related plant proteins have been discovered in the c. elegans genome (brandazza et al., 2004; shatters et al., 2006). thaumatins were originally isolated from the fruits of thaumatococcus danielli and extensively studied because of its sweetening properties. later studies have demonstrated antifungal activity for thaumatin-like proteins such as stimulating microbial membrane permeability (vigers et al., 1991), beta-1,3glucanase activity (grenier et al., 1999) and alphaamylase inhibiting properties (svensson et al., 2004). moreover, recent work from the tan group has implicated an antimicrobial role for a member of the thaumatin family: thn-2. knockdown of thn-2 by rnai enhances the susceptibility of c. elegans to a p. aeruginosa infection (evans et al., 2008). as for spp-1 (see above), the expression of thn-2 is downregulated upon infection with this pathogen (shapira et al., 2006; evans et al., 2008). these findings suggest that homologs of these plant proteins could well represent potential antimicrobial proteins in c. elegans. note that thaumatins are not indisputably antimicrobial peptides as they count around 200 amino acid residues. conclusion to feed on and fight off a smorgasbord of bacteria and pathogens, c. elegans developed a diverse armory of immune defence molecules. this feed versus fight paradox is most certainly an intriguing issue. whilst on the one hand worms are completely dependent on bacteria/fungi for their survival, some of these micro-organisms might on the other hand contribute to their death. numerous chemoreceptors allow c. elegans to distinguish between benign and harmful bacteria. however, a continuous cost-benefit analysis is necessary for making the correct choice between for example a slightly pathogenic bacterium with a high nutritional value or a non-pathogenic bacterium with low nutritional value. the amps constitute the most numerous and versatile group of immune effector molecules, allowing specific and effective responses against different pathogens. even the expression of related amp genes can be regulated by very distinct pathways, providing the host specific alternative defence possibilities against the potentially detrimental effects of different types of pathogen invasions. the important role of amps in the early immune response has already been proven by the isolation of numerous animal amps, mostly from higher organisms such as vertebrates and arthropods. nonetheless, the c. elegans amps still form a poorly studied group. as described earlier in this review many putative amps were yet identified thanks to their induced expression upon infection or their structure/sequence similarities with closely related amps (kato et al., 2002; mallo et al., 2002; couillault et al., 2004). the strategy of homologybased searches starting from known amp sequences (e.g. vertebrate or arthropod amps), however, is limited due to the short length and rapid molecular evolution of these peptides (kato et al., 2002). this constant evolution of the immune defences compensates for the renewing virulence mechanisms of a pathogen and is a necessity to ensure the survival of a species. in fact, to our knowledge, none of the natural occurring amps were yet directly purified from c. elegans. therefore, we assume that not all c. elegans amps are identified yet which makes the identification of additional amps a first great challenge to better 49 understand the worm’s innate immunity. in addition, peptidomics based approaches for the identification of c. elegans amps might help to address the question of which amps might cooperate and contribute to target specific pathogens. since multiple amps are expressed in response to infection by a single pathogen a certain level of cooperativity might exist between different amps and/or other defense molecules. cases of cooperative activity among amps and/or other immune effectors have already been reported in mammals, e.g. the synergy between different murine defensins or the enhanced antibacterial effects of human β-defensin and lyzosyme (chen et al., 2005; wu et al., 2009). in c. elegans the field of synergistic effects in innate immunity thus remains an important research objective. to date, many aspects of the regulation of amp expression and their mode/site of action remain elusive. it was shown that c. elegans can activate multiple immune pathways upon encountering a single pathogen (alper et al., 2007; schulenberg et al., 2007) and assumed that this network of interacting signalling cascades results in the expression of an appropriate set of antimicrobial genes. indeed, another type of pathogen can elicit the expression of a different set of amps indicating that c. elegans distinguishes between pathogens and suppresses infections in a specific manner (alper et al., 2007; wong et al., 2007). yet, the exact role of many putative upstream regulators of amp expression, as suggested by e.g. genomewide transcriptomic analyses, has not yet been characterised. given that c. elegans is a versatile model with an extended experimental toolbox, e.g. the possibility to perform (large-scale) rnai and fluorescence studies (couillault et al., 2004; alper et al., 2007), this organism is ideally suited to address these and other challenges in innate immunity research. during evolution, c. elegans managed to survive a daily confrontation with bacteria, many of which can be harmful for humans. therefore, it is expected that future efforts will result in a deeper insight in how this worm resists bacteria in a sustainable way. acknowledgments pv benefits a post-doctoral fellowship and ab and ib a ph. d. fellowship of the research foundation flanders (fwo-vlaanderen). references abada ea, sung h, dwivedi m, park bj, lee sk, ahnn j. c. elegans behavior of preference choice on bacterial food. mol. cells 28: 209213, 2009. alegado ra, tan mw. resistance to antimicrobial peptides contributes to persistence of salmonella typhimurium in the c. elegans intestine. cell. microbiol. 10: 1259-1273, 2008. alper s, mcbride sj, lackford b, freedman jh, schwartz da. specificity and complexity of the caenorhabditis elegans innate immune response. mol. cell. biol. 27: 5544-5553, 2007. banyai l, patthy l. amoebapore homologs of caenorhabditis elegans. biochim. biophys. acta 1429: 259-264, 1998. brandazza a, angeli s, tegoni m, cambillau c, pelosi p. plant stress proteins of the thaumatinlike family discovered in animals. febs lett. 572: 3-7, 2004. brogden k. antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? nat. rev. microbiol. 3: 238-250, 2005. bruhn h. a short guided tour through functional and structural features of saposin-like proteins. biochem. j. 389: 249-257, 2005. bulet p, hetru c, dimarcq jl, hoffmann d. antimicrobial peptides in insects; structure and function. dev. comp. immunol. 23: 329-344, 1999. bulet p, stöcklin r, menin l. anti-microbial peptides: from invertebrates to vertebrates. immunol. rev. 198: 169-184, 2004. chen x, niyonsaba f, ushio h, okuda d, nagaoka i, ikeda s, et al. synergistic effect of antibacterial agents human beta-defensins, cathelicidin ll-37 and lysozyme against staphylococcus aureus and escherichia coli. j dermatol sci. 40: 123-132, 2005. couillault c, pujol n, reboul j, sabatier l, guichou jf, kohara y, et al. tlr-independent control of innate immunity in caenorhabditis elegans by the tir domain adaptor protein tir-1, an ortholog of human sarm. nat. immunol. 5: 488-494, 2004. evans ea, kawli t, tan mw. pseudomonas aeruginosa suppresses host immunity by activating the daf-2 insulin-like signaling pathway in caenorhabditis elegans. plos pathog. 4: 1000175, 2008. fjell cd, hancock re, cherkasov a. amper: a database and an automated discovery tool for antimicrobial peptides. bioinformatics 23: 11481155, 2007. froy o. convergent evolution of invertebrate defensins and nematode antibacterial factors. trends microbiol. 13: 314-319, 2005. grenier j, potvin c, trudel j, asselin a. some thaumatinlike proteins hydrolyse polymeric beta-1,3-glucans. plant j. 19: 473-480, 1999. jenssen h, hamill p, hancock re. peptide antimicrobial agents. clin. microbiol. rev. 19: 491-511, 2006. kato y. nematode antimicrobial peptides. curr. top. pept. protein res. 8: 1-10, 2007. kato y, aizawa t, hoshino h, kawano k, nitta k, zhang h. abf-1 and abf-2, asabf-type antimicrobial peptide genes in caenorhabditis elegans. biochem. j. 361: 221-230, 2002. kato y, komatsu s. asabf, a novel cysteine-rich antibacterial peptide isolated from the nematode ascaris suum. purification, primary structure and molecular cloning of cdna. j. biol. chem. 271: 30493-30498, 1996. kurz cl, tan mw. regulation of aging and innate immunity in c. elegans. aging cell 3:185-193, 2004. leippe m. antimicrobial and cytolytic polypeptides of amoeboid protozoa-effector molecules of 50 http://www.ncbi.nlm.nih.gov/pubmed?term=%22brandazza%20a%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22angeli%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22tegoni%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22cambillau%20c%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22pelosi%20p%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract javascript:al_get(this,%20'jour',%20'febs%20lett.'); javascript:al_get(this,%20'jour',%20'clin%20microbiol%20rev.'); primitive phagocytes. dev. comp. immunol. 23: 267-279, 1999. mallo gv, kurz cl, couillault c, pujol n, granjeaud s, kohara y, et al. inducible antibacterial defense system in c. elegans. curr. biol. 12: 1209-1214, 2002. means tk, mylonakis e, tampakakis e, colvin ra, seung e, puckett l, et al. evolutionarily conserved recognition and innate immunity to fungal pathogens by scavenger receptors scarf1 and cd36. j. exp. med. 206: 637653, 2009. minabi m, ueno s, pillai a, kato y. evolution of asabf-type antimicrobial peptides in nematodes: putative rearrangement of disulfide bonding patterns. dev. comp. immunol. 33: 1147-1150, 2009. muir re, tan mw. virulence of leucobacter chromiireducens subsp. solipictus to caenorhabditis elegans: characterization of a novel host-pathogen interaction. appl. environ. microbiol. 74: 4185-4195, 2008. murphy ct, mccarroll sa, bargmann ci, fraser a, kamath rs, ahringer j, et al. genes that act downstream of daf-16 to influence the lifespan of caenorhabditis elegans. nature 424: 277283, 2003. mysliwy j, dingley aj, stanisak m, jung s, lorenzen i, roeder t et al. caenopore-5: the three-dimensional structure of an antimicrobial protein from caenorhabditis elegans, dev. comp. immunol. [in press]. nathoo an, moeller ra, westlund ba, hart ac. identification of neuropeptide-like protein gene families in caenorhabditis elegans and other species. proc. natl. acad. sci. usa 98: 1400014005, 2001. patthy l. consensus approaches in detection of distant homologies. method enzymol. 266: 184198, 1996. pillai a, ueno s, zhang h, kato y. induction of asabf (ascaris suum antibacterial factor)-type antimicrobial peptides by bacterial injection: novel members of asabf in the nematode ascaris suum. biochem. j. 371: 663-668, 2003. pradel e, zhang y, pujol n, matsuyama t, bargmann ci, ewbank jj. detection and avoidance of a natural product from the pathogenic bacterium serratia marcescens by caenorhabditis elegans. proc. natl acad. sci. usa 104: 2295-2300, 2007. pujol n, link em, liu lx, kurz cl, alloing g, tan mw et al. a reverse genetic analysis of components of the toll signaling pathway in caenorhabditis elegans. curr. biol. 11: 809821, 2001. pujol n, cypowyj s, ziegler k, millet a, astrain a, goncharov a, et al. distinct innate immune responses to infection and wounding in the c. elegans epidermis. curr. biol. 18: 481-489, 2008a. pujol n, zugasti o, wong d, couillault c, kurz cl, schulenburg h, et al. anti-fungal innate immunity in c. elegans is enhanced by evolutionary diversification of antimicrobial peptides. plos pathogens 4, 2008b. ren m, feng h, fu y, land m, rubin cs. protein kinase d is an essential regulator of c. elegans innate immunity. immunity 30: 521-532, 2009. rodrígeuz de le vega rc, possani ld. on the evolution of invertebrate defensins. trends genet. 19: 684-687, 2005. roeder t, stanisak m, gelhaus c, bruchhaus i, grotzinger j, leippe m. caenopores are antimicrobial peptides in the nematode caenorhabditis elegans instrumental in nutrition and immunity. dev. comp. immunol. 34: 203209, 2010. schulenburg h, ewbank jj. the genetics of pathogen avoidance in caenorhabditis elegans. mol. microbiol. 66: 563-570, 2007. shapira m, hamlin bj, rong j, chen k, ronen m, tan mw. a conserved role for a gata transcription factor in regulating epithelial innate immune responses. proc. natl. acad. sci. usa 103: 14086-14091, 2006. shatters rg jr, boykin lm, lapointe sl, hunter wb, weathersbee aa. phylogenetic and structural relationships of the pr5 gene family reveal an ancient multigene family conserved in plants and select animal taxa. j. mol. evol. 63: 12-29, 2006. shtonda bb, avery l. dietary choice behaviour in caenorhabditis elegans. j. exp. biol. 209: 89102, 2006. styer kl, singh v, macosko e, steele se, bargmann ci, aballay a. innate immunity in caenorhabditis elegans is regulated by neurons expressing npr-1/gpcr. science 322: 460464, 2008. svensson b, fukuda k, nielsen pk, bonsager bc. proteinaceous alpha-amylase inhibitors. biochim. biophys. acta 1696: 145–156, 2004. tenor jl, aballay a. a conserved toll-like receptor is required for caenorhabditis elegans innate immunity. embo rep. 9: 103-109, 2008. troemel er, chu sw, reinke v, lee ss, ausubel fm, kim dh. p38 mapk regulates expression of immune response genes and contributes to longevity in c. elegans. plos genet. 2: 183, 2006. vigers aj, roberts wk, selitrennikoff cp. a new family of plant antifungal proteins. mol. plantmicrobe interact. 4: 315–323, 1991. wong d, bazopoulou d, pujol n, tavernarakis n, ewbank jj. genome-wide investigation reveals pathogen-specific and shared signatures in the response of caenorhabditis elegans to infection. genome biol. 8: r194, 2007. wu m, mcclellan sa, barrett rp, zhang y, hazlett ld. beta-defensins 2 and 3 together promote resistance to pseudomonas aeruginosa keratitis. j immunol. 183: 8054-60, 2009. zhang h, kato y. common structural properties specifically found in the csalphabeta-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? dev. comp immunol. 27: 499-503, 2003. zhang h, yoshida s, aizawa t, murakami r, suzuki m, koganezawa n, et al. in vitro antimicrobial properties of recombinant asabf, an antimicrobial peptide isolated from the 51 http://www.ncbi.nlm.nih.gov/pubmed/11516642?itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum&ordinalpos=6 http://www.ncbi.nlm.nih.gov/pubmed/11516642?itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum&ordinalpos=6 http://www.ncbi.nlm.nih.gov/pubmed/11516642?itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum&ordinalpos=6 http://www.ncbi.nlm.nih.gov/pubmed?term=%22shatters%20rg%20jr%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22boykin%20lm%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22lapointe%20sl%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22hunter%20wb%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22hunter%20wb%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22weathersbee%20aa%203rd%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract javascript:al_get(this,%20'jour',%20'j%20mol%20evol.'); nematode ascaris suum. antimicrob. agents chemother. 44: 2701-2705, 2000. zhang y, lu h, bargmann ci. pathogenic bacteria induce aversive olfactory learning in caenorhabditis elegans. nature 438: 179-184, 2005. ziegler k, kurz cl, cypowyj s, couillault c, pophillat m, pujol n, et al. antifungal innate immunity in c. elegans: pkcdelta links g protein signaling and a conserved p38 mapk cascade. cell host microbe 5: 341-352, 2009. zugasti o, ewbank jj. neuroimmune regulation of antimicrobial peptide expression by a noncanonical tgf-beta signaling pathway in caenorhabditis elegans epidermis. nat. immunol. 10: 249-256, 2009. 52 isj 5: 1-xx, 2008 isj 5: 1-11, 2008 issn 1824-307x review nongenomic and genomic actions of an insect steroid coordinately regulate programmed cell death of anterior silk glands of bombyx mori m manaboon, m iga, s sakurai division of life sciences, graduate school of natural science and technology, kanazawa university, japan accepted february 4, 2008 abstract the insect steroid hormone 20-hydroxyecdysone (20e) induces programmed cell death of larvaspecific tissues at pupal metamorphosis. in the silkworm bombyx mori, the anterior silk gland undergoes cell death in response to the metamorphic peak titer of ecdysteroids in vivo and also to 20e in vitro. although 20e elicits early gene activation, an additional 20e stimulus is required for completion of cell death. this additional stimulus involves caspase-3-like protease activation, indicating that 20e also acts through a nongenomic mechanism. studies using various inhibitors, agonists, and antagonists have shown that cell condensation is under the control of 20e genomic action, and that 20e nongenomic action begins with 20e binding to the putative membrane-bound ecdysone receptor, which is probably a g-protein-coupled receptor. this step is followed by a signaling pathway comprising phospholipase c/inositol 3,4,5-triphosphate/ca2+/protein kinase c/caspase-3-like protease, which induces dna and nuclear fragmentation. nuclear condensation is regulated by signaling of calmodulin/calmodulin-dependent protein kinase ii (camkii), but camkii activation is independent of intracellular ca2+ elevation. in addition, the genomic action of 20e is indispensable for driving its nongenomic action, indicating that crosstalk between genomic and nongenomic action plays a significant role in 20e-induced cell death. key words: ecdysone; programmed cell death; nongenomic; genomic; membrane ecdysone receptor; calcium; protein kinases introduction steroid hormones regulate development, reproduction, metabolism, and homeostasis in insects and mammals. in vertebrates, steroids including estrogens, androgens, progesterone and glucocorticoids control cell death (herold et al., 2006), and in insects, the steroid 20hydroxyecdysone (20e) induces apoptosis (terashima et al., 2000; fahrbach et al., 2005). regulation of those steroids has been studied primarily in conjunction with steroid receptor function. steroids regulate cell death by two major mechanisms. some, like estrogens, act as survival factors that trigger cell death when withdrawn, while ___________________________________________________________________________ corresponding author: sho sakurai, ph.d. division of life sciences graduate school of natural science and technology kanazawa university kakumamachi, kanazawa 920-1192, japan e-mail: ssakurai@kenroku.kanazawa-u.ac.jp others, such as glucocorticoids and 20e, actively trigger cell death. although little is known about the mode of action of steroids in cell death, recent studies have provided insight into the genetic mechanisms underlying 20e-induced programmed cell death in the salivary glands of drosophila melanogaster. it is well understood that steroid hormones regulate gene expression by binding to a nuclear receptor. thus, this genetic mechanism has been the major topic of study in relation to cell death. the genetic aspects, however, do not sufficiently account for the molecular mechanisms underlying steroid-induced cell death, which is accompanied at the later stages by caspase-3 activation (woo et al., 1998). parts of this pathway are also used in liganddependent cell death, such as that observed for fas-ligand-dependent cell death in lymphocytes (winoto and littman, 2002). similarly, 20e-induced cell death in drosophila salivary glands includes serial activation/inhibition of a protease cascade, including diap1/dronc (a homologue of caspase 1 mailto:ssakurai@kenroku.kanazawa-u.ac.jp 9) pathway leading executioner caspase activation such as drice and decay (dorstyn et al., 1999; yu et al., 2002; leulier et al., 2006; callus and vaux, 2007). both the genomic pathway that regulates early genes, leading to activation of death genes (yin and thummel, 2005), and the nongenomic pathway that activates caspase-3, are required for successful execution of programmed cell death (martin and baehrecke, 2004); however, little is known about how the two pathways are linked to each other. regulation of programmed cell death by 20e post-embryonic development of insects is associated with several molting cycles. in holometabolous insects, the larvae undergo larvalpupal ecdysis after growth. bombyx mori (silkworm) and d. melanogaster undergo four and two larvallarval molts, respectively. pupal ecdysis is accompanied by various developmental changes in tissues at the cellular and molecular levels (riddiford, 1994; henrich et al., 1999). larval-pupal transformation begins at the end of the penultimate instar (fourth larval instar in bombyx), when wing imaginal discs and leg primordia are committed to undergoing pupal metamorphosis at the following ecdysis (obara et al., 2002; koyama and sakurai, unpublished). in the feeding period of the last larval instar (fifth instar), wing discs and epidermal cells are pupally committed (riddiford, 1996; obara et al., 2002), while silk glands are committed to die after pupal ecdysis (kakei et al., 2005). all of these events are under the control of a single steroid hormone, 20e. silk glands, which are the largest tissues in bombyx last instar larvae, degenerate soon after pupation through 20e-induced programmed cell death. at the end of the feeding stage, the ecdysteroid concentration in the hemolymph increases slightly to a small peak known as the “commitment peak”. this rise causes feeding to cease and induces spinning of silk thread from the silk glands. silk glands consist of three parts: the anterior, middle, and posterior silk glands. the middle silk gland produces sericin, the “glue” protein, and the posterior silk gland produces fibroin, the silk thread protein itself. in the anterior silk gland (asg), the middle and posterior glands join to form a duct where the silk thread is spun by coating of the amorphous fibroin protein with sericin. the asg is entirely covered with a thick basement lamina and lined with a thin cuticle layer (cuticular intima) on the lumen-side surface (akai, 1983). it consists of hundreds of a single type of cell, which is rather flat and irregularly hexagonal and possesses highly branched, thread-like nuclei (fig. 1). the asg undergoes programmed cell death in response to a large metamorphic peak of ecdysteroid in the hemolymph. detachment at the cell boundary is the first morphological change (chinzei, 1975; terashima et al., 2000) and occurs on the third day (g2) of the prepupal period that begins with gut purge and lasts for 3-4 days. then, the cells condense and become round in shape (cell condensation), and the nuclear branches thicken (nuclear condensation). this step is followed by dna fragmentation, which is detectable as a ladder pattern on agarose gel electrophoresis, and by nuclear fragmentation (iga et al., 2007). finally, small granules containing the fragmented dna, which probably correspond to apoptotic bodies, are formed (fig. 2). the cell death process begins in response to the small ecdysteroid peak at the molecular level and is fully activated by the metamorphic peak, when the cells are slightly rounded in shape. further 20e stimulus is not needed after this point for cell death, ending with apoptotic body formation (terashima et al., 2000). although the drosophila salivary gland is not a homologue of the silk gland, its cells do undergo cell death in response to a metamorphic ecdysteroid peak in the prepupal period. bombyx larvae have salivary glands that survive until adult stage. these glands produce cocoonase, which opens a window in the cocoon at eclosion through which the adult escapes (kafatos et al., 1967). the salivary gland cells of drosophila are small and have round nuclei, which is disadvantageous for following changes in cellular and nuclear morphologies. hence, morphological descriptions of cell death in these glands have been focused on dna fragmentation as indicated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (tunel) method (daish et al., 2004), since the dna is difficult to recover in sufficient quantity to observe the ladder pattern on a gel (see martin and baehrecke, 2004). genetic regulation of 20e-induced programmed cell death in drosophila salivary glands has been the subject of extensive study. however, only the pathway leading to dna fragmentation has been studied. in contrast, the flat shape and highly branched nuclei of asg cells are conducive to the following changes in cellular and nuclear morphology and allow discrimination of the pathways leading to changes in cell morphology, nuclear condensation, dna fragmentation, nuclear fragmentation, and apoptotic body formation (fig. 1). ecr isoform involved in programmed cell death the nuclear receptor for 20e is a heterodimer of the ecdysone receptor, ecr, and its partner protein, ultraspiracle (usp) (yao et al., 1992; henrich et al., 1999). binding of 20e to its receptor regulates the expression of early genes and then of effecter genes (yin and thummel, 2005). the genomic effects of 20e action, beginning with serial activation/inhibition of gene expression, have been studied extensively using drosophila salivary glands (lee and baehrecke, 2001; baehrecke, 2003; yin and thummel, 2005), which are larva-specific. at the time of pupal metamorphosis, these glands undergo cell death in response to 20e, which induces pupariation and then pupation. the hierarchical control of expression of early genes in the salivary glands begins with 20e activation of ecr-b1, which encodes an ecr isoform involved in cell death. in drosophila, knockout of individual ecr isoforms has shown that mutations in ecr-a arrest development at the end of the last (third) larval instar 2 fig. 1 (a) in vivo progression of programmed cell death of bombyx anterior silk glands (asgs) after gut purge. g0-g3, 0-3 days after gut purge; p0-p2, 0-2 days after pupation. for each pair of panels representing an individual day, images were obtained by light microscopy (left panel) and by fluorescence microscopy after staining with acridine orange (right panel). note that the g0 cells are hexagonal in shape with finely branched nuclei. from g3 to p1, cellular and nuclear condensation occurs. finally, in p2, numerous granules are formed. inset: enlargement of acridine orange-stained image showing the small granules. at p2, the asgs are lined with thick basal membranes, which serve to maintain the outer shape, but soon after p2, the asgs degenerate completely and disappear from the pupal body. (b) in vitro progression of programmed cell death. asgs were cultured with 20e (0.5 µg/ml) and stained with dapi. in the far right panels for individual scores, bottom row of panels, the dapi-stained images are enlarged to show the shape of the nuclei. see fig. 2 for detailed progression. and prevent pupation, with the tissues that normally form adult structures remaining in their larval forms (davis et al., 2005). ecr-b1 mrna predominates in tissues (including salivary glands) destined to undergo programmed cell death at pupal metamorphosis (talbot et al., 1993). in an ecr-b1 loss-of-function drosophila mutant, the ecdysoneinducible genes in the larval salivary glands failed to activate (bender et al., 1997). furthermore, an ecrb1 knockout mutation prevents programmed cell death 3 fig. 2 death sequence in vitro. solid lines indicate the time period during which individual cellular events occur (see terashima et al., 2000 and iga et al., 2007 for details). modified from iga et al. (2007). in pupal bodies but does not affect the larval-pupal transformation. thus, ecr-b1 is the ecr isoform involved in metamorphic changes in larva-specific tissues that undergo cell death. in insect species other than drosophila, the roles of the ecr isoforms appear to be reversed. swallowtail butterflies commonly possess wings with intricately “cut” edges, especially in the posterior region of the hind wings. immediately after elongation of the wing imaginal discs at pupation, the wing edge is smooth. the swallowtail shape is then formed by elimination of the outer regions of the pupal wings by programmed cell death (suyama et al., 2003). in the pupal wings of the swallowtail papilio xuthus, two ecr isoforms are expressed: ecr-b1 is expressed exclusively in the proximal part of the margins between the cells that survive and form adult wings and the cells that undergo cell death, while ecr-a is expressed in the distal region that is eliminated in response to 20e. thus, ecr-a is the ecr isoform involved in region-specific programmed cell death in the pupal wings of p. xuthus. similarly, in blattella germanica (cockroach), ecr-a mediates programmed cell death of the prothoracic glands (cruz et al., 2006). in bombyx asgs, ecr-a is induced at the beginning of the prepupal period, and its mrna level increases until day 2 of the prepupal period (kamimura et al., 1997; sekimoto et al., 2006). in other tissues, such as wing discs, epidermis and midguts that do not undergo cell death, ecr-b1 is the predominant ecr isoform (kamimura et al., 1997). accordingly, although there is no knockout or knockdown data in lepidopterans, ecr-a is likely to be the isoform responsible for induction of programmed cell death in lepidopteran insects. genetic hierarchies downstream of ecr genetic regulation downstream of ecr is well documented in drosophila salivary glands (jiang et al., 2000; yin and thummel, 2005). the genes thus far shown to be involved in programmed cell death are the early genes ecr, broad complex (br-c), e74, e75, and e93; the late gene βftz-f1 (the drosophila homologue of fushitarazu); the death activator genes reaper (rpr) and head involution (hid); and dronc and drice, which are involved in the pathway leading to caspase activation (jiang et al., 2000; yin and thummel, 2005, 2007). there are two ecdysteroid surges during the drosophila third instar period, as in bombyx (sakurai et al., 1998); the first surge induces pupariation, and the second, large surge induces pupation. the surge that begins shortly before pupariation induces br-c, e74a, and e75a, but not the death activators, rpr and hid. the genes e75a, e75b, e74a, and br-c are upregulated in response to the second ecdysone surge, leading to death activator gene induction (jiang et al., 2000; dubrovsky, 2005; yin and thummel, 2005). in e93, and br-c mutants, the salivary glands fail to undergo cell death in response to 20e. this dysfunction is probably due to the lack of active caspase-3/drice, as indicated by reduction of drice expression in e74 mutant salivary glands (lee et al., 2003) and by suppression of dna/nuclear fragmentation (martin and baehrecke, 2004). these data provide further support for an interaction between genomic and nongenomic actions of 20e. direct evidence for early gene regulation has also been found in b. germanica (cruz et al., 2006, 2007). knockdown of b. germanica hormone 4 receptor 3 (bghr3) by a single injection of doublestranded rna into early last (sixth) instar b. germanica nymphs induces an arrest of developmental to adult, showing that bghr3 is involved in hierarchical stimulation of the early genes. bgecr-a knockdown by rna interference prevents programmed cell death of the prothoracic glands, which normally occurs after adult eclosion, at the end of the same instar. it also markedly reduces the bghr3 level in the glands, indicating an involvement of bghr3 in 20e-induced programmed cell death. e75 knockdown induces premature degeneration of the prothoracic glands, indicating that e75 acts as a “suppressor” of programmed cell death (mane-padros et al., 2008). therefore, in b. germanica, 20e stimulates the ecra/bghr3 pathway to activate the death activator genes, while also suppressing e75 expression in the prothoracic glands. this mechanism is similar to that found in drosophila salivary glands, where e75 is required to repress the death inhibitor gene diap2 (jiang et al., 2000; palanker et al., 2006). in bombyx asgs, the temporal profiles of e75 and bombyx hormone receptor 3 (bhr3) gene expression after 20e challenge (sekimoto et al., 2006) are very similar to those in drosophila salivary glands (lam et al., 1999), an indication for the presence of a similar transcription hierarchical control to b. germanica prothoracic glands and drosophila salivary glands. gene expression profiling has provided insight into the early gene regulation of 20e-induced cell death of bombyx asgs. the developmental expression profiles of early and early-late genes indicate that early gene regulation in bombyx is similar to that in drosophila (jiang et al., 2000; sekimoto et al., 2006, 2007). the times at which upregulation begins and expression peaks during the feeding and prepupal periods of bombyx are very similar to those in the drosophila prepupal period, with two exceptions described below (sekimoto et al., 2006) in drosophila, there are two peaks in the hemolymph ecdysteroid titer, one for pupariation and the other for pupation (riddiford, 1996). in bombyx, there are also two peaks, one for entering the prepupal period and the other for pupation. however, the two peaks are not separated by a distinct time interval; rather, the titer gradually increases with substantial fluctuations from the rise of the small peak to the maximum peak titer (sakurai et al., 1998). nongenomic action of vertebrate steroid hormones the various tissues and cells of vertebrates respond rapidly to steroid hormones (wehling, 1997; lösel and wehling, 2003; lösel et al., 2003; sergeev, 2005; tasker et al., 2006). on the other hand, the genomic effects of steroid hormones are not fully realized for several hours or days. the rapid responses are mediated by ligand-dependent channel proteins in the plasma membrane (qiu et al., 2006), by nuclear receptors localized to the plasma membrane (simoncini et al., 2002, 2004), or by membrane-bound ligand receptors (revankar et al., 2005; boonyaratanakornkit and edward, 2007). although the nongenomic actions of glucocorticoids have been well elucidated (herr et al., 2007), the identity of the glucocorticoid receptor is, as yet, unknown. it is assumed to be a g-protein coupled receptor (gpcr). studies of the novel mechanisms by which glucocorticoids suppress the immune response of t-lymphocytes have suggested that glucocorticoids have rapid effects on transmembrane currents, the t-cell receptor complex, and mitogen-activated protein (map) kinase signaling pathways, in addition to elevating the level of intracellular ca2+ (löwenberg et al., 2007). the steroid compound 17α,20β-dihydroxy-4pregnen-3-one (17α,20β-dp) acts on the sea trout oocyte to stimulate ovarian maturation (zhu et al., 2003). it brings about a decrease in the level of camp via binding to the membrane-bound progestin receptor (mpr), the first steroid membrane receptor identified as a gpcr (zhu et al., 2003; thomas et al., 2007). by coupling to g-protein αi subunit, mpr inhibits adenylyl cyclase, thus decreasing the intracellular camp level (pace and thomas, 2005). unlike other steroids with specific nuclear receptors, 17α,20β-dp does not have a nuclear receptor. therefore, it must elicit its effects only through nongenomic action. the nongenomic action of estrogen is mediated by a membrane-bound receptor or by an alternative pathway initiating with the cytoplasmic estrogen receptor. binding of estrogen to the membranebound estrogen receptor (gpr30) increases intracellular camp levels by activating adenylyl cyclase (lösel et al., 2003; rønnekleiv and kelly, 2005; thomas et al., 2005; zivadinovic et al., 2005). estrogen also binds with estrogen receptor α in the cytoplasm and activates phosphatidylinositol 3kinase (pi3k), thus activating a protein kinase cascade (simoncini et al., 2002). in insects, immunohistochemical analysis in rhodnius prolixus (schlattner et al., 2006) and bombyx (hossain et al., 2006) has identified an ecdysone receptor in the cytoplasm beneath the plasma membrane of brain neurosecretory cells, suggesting that ecr is localized close to the plasma membrane of other tissue cells, and therefore would be involved in asg cell death. however, inhibitors of pi3k and the map kinase/extracellular signal-regulated kinase (erk) kinase do not interfere with the 20e-triggered death sequence (iga et al., 2007; iga and sakurai, unpublished). thus, 20e-induced programmed cell death might not involve the ecr/pi3k pathway, although this pathway may be present in insect cells. possible nongenomic action of 20e arthropods respond rapidly to 20e, which is believed to affect na+-k+ atpase, na+-h+ exchangers, k+ channels, ecdysone transport, electrolyte (na+, k+, h+) transport, and second messenger (camp, ca2+) levels, and acts as a neuromodulator (see tomaschko, 1999 for review). in the wing imaginal discs of hyalophora gloveri (giant silk moth), 20e increases camp levels (applebaum and gilbert, 1972), indicating a 5 fig. 3 possible interaction between genomic and nongenomic actions of 20e. see text for details. modified from iga et al. (2007). nongenomic activity of 20e. it stimulates nomediated cell proliferation in the pupal eye of manduca sexta (tobacco hornworm) (champlin and truman, 1998, 2000). the no-mediated action of 20e is thought to involve activation of the nuclear ecr, which upregulates the gene for no synthase (nos), thereby increasing the activity of the enzyme. in the wing imaginal discs of bombyx fifth instar larvae, 20e stimulates cell proliferation (koyama et al., 2003). the presence of α-amanitin, a transcription inhibitor, does not affect this action of 20e (koyama and sakurai, unpublished data), indicating that de novo gene expression is unnecessary. thus, 20e probably elicits its effect on cell proliferation through a nongenomic pathway, like the estrogen/erα/pi3k/akt/nos pathway (simoncini et al., 2002). the molecular mechanisms that stimulate the 20e nongenomic pathway are largely unknown, but in drosophila, stimulation of this pathway is known to involve the catecholamine receptor (dopamine/ecdysone receptor; dmdopecr) (srivastava et al., 2005). dmdopecr is a gpcr that binds 20e and dopamine in different binding pockets; binding of 20e to its pocket on dmdopecr elevates the intracellular camp level. although dmdopecr acts as a 20e membrane receptor, it does not appear to be the membrane receptor involved in 20e-induced programmed cell death, since neither camp nor protein kinase a mediates 20e signaling up to asg cell death (iga et al., 2007). in the sections that follow below, i will present some details of the programmed cell death of bombyx asgs and discuss the 20e signaling pathway. because our information concerning 20e signaling derives entirely from our own studies, the following discussion is based on our unpublished data, except where a reference is cited (see fig. 3 for the following sections). involvement of the ecdysone membrane receptor in completion of cell death exposure to 20e induces programmed cell death of bombyx asgs. apoptotic body formation occurs 120 to 144 h after 20e challenge (fig. 1), but timed additions of α-amanitin have shown that de novo expression of the genes needed for execution of cell death is complete within 8 h of challenge (terashima et al., 2000). similarly, studies using cycloheximide, a translation inhibitor, have shown that protein synthesis is completed within 18 h of 20e challenge (terashima et al., 2000). in the classical model for steroid hormone action through nuclear receptor binding, these data would suggest that, after 18 h of challenge, the 20e stimulus is no longer needed for the cell death to occur. nevertheless, cell death is not fully realized unless 20e is present continuously for 42 h (terashima et al., 2000), suggesting that 20e activates a nongenomic pathway in addition to exerting its genomic action (fig. 2). since activation of the genetic pathway required for programmed cell death of bombyx asgs is complete within 18 h of the 20e challenge (fig. 2), we examined the nongenomic action of 20e using asgs that had been incubated with 20e for 18 h. under these culture conditions, 20e increased the intracellular level of camp (elmogy et al., 2006), suggesting the presence of an ecdysone membrane receptor (mecr; elmogy et al., 2004, 2007). [subsequently, cell death was found not to involve camp (iga et al., 2007).] the plasma membrane fraction prepared from the pre-cultured asgs bound to ponasterone a, a plant ecdysteroid with a km of 18 nm (elmogy et al., 2004), a value being comparable to the km for dmdopecr (srivastava et al., 2005). this value is approximately 10 times higher than the km for the lepidopteran ecr/usp complex (minakuchi et al., 2002) the binding protein(s) in bombyx asgs are integral plasma membrane proteins (elmogy et al., 2004, 2007). in addition, the bisacrylkydrazine ecdysone agonists, which bind with a high affinity to ecr/usp, exhibit very low affinities to the putative mecr and also dmdopecr, showing that the mecr is not the classical ecr. these biochemical and topological evidence indicates the presence of mecr in bombyx asgs. calcium mobilization and inositol triphosphate agonists, antagonists, and inhibitors are powerful tools for studying signal transduction pathways. since serial activation of protein kinases is a major feature of these pathways, we used various kinase inhibitors to search for the kinase involved in 20e signaling. inhibition of protein kinase c (pkc) suppressed the progression of death sequence except for nuclear condensation, suggesting that ca2+ is the second messenger. in fact, a ca2+ ionophore added to the asg culture after 18 h of 20e challenge mimicked the dna and nuclear fragmentation-inducing effects of 20e. when the ionophore was added at the beginning of the culture, however, it elicited no change in the asgs, indicating that the genomic effects of 20e are 6 a prerequisite for driving the ca2+ pathway (iga et al., 2007). verapamil, which blocks voltage-activated ca2+ channels, had no effect on the death sequence, indicating that 20e does not activate ltype ca2+ channels (lieberherr and grosse, 1994). flunarizine, which blocks ligand-dependent (ttype) ca2+ channels (qui et al., 2006), inhibited dna and nuclear fragmentation, but allowed other cellular responses (cell and nuclear condensation) to occur normally. the inhibitory effect of flunarizine indicates that the mecr could be a ltype ca2+ channel-coupled receptor (manaboon and sakurai, unpublished). in this case, the ca2+ influx could come from the medium. however, 20e induced complete cell death in ca2+-free medium, and flunarizine exhibited the same inhibitory effects in ca2+-free medium as in normal medium. thus, the ca2+ reservoir must be inside the cell, most likely in the endoplasmic reticulum (er), which provides ca2+ for many signal transduction pathways. in fact, 2-aminoethyl diphenylborinate, an inhibitor of the inositol 3,4,5triphosphate receptor (ip3r) on the er membrane inhibits dna and nuclear fragmentation (manaboon and sakurai, unpublished), consistent with the er being the reservoir for intracellular ca2+ elevation. flunarizine may not act as a ca2+ channel blocker in bombyx asgs but inhibit calmodulin/camkii pathway, which may mediate the 20e-induced dna and nuclear fragmentations (see below), as demonstrated in bovine brains (kubo et al., 1984). upstream of ca2+ suramin, an inhibitor of g-proteins, suppresses the cellular responses to 20e (dna and nuclear fragmentation) underlying the nongenomic pathway. u73122, an inhibitor of phospholipase c (plc), also inhibits these responses, indicating that the gprotein αq subunit (gαq)/plc/ip3 pathway acts downstream of the mecr (manaboon and sakurai, unpublished). in the prepupal period, the intracellular camp level increases transiently at the third day of gut purge, when the asgs are fully stimulated to complete programmed cell death with no further 20e stimulus (elmogy et al., 2007). in asgs cultured with 20e, intracellular camp also increases, beginning 24 h after 20e challenge, indicating an involvement of g-protein αs subunit (gαs). however, dibutyl camp, a membrane-permeable camp analogue, does not mimic 20e action, and an inhibitor of protein kinase a (pka) has no effect on 20e signaling (iga et al., 2007), demonstrating camp/pka is not involved in induction of cell death. in chinese hamster ovary cells expressing histamine h1 receptor, histamine increases intracellular camp levels through activation of the receptor, which releases g-protein βγ subunits (gβγ) that activate adenylyl cyclase (maruko et al., 2005). if gβγ/adenylyl cyclase pathway occur in the asgs, the mecr may be coupled with both gαq, but the role of gβγ and diacylglyceride in cell death remains to be seen. downstream of ca2+ as described above, inhibition of pkc suppresses dna and nuclear fragmentation. pkc inhibitor suppresses these cellular responses when added 24 h, but not 48 h, after 20e challenge, showing that the pkc activation required for inducing these responses is complete by 48 h. however, when pkc activity was assessed using a fluorescence labeled pkc substrate, it was shown to be at substantial levels with small fluctuations (iga et al., 2007). since the substrate is phosphorylated by a broad spectrum of pkc isozymes, the isozyme involved in 20e signaling remains to be identified. in rat, phorbol esters increase intracellular antiapoptotic protein, phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (ped/pea)-15 by reducing its proteasomal degradation, thereby enhancing its anti-apoptotic action. pkc-ζ and calmodulin/calmodulin-dependent protein kinase ii (camkii) activities are necessary for phorbol ester-dependent phosphorylation of ped/pea-15 (perfetti et al., 2007), indicating that whether pkc induces or inhibits apoptosis depends on the particular subtype. therefore, subtype identification is important for understanding the 20e signaling pathway. caspase-3, an apoptosis-related cysteine protease, is a key enzyme commonly found in the cell death pathways that respond to extracellular signals (martin and baehrecke, 2004). in vertebrates, caspase-3 activation releases caspaseactivated dnase (cad) from its inhibitor (inhibitor of caspase-activated dnase; icad), and the liberated cad induces dna fragmentation (liu et al., 1997; enari et al., 1998; sakahira et al., 1998). since caspase-3 is highly conserved throughout the animal kingdom, caspase-3-like protease activity can be measured using a colorimetric substrate for human caspase-3 (ilangovan et al., 2003). an antihuman caspase-3 antibody exhibits strong crossreactivity to the caspase-3-like protease of drosophila (yu et al., 2002). using these tools, we showed that 20e-induced cell death includes activation of the caspase-3-like protease. a caspase-3 inhibitor inhibited dna and nuclear fragmentation, showing that the caspase-3like protease is involved in 20e signaling, as it is in cell death in other animals. timed addition of the inhibitor showed that addition at 72 h, but not at 96 h, of 20e challenge prevented dna and nuclear fragmentation. caspase-3 activity measured using a colorimetric substrate also began to increase after 72 h and peaked at 96 h. thus, the caspase-3-like protease may be activated between 72 and 96 h, in accordance with the timing of dna fragmentation beginning at 96 h (iga et al., 2007). the caspase-3-like protease involved in 20einduced cell death appears somewhat different from human caspase-3. western blot analysis of cultured bombyx asgs using anti-human caspase-3 antibody that recognizes the active fragment of caspase-3 revealed a single immunoreactive band. the intensity of this band increased dramatically between 72 and 96 h of 20e challenge, in accordance with the above-mentioned results. in human and drosophila, proteolytic cleavage of 7 caspase-3 yields active fragments of 17 kda (han et al., 1997) and 45 kda, respectively; in bombyx, the molecular weight of the fragment, estimated based on western blotting, was 66 kda (kaneko and sakurai, unpublished). these data indicate that the bombyx caspase-3-like protease functions similarly to human caspase-3, but further understanding of the caspase-3-like protease awaits its identification and characterization. calmodulin/calmodulin-dependent protein kinase pathway the 20e signaling pathway leading to nuclear (chromatin) condensation in bombyx asgs is distinct from the pathway leading to caspase-3-like protease-dependent dna and nuclear fragmentation (iga et al., 2007). similarly, in the drosophila adult egg chamber, chromatin condensation occurs independently of any caspase-3-like protease activation in ovarian nurse or follicle cells and is controlled independently of dna fragmentation (nezis et al., 2006). induction of the apoptotic chromatin condensation has been suggested to result from acinus activation by active caspase-3 (sahara et al., 1999). in bombyx asgs, the calmodulin antagonist w7, when added at 18 h of 20e challenge, inhibits induction of nuclear condensation as well as of dna and nuclear fragmentation. similarly, kn-93, an inhibitor of camkii, inhibited the condensation, indicating that calmodulin/camkii activation leads to nuclear condensation (manaboon and sakurai, unpublished). calmodulin/camkii is usually activated by ca2+, but in 20e-induced cell death, a ca2+ ionophore mimicks 20e action by inducing dna and nuclear fragmentation but not nuclear condensation. this result indicates that 20e signaling activates calmodulin/camkii independently of ca2+ to regulate nuclear condensation. a calmodulin agonist and camkii inhibitor suppresses dna and nuclear fragmentation to the same degree as that of the caspase-3 inhibitor, indicating that activation of the caspase-3-like protease in bombyx may be dually regulated through pkc and camkii, although this issue is far from settled. the calmodulin/camkii pathway is involved in cell death in mammals and insects. camkii activation is regulated by ca2+/calmodulindependent protein (cam) binding or by autophosphorylation, which persists independently of ca2+. although camkii autophosphorylation is initiated by binding of ca2+/cam, ca2+/camindependent activity is substantial and is postulated to be important for synaptic and cellular plasticity in rat and drosophila (griffith, 2004; elgersma et al., 2004). in the central nervous system of drosophila, the autophosphorylation ability of ca2+/camkii allows camkii to become ca2+-independent (mehren and griffith, 2004). the protein phosphatase 1 and 2a (pp1/pp2) inhibitor calyculin a induces apoptosis, and the camkii inhibitor kn-93 blocks this effect of calyculin a. calyculin a induces apoptosis by hyperphosphorylating camkii, suggesting that stringent limitation of camkii autophosphorylation restricts apoptosis (fährmann et al., 2007). although the mechanism of caspase-3-like protease activation in asgs is unknown, ca2+-independent, cam-dependent activation of camkii may be involved. isoform-specific suppression of camkiiδc with the dominant negative-camkiiδc mutant, as well as nonselective camkii inhibition by kn-93, inhibits β1adrenergic receptor-mediated stimulation of camkiiδc-mediated apoptosis in rat cardiomyocytes (zhu et al., 2007). in drosophila, dmdopecr is a homolog of the vertebrate γ-adrenergic receptors and is activated by 20e as well as dopamine. it has both a 20e-binding pocket and a dopamine-binding pocket. the receptor is coupled with gαs, and 20e activates the receptor as powerfully as dopamine (srivastava et al., 2005). these independent results indicate an involvement of mecr in activation of camkii pathway in bombyx asg cell death. camkii activation obviously mediates extracellular signal transduction in the cell death sequence, and ca2+/cam-independent activation is brought about through autophosphorylation of camkii, although the initial phase of autophosphorylation requires mobilization of intracellular ca2+. in the death of bombyx asgs, a ca2+ ionophore may not activate camkii, since it does not mimic 20e by inducing nuclear condensation, but inhibition of either calmodulin or camkii suppresses nuclear condensation (iga et al., 2007; manaboon and sakurai, unpublished), indicating that the mechanism involves ca2+independent, calmodulin-dependent camkii activation. concluding remarks although our understanding of the nongenomic action of steroid hormones is limited, elucidation of its molecular mechanisms is important for understanding how steroid hormones exert their effects at quite different levels of biological events, including embryonic and post-embryonic development, reproduction, metabolism, homeostasis, and biological defense. in eliciting its effects, 20e has interacting nongenomic and genomic actions. in choristoneura fumiferana (spruce budworm), dequalinium-14;1,1′decamethylenebis-4-aminoquinaldinium diiodide (deca), an inhibitor of receptor of activated c kinase 1 (rack1) binding to pkc, blocks 20einduced expression of the transcription factor chr3 by inhibiting translocation of ecr, probably by preventing its phosphorylation (quan et al., 2006). chr3 is an early-late gene and is a homologue of bhr3 in bombyx and bghr3 in b. germanica, the latter of which is involved in prothoracic gland cell death (cruz et al., 2006, 2007). activation of the rack1/pkc pathway is probably one of the nongenomic actions of 20e mediated by mecr, and an example of the interaction between the genomic and nongenomic actions of 20e. nongenomic action of steroids may be involved, directly or indirectly, in regulating a variety of biological events. elucidation of its underlying molecular mechanisms will contribute to the development not only of chemicals for insect 8 population control but also of therapies and drugs for syndromes that respond to steroid hormones. until now, the molecular mechanisms underlying steroid action have been mostly understood as separate genomic and nongenomic mechanisms. we now recognize that this paradigm is inadequate. the elucidation of the interaction between the two signaling pathways may herald a new era for the study of steroid hormones. references akai h. the structure and ultrastructure of the silk gland. experientia 39: 443-449, 1983. applebaum sw, gilbert li. stimulation of adenyl cyclase in pupal wing epidermis by β-ecdysone. dev. biol. 27: 165-175, 1972. baehrecke eh. autophagic programmed cell death in drosophila. cell death differ. 10: 940-945, 2003. bender m, imam fb, talbot ws, ganetzky b, hogness ds. drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms. cell 91: 777-788, 1997. boonyaratanakornkit v, edwards dp. receptor mechanisms mediating non-genomic actions of sex steroids. semin. reprod. med. 5: 139-153, 2007. callus ba, vaux dl. caspase inhibitors: viral, cellular and chemical. cell death differ. 14: 7378, 2007. champlin dt, truman jw. ecdysteroid control of cell proliferation during optic lobe neurogenesis in the moth manduca sexta. development 125: 269-277, 1998. champlin dt, truman jw. ecdysteroid coordinates optic lobe neurogenesis via a nitric oxide signaling pathway. development 127: 35433551, 2000. chinzei y. induction of histolysis by ecdysterone in vitro. breakdown of anterior silk gland in silkworm, bombyx mori (lepidoptera, bombycidae). appl. ent. zool. 10: 136-138, 1975. cruz j, mane-padros d, belles x, martin d. functions of the ecdysone receptor isoform-a in the hemimetabolous insect blattella germanica revealed by systemic rnai in vivo. dev. biol. 297: 158-171, 2006. cruz j, martin d, belles x. redundant ecdysis regulatory functions of three nuclear receptor hr3 isoforms in the direct-developing insect blattella germanica. mech. dev. 124: 180-189, 2007. daish tj, mills k, kumar s. drosophila caspase dronc is required for specific developmental cell death pathways and stress-induced apoptosis. dev. cell 7: 909-915, 2004. davis mb, carney ge, robertson ae, bender m. phenotypic analysis of ecr-a mutants suggests that ecr isoforms have unique functions during drosophila development. dev. biol. 282: 385-396, 2005. dorstyn l, read sh, quinni lm, richardsoni h, kumar s. decay, a novel drosophila caspase related to mammalian caspase-3 and caspase-7. j. biol. chem. 274: 30778–30783, 1999. dubrovsky eb. hormonal cross talk in insect development. trends endocrinol. metab. 16: 611, 2005. elgersma y, sweatt jd, giese kp. mouse genetic approaches to investigating calcium/calmodulin-dependent protein kinase ii function in plasticity and cognition. j. neurosci. 24: 8410-8415, 2004. elmogy m, iwami m, sakurai s. presence of membrane ecdysone receptor in the anterior silk gland of the silkworm bombyx mori. eur. j. biochem. 271: 3171-3179, 2004. elmogy m, iwami m, sakurai s. solubilization of the ecdysone binding protein from anterior silk gland cell membranes of the silkworm, bombyx mori. zool. sci. 24: 971-977, 2007. elmogy m, terashima j, iga m, iwami m, sakurai s. a rapid increase in camp in response to 20hydroxyecdysone in the anterior silk glands of the silkworm, bombyx mori. zool. sci. 23: 715719, 2006. enari m, sakahira h, yokoyama h, okawa k, iwamatsu a, nagata s. a caspase-activated dnase that degrades dna during apoptosis, and its inhibitor icad. nature 391: 43-50, 1998. fahrbach se, nambu jr, schwartz lm. programmed cell death in insect neuromuscular system during metamorphosis. in: gilbert li, iatrou k, gill ss (eds), comprehensive molecular insect science, vol. 2. elsevier, oxford, pp 165-198, 2005. fährmann m, honisch s, kaufhold ma, leitges m, beil w. stringent time-dependent transregulation of calcium calmodulin kinase ii (camkii) is implicated in anti-apoptotic control. biochim. biophys. acta [in press], 2007. griffith lc. regulation of calcium/calmodulindependent protein kinase ii activation by intramolecular and intermolecular interactions. j. neurosci. 24: 8394-8398, 2004. han z, hendrickson ea, bremner ta, wyche jh. a sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro. j. biol. chem. 272: 1343213436, 1997. henrich vc, rybczynski r, gilbert li. peptide hormones, steroid hormones, and puffs: mechanisms and models in insect development. vitam. horm. 55: 73-125, 1999. herold mj, mcpherson kg, reichardt hm. glucocorticoids in t cell apoptosis and function. cell. mol. life sci. 63: 60–72, 2006. herr i, gassler n, friess h, büchler mw. regulation of differential proand anti-apoptotic signaling by glucocorticoids. apoptosis 12: 271-291, 2007. hossain m, shimizu s, fujiwara h, sakurai s, iwami m. ecr expression in the prothoracicotropic hormone-producing neurosecretory cells of the bombyx mori brain. febs j. 273: 3861-3868, 2006. iga m, iwami m, sakurai s. nongenomic action of an insect steroid hormone in steroid-induced programmed cell death. mol. cell. endocrinol. 263: 18-28, 2007. ilangovan r, marshall wl, hua y, zhou j. inhibition of apoptosis by z-vad-fmk in smn-depleted s2 cells. j. biol. chem. 278: 30993-30999, 2003. 9 jiang c, lamblin af, steller h, thummel cs. a steroid-triggered transcription hierarchy controls salivary gland cell death during drosophila metamorphosis. mol. cell 5: 445-455, 2000. kafatos fc, tartakoff am, law jh. cocoonase. i. preliminary characterization of a proteolytic enzyme from silk moth. j. biol. chem. 242: 1477-1487, 1967. kakei m, iwami m, sakurai s. death commitment in the anterior silk gland of the silkworm, bombyx mori. j. insect physiol. 51: 17-25, 2005. kamimura m, tomita s, kiuchi m, fujiwara h. tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms. ecdysteroid-dependent transcription in cultured anterior silk glands. eur. j. biochem. 248: 786793, 1997. koyama t, iwami m, sakurai s. ecdysteroid control of cell cycle and cellular commitment in insect wing imaginal discs. mol. cell. endocrinol. 213: 155-166, 2003. kubo k, matsuda y, kase h, yamada k. inhibition of calmodulion-dependent cyclic nucleotide phosphodiesterase by flunarizine, a calciumentry blocker. biochem. biophys. res. commun. 124: 315-321, 1984. lam g, hall bl, bender m, thummel cs. dhr3 is required for the prepupal-pupal transition and differentiation of adult structures during drosophila metamorphosis. dev. biol. 212: 204-216, 1999. lee cy, baehrecke eh. steroid regulation of autophagic programmed cell death during development. development 28: 1443-1455, 2001. lee cy, clough ea, yellon p, teslovich tm, stephan da, baehrecke eh. genome-wide analyses of steroidand radiation-triggered programmed cell death in drosophila. curr. biol. 13: 350-357, 2003. leulier f, ribeiro ps, palmer e, tenev t, takahashi k, robertson d, et al. systematic in vivo rnai analysis of putative components of the drosophila cell death machinery. cell death differ. 13: 1663-1674, 2006. lieberherr m, grosse b. androgens increase intracellular calcium concentration and inisitol 1,4,5-phosphate and diacylglycerol formation via a pertussis toxin-sensitive g-protein. j. biol. chem. 269: 7217-7223, 1994. liu x, zou h, slaughter c, wang x. dff, a heterodimeric protein that functions downstream of caspase-3 to trigger dna fragmentation during apoptosis. cell 89: 175184, 1997. lösel r, falenstein e, feuring m, schltz a, tillmann hc, rossel-haseroth k, et al. nongenomic steroid action: controversies, questions, and answers. physiol. rev. 83: 965-1016, 2003. lösel r, wehling m. nongenomic actions of steroid hormones. nat. rev. mol. cell biol. 4: 46-56, 2003. löwenberg m, verhaar ap, van den brink gr, hommes dw. glucocorticoid signaling: a nongenomic mechanism for t-cell immunosuppression. trends mol. med. 13: 157163, 2007. mane-padros d, cruz j, vilaplana l, pascual n, belles x, martin d. the nuclear hormone receptor bge75 links molting and developmental progression in the direct-developing insect blattella germanica. dev. biol. [in press], 2008. martin dn, baehrecke eh. caspases function in autophagic programmed cell death in drosophila. development 131: 275-284, 2004. maruko t, nakahara t, sakamoto k, saito m, sugimoto n, takuwa y, et al. involvement of the subunits of g proteins in the camp response induced by stimulation of the histamine h1 receptor. naunyn-schmiedegerg's arch. pharmacol. 372: 153-159, 2005. mehren je, griffith lc. calcium-independent calcium/calmodulin-dependent protein kinase ii in the adult drosophila cns enhances the training of pheromonal cues. j. neurosci. 24: 10584-10593, 2004. minakuchi c, nakagawa y, kiuchi m, tomita s, kamimura m. molecular cloning, expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer chilo suppressalis. insect biochem. mol. biol. 32: 999-1008, 2002. nezis ip, stravopodis dj, margaritis lh, papassideri is. chromatin condensation of ovarian nurse and follicle cells is regulated independently from dna fragmentation during drosophila late oogenesis. differentiation 74: 293-304, 2006. obara y, miyatani m, ishiguro y, hirota k, koyama t, izumi s, et al. pupal commitment and its hormonal control in wing imaginal discs. j. insect physiol. 48: 933-944, 2002. pace mc, thomas p. activation of a pertussis toxinsensitive, inhibitory g-protein is necessary for steroid-mediated oocyte maturation in spotted seatrout. dev. biol. 285: 70-79, 2005. palanker l, necakov as, sampson hm, ni r, hu c, thummel cs, et al. dynamic regulation of drosophila nuclear receptor activity in vivo. development 133: 3549-3562, 2006. perfetti a, oriente f, iovino s, alberobello at, barbagallo ap, esposito i, et al. phorbol esters induce intracellular accumulation of the antiapoptotic protein ped/pea-15 by preventing ubiquitinylation and proteasomal degradation. j. biol. chem. 282: 8648-8657, 2007. qiu j, bosch ma, jamali k, xue c, kelly mj, rønnekleiv ok. estrogen upregulates t-type calcium channels in the hypothalamus and pituitary. j. neurosci. 26: 11072-11082, 2006. quan gx, krell pj, arif bm, feng q. receptor of activated c kinase 1 (rack1) is necessary for the 20-hydroxyecdysone-induced expression of the transcription factor chr3 in the spruce budworm choristoneura fumiferana. insect mol. biol. 15: 79-87, 2006. revankar cm, cimino df, sklar la, arterburn jb, prossnitz er. a transmembrane intracellular estrogen receptor mediates rapid cell signaling. science 307: 1625-1630, 2005. riddiford lm. cellular and molecular actions of juvenile hormone i. general considerations and premetamorphic actions. adv. insect physiol. 24: 213-274, 1994. 10 riddiford lm. juvenile hormone: the status of its "status quo" action. arch. insect biochem. physiol. 32: 271-286, 1996. tasker jg, di s, malcher-lopes r. rapid glucocorticoid signaling via membraneassociated receptors. endocrinol. 147: 55495556, 2006. rønnekleiv ok, kelly mj. diversity of ovarian steroid signaling in the hypothalamus. front. neuroendocrinol. 26: 65-84, 2005. terashima j, yasuhara n, iwami m, sakurai s. programmed cell death triggered by insect steroid hormone, 20-hydroxyecdysone, in the anterior silk gland of the silkworm, bombyx mori. dev. genes evol. 11: 545-558, 2000. sahara s, aoto m, eguchi y, imamoto n, yoneda y, tsujimoto y. acinus is a caspase-3-activated protein required for apoptotic chromatin condensation. nature 401: 168-173, 1999. thomas p, pang y, filardo ej, dong j. identity of an estrogen membrane receptor coupled to a g protein in human breast cancer cells. endocrinol. 146: 624-632, 2005. sakahira h, enari m, nagata s. cleavage of cad inhibitor in cad activation and dna degradation during apoptosis. nature 391: 9699, 1998. thomas p, pang y, groenen p, kelder j, dong j, zhu y, et al. steroid and g protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins. endocrinol. 148: 705-718, 2007. sakurai s, kaya m, satake s. hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm, bombyx mori: role of low ecdysteroid titer in larval-pupal metamorphosis and a reappraisal of the head critical period. j. insect physiol. 44: 867-881, 1998. tomaschko kh. nongenomic effects of ecdysteroids. arch. insect biochem. physiol. 41: 89-98, 1999. schlattner u, vafopoulou x, steel cg, hormann re, lezzi m. non-genomic ecdysone effects and the invertebrate nuclear steroid hormone receptor ecr--new role for an "old" receptor? mol. cell endocrinol. 247: 64-72, 2006. wehling m. specific, nongenomic actions of steroid hormones. ann. rev. physiol. 59: 365-393, 1997. winoto a, littman dr. nuclear hormone receptors in t lymphocytes. cell 109 (suppl): s57-66, 2002. sekimoto t, iwami m, sakurai s. coordinate responses of transcription factors to ecdysone during programmed cell death in the anterior silk gland of the silkworm, bombyx mori. insect mol. biol. 15: 281-292, 2006. woo m, hakem r, soengas ms, duncan gs, shahinian a, kagi d, et al. essential contribution of caspase 3/cpp32 to apoptosis and its associated nuclear changes. genes dev. 12: 806-819, 1998. sekimoto t, iwami m, sakurai s. 20hydroxyecdysone regulation of two isoforms of the ets transcription factor e74 gene in programmed cell death in the silkworm anterior silk gland. insect mol. biol. 16: 581-590, 2007. yao tp, segraves wa, oro ae, makeown m, evans rm. drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. cell 71: 63-72, 1992. yin vp, thummel cs. mechanisms of steroidtriggered programmed cell death in drosophila. semin. cell dev. biol. 16: 237-243, 2005. sergeev in. calcium signaling in cancer and vitamin d. j. steroid biochem. mol. biol. 97: 145-151, 2005. yin vp, thummel cs. down-regulation of inhibitor of apoptosis levels provides competence for steroid-triggered cell death. j. cell biol. 178: 85-92, 2007. simoncini t, fornari l, mannella p, varone g, garuso a, liao jk, et al. novel nontranscriptional mechanisms for estrogen receptor signaling in the cardiovascular system. interaction of estrogen receptor alpha with phosphatidylinositol 3-oh kinase. steroids 67: 935-939, 2002. yu sy, yoo sj, yang l, zapata c, srinivasan a, hay ba, et al. a pathway of signals regulating effector and initiator caspases in the developing drosophila eye. development 129: 3269-3278, 2002. simoncini t, mannella p, fornari l, caruso a, varone g, genazzani ar. genomic and nongenomic effects of estrogens on endothelial cells. steroids 69: 537-542, 2004. zhu w, woo ay, yang d, cheng h, crow mt, xiao rp. activation of camkiiδc is a common intermediate of diverse death stimuli-induced heart muscle cell apoptosis. j. biol. chem. 282: 10833-10839, 2007. srivastava dp, yu ej, kennedy k, chatwin h, reale v, hamon m, et al. rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel drosophila g-proteincoupled receptor. j. neurosci. 25: 6145-6155, 2005. zhu y, rice cd, pang y, pace m, thomas p. cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. proc. natl. acad. sci. usa 100: 22312236, 2003. suyama e, matsunaga t, shirai h, fujiwara h. pupal wing morphogenesis controlled by region-specific expression of ecdysone receptor (ecr) isoforms. zool. sci. 20: 1609, 2003. zivadinovic d, gametchu b, watson cs. membrane estrogen receptor-α levels in mcf-7 breast cancer cells predict camp and proliferation responses. breast cancer res. 7: r101-112, 2005. talbot ws, swyryd ea, hogness ds. drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. cell 73: 1323-1337, 1993. 11 isj 5: 97-xxx, 2008 isj 5: 97-102, 2008 issn 1824-307x research report resistance mechanisms to oxydemeton-methyl in tetranychus urticae koch (acari: tetranychidae) m ghadamyari, jj sendi department of plant protection, college of agriculture, university of guilan, rasht, iran accepted july 21, 2008 abstract the resistance mechanisms to oxydemeton-methyl were surveyed in two iranian strains of the two spotted spider mite, tetranychus urticae koch. bioassay was carried out on two strains, collected from tehran and rasht using dipping method. the results of bioassay indicated that resistance ratio was 20.47 for resistant strain. the activity of esterase and glutathione s-transferase in resistant and susceptible strains showed that one of resistance mechanisms to oxydemeton-methyl was esterasebased resistance and glutathione s-transferase. the esterase activity of the resistant strain was 2.5 and 2.14-fold higher than those of the susceptible strain for α-naphtyl acetate (α-na) and β-naphtyl acetate (β-na) respectively. the kinetic characteristics acetylcholinesterase (ache) showed that the ache of resistant strain had lower affinity to artificial substrates; acetylthiocholine and butyrylthiocholine than that of susceptible strain. i50 of oxydemeton-methyl for resistant and susceptible strains were 2.68×10-6 m and 7.79×10-7 m respectively. the results suggested that ache of resistant strain is insensitive to oxydemeton-methyl and ratio of ache insensitivity of resistant to susceptible strain were 3.49 and 7.8-fold to oxydemeton-methyl and paraoxon, respectively. key words: tetranychus urticae; oxydemeton-methyl; esterase; insensitive acetylcholinesterase; glutathione s transferase introduction the two-spotted spider mite, tetranychus urticae koch (acari: tetranychidae), is an important agricultural pest with a global distribution. its phytophagous nature, high reproductive potential and short life cycle facilitate rapid resistance development to many acaricides often after a few applications (cranham and helle 1985; keena and granett, 1990; devine et al., 2001; stumpf and nauen, 2001). so far resistance have been reported in several countries for compounds, such as organophosphates (ops) (sato et al, 1994; anazawa et al., 2003), dicofol (fergusson-kolmes et al., 1991), organotins (edge and james, 1986); hexythiazox (herron and rophail, 1993), clofentezine (herron et al., 1993); fenpyroximate (sato et al., 2004) and abamectin (beers et al., 1998). ___________________________________________________________________________ corresponding author: m ghadamyari department of plant protection college of agriculture university of guilan, rasht, iran e-mail: ghadamyari@guilan.ac.ir insensitive ache causing op resistance is widespread and has been detected in t. urticae strains from germany (matsumura and voss, 1964; smissaert et al., 1970), japan (anazawa et al., 2003) and new zealand (ballantyne and harrison, 1967) and in a few other tetranychid pest species, including t. cinnabarinus from israel (zahavi and tahori, 1970) and t. kanzawai from japan (kuwahara, 1982 and aiki et al., 2005). also the insensitivity of ache to demeton-s-methyl, ethyl paraoxon, chlorpyrifos oxon and carbofuran was identified in a german laboratory strain of t. urticae and a field collected strain from florida (stumpf et al., 2001). however, insensitive ache was not the only mechanism of op resistance in spider mites described, as some resistant strains of t. urticae showed an enhanced degradation of malathion, malaoxon, and ethyl parathion to nontoxic products (matsumura and voss, 1964; herne and brown, 1969). op-resistant strains of t. kanzawai rapidly degraded malathion in vitro and the resistance was obviously attributed to high nonspecific esterase activity (kuwahara, 1981, 1982). pilz et al. 97 mailto:ghadamyari@guilan.ac.ir (1978) showed that a german dimethoateselected laboratory strain of t. urticae possessed multiple mechanisms of op resistance. in addition to an ache insensitive to dimethoxon, the toxicity of dimethoate was enhanced by synergists, such as piperonyl butoxide indicating the involvement of cytochrome p-450-mediated oxidative detoxication. oxydemeton-methyl is currently used in iran to control some pests, such as aphids and t. urticae in several crops. the intensive use of oxydemetonmethyl to control of t. urticae and aphids in greenhouse facilitates resistance development in some populations of t. urticae in iran. there is no information about oxydemeton-methyl resistance in this pest in iran. resolution of the underlying biochemical mechanisms of resistance can play an important role in circumventing problems associated with pesticide resistance and assist in rational choices of chemicals for pesticide mixtures and rotations. the purpose of this study was to collect information about the presence of esterases, gluthathion s-transferase and insensitive acetylcholinesterases in the resistance of t. urticae by bioassays and biochemical assays. material and methods two spotted spider mite strains the resistant strain was collected from infected been plants grown in the research greenhouse in plant pests and disease research institute of iran, tehran. a strain from rasht was considered as a strain susceptible to oxydemeton-methyl which had no previous exposure to pesticides and was collected from convolvulus sp. in university of guilan. the mites were reared routinely on been plants (phaseolus vulgaris) grown under greenhouse conditions [25 ± 4 °c, 60 ± 20 rh (relative humidity)]. pesticide oxydemeton-methyl was used as the commercial formulation in the bioassay (ec 25 %) and was purchased from bayer crop science, germany. chemicals acetylthiocholine iodide (atc), sbutyrylthiocholine iodide (btc), 5,5 ́-dithiobis-(2nitrobenzoic acid, dtnb), triton x-100 were purchased from sigma. fast blue rr salt, α-naphtyl acetate (α-na) and β-naphthyl acetate (β-na) were obtained from fluka, and oxydemeton-methyl from accustandard. 1-chloro-2,4-dinitrobenzene (cdnb), 1,2-dichloro-4-nitrobenzene (dcnb) were purchased from merck, germany. bioassay the toxicities of oxydemeton-methyl to the susceptible and resistant strains of two-spotted spider mite were assayed using the dipping method. the formulated oxydemeton-methyl was diluted with distilled water to generate five serial dilutions. the leaf disc (diameter 3.5 cm) was immersed in the dilutions for 45 s. after drying, adult mites were placed on each treated leaf disk on wet cotton in a petri dish. up to 10 adults were placed on each leaf disk. mortality was assessed after the treated mites were maintained at 25 ± 2 °c, 70 ± 10 rh and 16:8 (light: dark) for 48 h. mites that could walk at least one body length after a gentle probe with a fine brush were scored alive. bioassay data were analyzed for ld50 values and their 95 % confidence intervals (95 % cl) using the polo-pc computer program (leora software 1987). resistance factors (rf) were calculated by dividing the ld50 value of the resistant strain by the ld50 value of the susceptible strain. determination of esterase activity adults were homogenized in ice-cold 0.2 m phosphate buffer (ph 7.0) containing 0.05 % triton x-100. after the homogenates were centrifuged at 10000 g for 12 min at 4 °c. the esterase activity was measured according to van asperen’s method (van asperen, 1962). the substrate was α-na and β-na. fifteen µl of supernatant was added to a microplate containing 35 µl 0.2 m, ph 7.0, phosphate buffer per well. the addition of 100 µl substrate per well (0.65 mm in buffer) initiated a reaction. after incubation for exactly 10 min at room temperature, 50 µl of fast blue rr salt was added and the microplate left in the dark for 30 min. absorbance at 450 nm (od450) was then measured in a microplate reader (awareness stat fax® 3200). determination of glutathione s-transferase (gst) adults were homogenized in ice-cold 0.2 m phosphate buffer (ph 7.0). after the homogenates were centrifuged at 10,000xg for 12 min at 4 °c. gst activity was measured using 1-chloro-2,4dinitrobenzene (cdnb), 1,2-dichloro-4-nitrobenzene (dcnb) and reduced gsh as substrates with slight modifications according to habig et al. (1974) in 96well microplates. the total reaction volume per well of a 96-well microplate was 300 µl, consisting of 100 µl, supernatant, cdnb (or dcnb) and gsh in buffer, giving final concentrations of 0.4 and 4 mm of cdnb (or dcnb) and gsh, respectively. the non-enzymatic reaction of cdnb (or dcnb) with gsh measured without supernatant served as control. the change in absorbance was measured continuously for 10 min at 340 nm in a thermomax kinetic microplate reader (awareness stat fax® 3200). ache kinetics mites were homogenized in ice-cold 0.2 m phosphate buffer (ph 7.0) containing 0.1 % triton x-100. after the homogenates were centrifuged at 10000 g for 15 min at 4 oc. ache activity was measured according to the methods of stumpf et al (2001) with some modifications. fifty microliters of the enzyme source was added to each well of microplate containing 140 µl of 0.2 m phosphate buffer (ph 7.0) and 20 µl dtnb solution. then 40 µl of atc was added to each well. the concentrations of the substrate were changed from 0.01 mm to 10 mm to evaluate the michaels’s constant (km). optical density was measured at 415 nm with a microplate reader (awareness stat fax® 3200). 98 table 1 log dose probit-mortality data for oxydemeton-methyl against susceptible and resistant strain of t. urticae strain insecticide n ld50 (95 % ci) a slope ± se χ2 b rrc resistant oxydemeton-methyl 245 4675.9 (44734892) 10.79±1.36 0.88 20.47 susceptible oxydemeton-methyl 250 228.6 (191-268) 2.5 ± 0.27 1.11 ald50 values and their ci are expressed in ppm formulated pesticide bvalues of χ2 smaller than 7.81 (p < 0.05) considered to be represented satisfactory agreement between observed and expected results cresistance ratio, ld50 of resistant strain/ld50 of susceptible strain inhibition assay the enzyme was preincubated with inhibitor at 37 °c for 15 min. after preincubation, the atc substrate was added to the mixture (containing 0.2 m phosphate buffer (ph 7.0) and dtnb). the remaining activity was determined at 30 min following preparation of the reaction mixture. optical density was measured at 415 nm with a microplate reader (awareness stat fax® 3200). i50 values for the ache of susceptible and resistant strains were estimated by probit analysis using the polo-pc computer program. results resistance levels in bioassay table 1 summarizes the toxicological data for susceptible and resistant strains exposed to oxydemeton-methyl. the resistance ratio of the resistant strain was 20.47. fig. 1 esterase activity in resistant and susceptible strains of t. urticae. the asterisk (*) indicates significant differences between the two strains at p<0.01 (t-test) activity of esterase the measured esterase activity of the resistant strain was significantly higher than that of the susceptible strain (t-test p < 0.001). the esterase activity of the resistant strain was 2.5 and 2.14-fold higher than those of the susceptible strain for α-na and β-na respectively (fig. 1). activity of gst the measured glutathione s-transferase activity of the resistant strain was significantly higher than that of the susceptible strain (t-test p < 0.001). the glutathione s-transferase activity of the resistant strain was 1.75 and 1.27-fold higher than those of the susceptible strain for cdnb and dcnb, respectively (fig. 2). kinetic analysis of ache the effect of substrate concentrations on ache activity were investigated using atc and btc. the different specificities of ache in resistant and susceptible strains toward two substrates are summarized in table 2. km values suggest that ache in resistant strain was kinetically different from that in susceptible strain, indicating qualitative differences among enzymes in two strains. the kinetic study indicated that ache from the resistant strain had 1.55 and 2.16fold lower affinities to substrates atc and btc than susceptible strain respectively. ache of susceptible strain showed significantly higher affinity toward btc than ache of resistant strain, suggesting that a modification of the enzyme catalytic site might be present in the ache from the resistant mite. inhibition of ache by oxydemeton-methyl and paraoxon a comparison of the i50 values of the susceptible and resistant strains showed 3.49 and 7.8-fold resistance to oxydemeton-methyl and ethyl paraoxon, respectively (table 3; fig. 3). discussion metabolic resistance mechanisms seem to be most important in arthropod species exhibiting resistance to organophosphate and carbamate pesticides (devonshire et al., 1982; kono and tomita, 1992; moores et al., 1994; ghadamyari et 99 table 2 km and vmax values of ache in resistant and susceptible strains of t. urticae vmax (δod/30 min/mite) (±sd) km (µm) (±sd) strain substrate 5 ± 0.4 95± 5.2* resistant atc 4.33 ± 0.31 61± 4.1 susceptible 3.2±0.27 337±32* resistant btc 2.9±0.23 156±23 susceptible the asterisk (*) indicates significant differences between the two strains at p<0.01 (t-test) al., 2008a, b). our results showed that probably glutathione s-transferase was related to oxydemeton-methyl resistance in t. urticae, and there is 1.75and 1.27-fold increase in glutathione s-transferase activity in the resistant strain, when cdnb and dcnb were used as substrate respectively. gsts are detoxification enzymes frequently associated with insecticides resistance, particularly op resistance (soderlund and bloomquist, 1990; yu, 1996). these enzymes may act as binding proteins increasing the activity of other pesticide detoxification enzymes such as esterases (grant and matsumura, 1994). also esterases have a role in resistance of t. urticae to oxydemeton-methyl (fig.1). these enzymes probably sequester or degrade insecticide esters before they reach their target sites in the nervous system. this mechanism seems to be important in the insecticide resistance of culex mosquitoes (mouches et al, 1986; kono and tomita, 1992; tomita et al., 1996) and aphis gossypii (suzuki et al., 1993). the relationship between the enzymes which catalyze hydrolysis of β-na and degradation of malathion was studied in resistance fig. 2 gst activity in resistant and susceptible strains of t. urticae. the asterisk (*) indicates significant differences between the two strains at p<0.01 (t-test) and susceptible strains of t. kanzawai kishida by kuwahara (1981). their results showed that resistance to malathion was associated with increased esterase activity at e3 and e4 bonds on which the main peak of malathion degradation was detected. further experimental data are required to evaluate the importance of these two degradation pathways and to clarify the existence of general esterase and glutathione transferase for oxydemeton-methyl resistance in t. urticae. although metabolic detoxification mechanisms are implicated, insensitive ache is considered one of the mechanisms of resistance to oxydemetonmethyl in t. urticae. the occurrence of pesticideinsensitive ache in spider mite was first demonstrated by smissaert (1964). the present study indicates that the resistant strain possesses an altered ache with decreased sensitivity to inhibition by oxydemeton-methyl and paraoxon and decreased affinity to atc and btc substrates. the km values for atc determined in our study were 95 and 61 µm for the insensitive and sensitive forms of ache, respectively (table 2). the maximum velocities of ache from resistant and susceptible strains were equal and only differed in terms of the affinity toward atc and btc (table 2). our results agree well with those reported by anazawa et al. (2003) with respect to the involvement of insensitive ache in conferring op resistance in t. urticae. because ache from the resistant strain had reduced affinity to atc and btc (i.e., increased km values) and reduced sensitivity to inhibition by oxydemetn-methyl and paraoxon (i.e., increased i50 values) compared with ache from susceptible strain, it is clear that the resistant strain possesses qualitatively altered ache. recent molecular investigations suggest that some amino acid substitutions in the ache of t. urticae may result in different responses of the altered aches to different substrates and inhibitors (anazawa et al., 2003). therefore the amino acid sequences of ache in iranian strains need to be analyzed. the mechanisms of oxydemeton-methyl resistance in t. urticae vary and belong to different classes of biochemical mechanisms and both detoxification and target alteration are involved in resistance of t. urticae to oxydemeton-methyl. zhu and gao (1999) showed that the modified ache alone was not sufficient to cause a high degree of resistance in insects. it seems that these two mechanisms (esterase, gst and ache insensitivity) have an additive interaction in t. urticae. on the basis of these data, it can not be 100 table 3 i50 values of oxydemeton-methyl and paraoxon on ache from susceptible and resistant strains of t. urticae i50 (m) (95 % ci) inhibitor resistant susceptible ir (95 % ci)a 2.68×10-6 (2.3×10-63.15×10-6) 7.79×10-7 (6.6×10-79×10-7) 3.49 (2.82-4.37)* oxydemeton-methyl paraoxon 6.5×10-6 (5.4×10-67.8×10-6) 8×10-7 (5.2×10-7-12.2×10-7) 7.8(5.2-11.8)* ainsensitivity ratio = i50 for resistant strain/susceptible strain and confidence interval (ci) the asterisk (*) indicates significant differences between the two strains at p<0.05 (t-test) decided which mechanism is the dominant factor in the oxydemeton-methyl resistance. resistant strain has high potential to develop cross-resistance to parathion since the ache from resistant strain showed 7.8-fold insensitivity to ethyl paraoxon and this strain had no previous exposure to parathion. it may be inferred that probably altered ache due to extensive use of oxydemeton methyl might have caused insensitivity to paraoxon. in conclusion, oxydemeton-methyl is the most commonly used insecticide and acaricide for controlling t. urticae and aphids in iran and therefore the present findings may be regarded as a future strategy for controlling t. urticae. fig. 3 inhibition of ache from t. urticae by oxydemeton-methyl and ethyl paraoxon acknowledgments this work was supported by university of guilan (rasht, iran). references anazawa y, tomita t, aiki y, kozaki t, kono y. sequence of a cdna encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite, tetranychus urticae, insect biochem. mol. biol. 33: 509-514, 2003. aiki y, kozaki t, mizuno h, kono y. amino acid substitution in ace paralogous acetylcholinesterase accompanied by organophosphate resistance in spider mite tetranychus kanzawai. pestic. biochem. physiol. 82: 154-161, 2005. ballantyne gh, harrison ra. genetic and biochemical comparisons of organophosphate resistance between strains of spider mites (tetranychus species: acari), entomol. exp. appl. 10: 231-239, 1967. beers eh, riedl h, dunley je. resistance to abamectin and reversion to susceptibility to fenbutatin oxide in spider mite (acari: tetranychidae) populations in the pacific northwest. j. econ. entomol. 91: 352-360, 1998. cranham je, helle w. pesticide resistance in tetranychidae. in: helle w, sabelis mw (eds), spider mites: their biology, natural enemies, and control. vol. 1b, amsterdam, elsevier, pp 405-421, 1985. devine gj, barber m, denholm i. incidence and inheritance of resistance to meti-acaricides in european strains of the two-spotted spider mite (tetranychus urticae) (acari: tetranychidae). pest manag. sci. 57: 443-448, 2001. devonshire al, moores gd. a carboxylesterase with broad substrate specificity causes organophosphorus, carbamate, pyrethroid resistance in peach potato aphid (myzus persicae). pestic. biochem. physiol. 18: 235246, 1982. edge ve, james, dg. organotin resistance in tetranychus urticae (acari: tetranychidae) in australia. j. econ. entomol. 79: 1477-1483, 1986. fergusson-kolmes la, scott jg, dennehy tj. dicofol resistance in tetranychus urticae (acari: tetranychidae): cross-resistance and pharmacokinetics. j. econ. entomol. 84: 41-48, 1991. 101 ghadamyari m, mizuno h, oh s, talebi k, kono y. studies on pirimicarb resistance mechanisms in iranian populations of the peach-potato aphid, myzus persicae. appl. entomol. zool. 43: 149157, 2008a. ghadamyari m, talebi k, mizuno h, kono y. oxydemeton-methyl resistance, mechanisms and associated fitness cost in green peach potato aphids (homopotera: aphididae). j. econ. entomol. [in press], 2008b. grant df, matsumura f. glutathione s-transferase 1 and 2 in susceptible and insecticide resistant aedes aegypti. pestic. biochem. physiol. 33: 132-143, 1994. habig wh, pabst mj, jakoby wb. glutathione stransferases, the first step in mercapturic acid formation. j. biol. chem. 249: 7130-7139, 1974. herne dhc, brown awa. inheritance and biochemistry of op-resistance in a new york strain of the two-spotted spider mite. j. econ. entomol. 62: 205-209, 1969. herron ga, edge v, rophail j. clofentezine and hexythiazox resistance in tetranychus urticae koch in australia. exp. appl. acarol. 17: 433440, 1993. herron ga, rophail j. genetics of hexythiazox resistance in two spotted spider mite, tetranychus urticae koch. exp. appl. acarol. 17: 423-431, 1993. keena ma, granett j. genetic analysis of propargite resistance in pacific spider mites and two spotted spider mites (acari: tetranychidae). j. econ. entomol. 83: 655-661, 1990. kono y, tomita t. characteristics of highly active carboxylesterases in insecticide-resistant culex pipiens quinquefasciatus. jpn. j. sanit. zool. 43: 297-305, 1992. kuwahara m. insensitivity of the acetylcholinesterase from the organophosphate-resistant kanzawa spider mite, tetranychus kanzawai kishida (acarina: tetranychidae), to organophosphorus and carbamate insecticide. appl. entomol. zool. 17: 486-493, 1982. kuwahara m, miyata t, saito t, eto m. relationship between high esterase activity and in vitro degradation of 14c-malathion by organophosphate-resistant and susceptible strains of the kanzawa spider mite, tetranychus kanzawai kishida (acarina: tetranychidae), and their inhibition with specific synergists. appl. entomol. zool. 16: 297 -305, 1981. leora software. polo-pc: a user guide to probit or logit analysis. leora software, berkeley, ca, 1987. matsumura t, voss g. mechanism of malathion and ethyl parathion resistance in the two-spotted spider mite, tetranychus urticae. j. econ. entomol. 57: 911-916, 1964. moores gd, devine j, devonshire al. insecticideacetylcholinesterase can enhance esterasebased resistance in myzus persicae and myzus nicotianae. pestic. biochem. physiol. 49: 114120, 1994. mouches c, pasteur n, berge jm, hyrien o, raymond m, de saint vincent br, de silvestri m, georgihiou gp. amplification of an esterase gene is responsible to insecticide resistance in a california culex mosquito. science 233: 778780, 1986. pilz r, pfeiffer g, otto d. untersuchungen zum resistenz mechanismus eines dimethoatresistenten spinnmilbenstammes (tetranychus urticae koch). arch. phytopathol. 14: 383391, 1978. sato me, miyata t, da silva m, raga a de souza filho mf. selections for fenpyroximate resistance and susceptibility, and inheritance, crossresistance and stability of fenpyroximate resistance in tetranychus urticae koch (acari: tetranychidae). appl. entomol. zool. 39: 293302, 2004. sato me, suplicy filho n, de souza filho mf, takematsu ap. resistência do ácaro rajado tetranychus urticae (koch, 1836) (acari: tetranychidae) a diversos acaricidas em morangueiro (fragaria sp.) nos municípios de atibaia-sp e piedadesp. ecossistema 19: 4046, 1994. smissaert hr. cholinesterase inhibition in spider mites susceptible and resistant to organophosphates, science 143: 129-131, 1964. smissaert hr, voerman s, oostenbrugge l, renooy n. acetylcholinesterase of organophosphate susceptible and resistant spider mites. j. agric. food chem. 18: 65-75, 1970. soderlund dm, bloomquist jr. molecular mechanisms of insecticide resistance. in: roush rt, tabashnik be (eds), pesticide resistance in arthropods, chapman & hall, new york, pp.58-96, 1990. stumpf n, nauen r. cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in tetranychus urticae (acari: tetranychidae). j. econ. entomol. 94: 1577-1583, 2001. stumpf n, zebitz cpw, kraus w, moores, gd, nauen r. resistance to organophosphates and biochemical genotyping of acetylcholinesterases in tetranychus urticae (acari: tetranychidae). pestic. biochem. physiol. 69: 131-142, 2001. suzuki k, hama h, konno y. carboxylesterase of the cotton aphid, aphis gossypii glover (homoptera: aphididae), responsible for fenitrothion resistance as a sequestering protein. appl. entomol. zool. 28: 439-450, 1993. tomita t, kono y, shimada t. chromosomal localization of amplified esterase genes in insecticide resistant culex mosquitoes. insect biochem. molec. biol. 26: 853-857, 1996. van asperen k. a study of housefly esterases by means of a sensitive colorimetric method. j. insect physiol. 8: 401-416, 1962. yu, sj. insect glutathione s-transferases. zoological studies. 35: 9-19, 1996. zahavi m, tahori as. sensitivity of acetylcholinesterase in spider mites to organophosphorous compounds. biochem. pharmacol. 19: 219-225, 1970. zhu yk, gao jr. increased activity associated with reduced sensitivity of acetylcholinesterase in organophosphate-resistant greenbug, schizaphis graminum (homoptera: aphididae). pestic. sci. 55: 11-17, 1999. 102 lectins and cytokines in celomatic invertebrates: two tales with the same end isj 7: 1-10, 2010 issn 1824-307x minireview lectins and cytokines in celomatic invertebrates: two tales with the same end d malagoli1, s sacchi2, e ottaviani1 1department of animal biology, university of modena and reggio emilia, modena, italy 2department of biological sciences, george washington university, washington dc, usa accepted december 18, 2009 abstract the paper presents the principle data regarding the presence and the roles of lectins and cytokines in invertebrates. the former have been described in the main invertebrate taxa, such molluscs, annelids, arthropods, echinoderms and tunicates, while convincing evidence for cytokines was found only in the insects, drosophila melanogaster and pseudaletia separata, and the freshwater crayfish, pacifastacus leniusculus. lectins and cytokines share convergent and common functions, and one of the multiples roles of these messenger molecules is their participation in fighting against non-self. kew words: invertebrate immunity; lectins; cytokines; evolution introduction according to barondes (1988) e yoshizaki (1990), lectins are sugar-binding proteins or glycoproteins bearing one or more sugar-binding sites and capable of agglutinating cells and/or precipitating glycoconjugates. the specificity of lectin is usually defined in terms of the monosaccharide(s) or simple oligosaccharides that inhibit lectin-induced agglutination. although lectins were first discovered in plants, they are present in all kingdoms, including bacteria and animals. cytokines constitute a more heterogeneous group of soluble mediators, but despite significant differences in terms of structure and function, some common characteristics are evident. cytokines are mainly produced by the cells of the immune or neuroendocrine systems (nisticò, 1993; schöbitz et al., 1994). described principally in mammalian models, cytokines are glycoproteins of a relatively small molecular weight, and in most cases they are synthesized de novo by activated cells during the efferent phase of immune response. the main role of cytokines is that of mediator and modulator of immune responses and inflammation, but they are also involved as signal molecules in the neuroendocrine system (nisticò, 1993; blalock, 1994; schöbitz et al., 1994). cytokines are characterized by pleiotropicity and redundancy (i.e., the same cytokine can have different effects on ______________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it diverse cellular targets, and the same function can be performed by different cytokines), they act on target cells by autocrine, paracrine and endocrine mechanisms, and they bind to specific plasma membrane receptors which show a certain degree of promiscuity (kishimoto et al., 1994; paul and seder, 1994). notably, several human cytokines also display a lectin-like activity, and this may be essential to explain some of their biological properties (cebo et al., 2002). immune recognition in celomatic invertebrates is principally carried out by cells and humoral components that include lectins and cytokines and are present in the hemolymph (ottaviani, 2005, 2006). from the literature, it emerges that comparative immunologists have devoted their attention mainly to invertebrate lectins (table 1) rather than to cytokines (table 2). this may be related to the fact that, usually, lectins are more abundant, stable and functionally recognizable than cytokines and it is possible to purify and characterize a lectin even in absence of a conspicuous molecular dataset. conversely, the isolation and characterization of a cytokine needs several molecular biology-based investigations. now that molecular databases are becoming available for several invertebrate models, cytokines are, not surprisingly, receiving the appropriate attention. lectins lectins may be classified by structural or functional criteria. lectins play an important role in cell-to-cell or cell-to-matrix interaction, glycoprotein 1 mailto:enzo.ottaviani@unimore.it table 1 examples of lectins described in invertebrates taxon species reference mollusca helix pomatia hammarström and kabat, 1969 helix pomatia hu et al., 2008 aplysia californica pauley et al., 1971 mercenaria mercenaria arimoto and tripp, 1977 biomphalaria glabrata stein and basch, 1979 biomphalaria glabrata boswell and bayne, 1984 mytilus edulis renwrantz et al., 1985 octopus vulgaris rögener et al., 1985 planorbarius corneus ottaviani and tarugi, 1986 planorbarius corneus ottaviani and tarugi, 1986 crassostrea virginica yamaura et al., 2008 annelida lumbricus terrestris stein et al., 1982 eisenia fetida eue et al., 1998 caenorhabditis elegans cooper and barondes, 1999 eisenia fetida bloc et al., 2002 arthropoda sarcophaga peregrina komano et al., 1980 sarchophaga peregrina takahashi et al., 1985 rhodnius prolixus pereira et al., 1981 limulus polyphemus rostam-abadi and pistole, 1982 limulus polyphemus muta et al., 1991 limulus polyphemus amstrong et al., 1996 aphonopelma chalcodes vasta and cohen, 1984 aphonopelma cochise vasta and cohen, 1984 aphonopelma chiricawa vasta and cohen, 1984 cancer antennarius ravindranaths et al., 1985 spodoptera exigua pendland and boucias, 1986 megabalanus rosa muramoto and kamiya, 1990 periplaneta americana jomori and natori, 1991 calliphora vomitoria mckenzie and preston, 1992 pacifastacus leniusculus kopáček et al., 1993 tachypleus tredenatus saito et al., 1997 tachypleus tredenatus kawabata and iwanaga, 1999 pinellia ternata yao et al., 2003 anopheles gambiae pace and baum, 2004 drosophila melanogaster pace and baum, 2004 echinodermata anthocidaris crassispina giga et al., 1985 anthocidaris crassispina giga et al., 1987 anthocidaris crassispina ozeki et al., 1991 holothuria polii canicattì and rizzi, 1991 asterina pectinifera kamiya et al., 1992 paracentrotus lividus canicattì et al., 1992 paracentrotus lividus drago et al., 2009 stichopus japonicus hatakeyama et al., 1993 stichopus japonicus himeshima et al., 1994 2 table 1 (continue) examples of lectins described in invertebrates taxon species reference echinodermata stichopus japonicus matsui et al., 1994 cucumaria echinata hatakeyama et al., 1994 cucumaria japonica bulgakov et al., 2000 strongylocentrotus purpuratus hibino et al., 2006 holothuria scabra gowda et al., 2008 tunicata botrylloides leachii coombe et al., 1982 didemnum candidum vasta et al., 1986 phallusia mamillata parrinello and arizza, 1989 styela clava kelly et al., 1992 clavelina picta elola and vasta, 1994 clavelina picta vasta et al., 1999 botryllus schlosseri ballarin et al., 1999 botryllus schlosseri gasparini et al., 2008 halocynthia roretzi sekine et al., 2001 pyura stolonifera pearce et al., 2001 ciona intestinalis azumi et al., 2003 ciona intestinalis parrinello et al., 2007 ciona intestinalis bonura et al., 2009 trafficking, protein folding, signal transduction, fertilization, development and self/non-self discrimination (vasta et al., 2004). with regards the structural composition, at least seven families have been identified in animals on the basis of the carbohydrate-recognition domain (crd): 1. p-type lectins; 2. s-type lectins; 3. c-type lectins; 4. pentraxins (sharon, 1993; drickamer and taylor, 1993); 5. i-type lectins (gabius, 1997; angata et al., 2002); 6. fucolectins (bianchet et al., 2002); 7. rhamnose-binding lectins (jimbo et al., 2007; terada et al., 2007). further studies have revealed others, e.g., galectins (formerly included among stype lectins) (barondes et al., 1994), calnexin, calreticulin (trombetta and helenius, 1998; parodi, 2000), collectins, ficolins (lu et al., 2002), immulectins (ascribable to c-type lectins) (yu et al., 2002) and mannose-binding lectins (ascribable to ctype lectins) (ip et al., 2009). vasta and colleagues (2004) report that only some of the above mentioned lectins are present in invertebrates. the c-type crds have been reported in several invertebrates, such as the flesh fly sarchophaga peregrina (takahashi et al., 1985), the sea urchin anthocidaris crassispina (giga et al., 1987), the acorn barnacle megabalanus rosa (muramoto and kamiya, 1990), the tunicate polyandrocarpa misakiensis (suzuki et al., 1990), the horseshoe crab limulus polyphemus (muta et al., 1991), the cockroach periplaneta americana (jomori and natori, 1991), the sea urchins paracentrotus lividus (canicattì et al., 1992) and strongylocentrotus purpuratus (smith et al., 1996) and the sea cucumber stichopus japonicus (himeshima et al., 1994). pentraxins have been reported in the tunicates clavelina picta (elola and vasta, 1994), the horseshoe crabs l. polyphemus (amstrong et al., 1996) and tachypleus tridenatus (saito et al., 1997). galectins were found in the dipterans drosophila melanogaster and anopheles gambiae (pace and baum, 2004), the nematode caenorhabditis elegans (cooper and barondes, 1999) and the ascidian, clavelina picta (vasta et al., 1999). fucolectins have been retrieved in bivalves (yamaura et al., 2008) and rhamnose-binding lectins have been observed in bivalves (naganuma et al., 2006), echinoderms (ozeki et al., 1991) and tunicates (gasparini et al., 2008). if the increased availability of molecular information has improved our knowledge of invertebrate cytokines, this is also the case for lectins. for instance, two galectins have been characterized in c. elegans and a screening of the genbank database ten years ago retrieved 26 putative galectins (cooper and barondes, 1999). in the fruit fly d. melanogaster a galectin homologue (dmgal, genbank accession number af338142) has been identified (pace et al., 2002), and in the solitary ascidian ciona intestinalis nine collectin-like genes have been retrieved (azumi et al., 2003). collectin gene expression has been found to change after lps injection in c. intestinalis (bonura et al., 2009). in the colonial ascidian botryllus schlosseri ballarin and collaborators have identified 5 transcripts from a cdna library, each with a complete coding sequence homologous to known rhamnose-binding lectins (gasparini et al., 2008). in the fully sequenced genome of s. purpuratus were identified 104 genes that encode for small c-type lectins composed of one or two domains that can 3 table 2 list of the cytokines described in invertebrates, including dhf taxon species cytokine name reference arthropoda drosophila melanogaster spätzle morisato and anderson, 1994 drosophila melanogaster upd-3 agaisse et al., 2003 drosophila melanogaster dhf malagoli et al., 2007 pseudaletia separata hcp nakatogawa et al., 2009 pacifastacus leniusculus astakine söderhäll et al., 2005 bind a wide range of oligosaccharide (hibino et al., 2006). one of these c-type lectins called spechinoidin was well characterized and it was shown a possible function in the immune defense of the sea urchin because its expression is exclusively in the phagocytes after lps-challenge (multerer and smith, 2004; terwilliger et al., 2004). molecular recognition is carried out by lectins through specific carbohydrate binding motifs. the carbohydrates exhibit several folds corresponding to different carbohydrate-binding motifs (vijayan and chandra, 1999). according to vasta and colleagues (1994), c-type lectins and pentraxins play an important role in innate immune functions, since they are probably the most ancient non-self recognition/defense mechanism. recently, a large number of c-type lectin domain-(ctld) containing proteins has been reported in c. elegans, many of which show a pathogen-specific response during infection (schulenburg et al., 2008). among the ctype lectins, mannose-binding lectins (mbl) deserve particular attention. indeed, mbl are involved in innate immune protection and work with epithelial barriers, cellular defenses such as phagocytosis (they can act as opsonins), and pattern-recognition receptors that trigger pro-inflammatory signalling cascades. in particular, ip and colleagues (2009) have found that mbl play a role as a co-receptor of toll-like receptors (tlrs), since they are linked by their spatial localization on the phagosome. furthermore, a novel involvement of mbl as a tlr co-receptor has been found, and a new paradigm for the role of these opsonins has been defined: mbl may function not only to increase microbial uptake but also to coordinate spatially, amplify, and synchronize innate immune defense mechanisms. chemical analysis and immunocytochemical reactions have demonstrated the presence of nacetylmuramic acid (nam) and the absence of sialic acid in the glycoconjugates in different tissue from mollusca gastropoda (bolognani et al., 1981; ottaviani and montagnani, 1989; bolognani fantin and ottaviani, 1990; ottaviani et al., 1990). accordingly, nam has also been found in the carbohydrate fraction of a lectin present in the freshwater snail, planorbarius corneus (ottaviani and tarugi, 1986). cytokines as far as cytokines in invertebrates are concerned, several authors have reported the presence of cytokine-like molecules in molluscs, insects, annelids, echinoderms and tunicates. together with morphological evidence, functional experiments have also suggested the presence of invertebrate cytokines that are homologues to those in mammals. indeed, several mammalian cytokines, e.g., il-2, il-8 and growth factors, stimulate cell motility, chemotaxis, phagocytosis, cytotoxicity, stress response, wound repair and the regulation of cell death in invertebrate immune cells (ottaviani et al., 2004). most of these findings were recorded in the 1980s and 1990s and principally concerned il-like molecules. however, the scientific community was sceptical about the existence of invertebrate homologues of vertebrate interleukins, given the absence of experimental evidence for the presence of a real gene similarity. since the immune molecules, celomic cytolitic factor (ccf), from the annelid eisenia foetida and human tumor necrosis factor (tnf)-α present a relevant functional similarity as a result of a shared lectin-like activity, beschin and colleagues (2001) surmized that the evidence of invertebrate immune molecules that were hypothetically homologous to vertebrate cytokines was essentially the consequence of a functional convergence on a lectin-like activity by both the invertebrate immune factors and the vertebrate cytokines. in other words, the elusive, invertebrate cytokine-like immune factors were suggested to be lectins or, alternatively, molecules endowed with lectin-like activity, whose effects were similar to those displayed by some vertebrate cytokines (beschin, 1999; beschin et al., 2001, cebo et al., 2002). this hypothesis was reinforced not only by the data on ccf, but also by the absence of molecular evidence (immunoblot or pcr-derived data (beschin et al., 2004) supporting the existence of invertebrate cytokines. a drawback of this analysis is, however, the extreme variability of the cytokine sequences, especially of interleukins, that makes the application of a typical sequence-based algorithm to find cytokine gene homologues almost impossible (huising et al., 2006). molecular biology and functional studies have demonstrated the presence of cytokines in invertebrates: spätzle (morisoto and anderson, 1994) and upd3 (harrison et al., 1998; agaisse et al., 2003) in d. melanogaster, hemocyte chemotactic peptide (hcp) from the moth pseudaletia separata (nakatogawa et al., 2009) and 4 fig. 1 distribution of helical motifs in the preprotein form of dhf (a) and a mammalian helical cytokine (b). the name of the helices (a to d) and their position between the first methionine (m) and the last aminoacid (stop) are given accordingly to conklin (2004) and conklin et al. (2005). the extension of the signal peptide (signal) is also reported. boxes of similar colors (e.g., azure bars and solid azure) indicate correspondent helices with unrelated amino acidic sequences. astakine 1 in the freshwater crayfish pacifastacus leniusculus (söderhäll et al., 2005). however, there is no indication of gene homology or structure similarity between these molecules and cytokines in vertebrate species. more precisely, the conformation of spätzle resembles that of vertebrate ngf and coagulogen in the horseshoe crab (mizuguchi et al., 1998). upd-3 and hcp has no homology or similarity with vertebrate cytokines and immune molecules while astakine 1 possesses a prokineticin (pk) domain found in vertebrates in molecules with many different functions, including angiogenesis and spermatogenesis (söderhäll et al., 2005). while the cited invertebrate cytokines show little or no conservations with their functional counterparts in vertebrates, signal transduction pathways appear to be well conserved. spätzle activates toll signalling that is considered to share significant similarity with the pathway activated by il-1 in mammals (lemaitre and hoffmann, 2007), while the hemocyte-derived upd-3 activates the jak/stat pathway in the fat body (agaisse and perrimon, 2004). in terms of function, the gene spätzle encodes for a secreted protein that requires proteolytic processing for activity (morisato and anderson, 1994). the protein spätzle acts immediately upstream of the receptor toll. spätzle mutant flies can recover the inducibility of drosomycin after injection of either recombinant full-lengh spätzle or hemolymph from wild-type flies. however, the recovery of drosomycin induction is always subsequent to an immune challenge with mycetes or gram positive bacteria. these results demonstrate that spätzle is a cytokine present in the hemolymph as an inactive precursor which is converted to its active form in response to infections (ferrandon et al., 2004). upd3 has been characterized as a member of the unpaired (upd) family that activates the jak/stat pathway during the embryogenesis of drosophila (harrison et al., 1998). upd3 is secreted by hemocytes and subsequently activates tota expression in the fat body (agaisse and perrimon, 2004). rna interference data suggest that upd-3 is not induced by activation of the imd-pathway, or at least not by the branch controlled by the kinase dtak1 (malagoli et al., 2008). hcp is a chemotactic factor that displays several characteristics of a mammalian chemokine. it is a small secreted peptide, present in epidermis, granulocytes and nervous system of the larvae of the lepidopteron p. separata. hcp displays a strong chemotactic activity and recruits circulating hemocytes to the wound where it is supposed to enhance clotting. at present no information is available on the receptor bound by hcp and on the signalling pathway activated by this insect chemokine (nakatogawa et al., 2009). finally, astakine 1 induces a strong hematopoietic response by interacting with a f1atp synthase receptor present exclusively on the hemopoietic tissue and not on the surface of circulating hemocytes (lin et al., 2009). a similar molecule, astakine 2, has been identified in another crustacean, the shrimp penaeus monodon, but there is still scant information on this finding (söderhall et al., 2005). the discovery of the above mentioned cytokines has contributed to the general acceptance of the existence of cytokines in invertebrates; however, the findings offer little help in understanding whether homologues for vertebrate interleukins can be retrieved in invertebrate models. a significant advance in this field was the discovery of the first gene predicted to encode for a helical cytokine in d. melanogaster labelled dhf (drosophila helical factor) (malagoli et al., 2007). dhf was recorded following the utilization of an algorithm (qt method) specifically developed to scan protein and cdna databases and recognize sequences encoding for helical cytokines (conklin, 2004). in vertebrates, helical cytokines represent one structural class of cytokines that include il-2, il-6, il-11, il-23, interferon α-1 and gm-csf (granulocyte-macrophage colony-stimulating factor). as mentioned above, helical cytokines are a divergent protein family, and their phylogenic relationship arises from the conserved protein structure, intron phases and broadly similar receptor families (conklin et al., 2005). accordingly, no sequences similar to that of dhf have been retrieved in databases of other insects such as a. gambiae and apis mellifera. 5 dhf (genpept accession no. aaf53861) is a peptide of 214 amino acids, and the qt method predicts that this sequence has a helical cytokine fold with 4 core amphipathic helices (fig. 1). functional experiments demonstrate that dhf expression is significantly increased after immune stimulation, suggesting the involvement of this putative helical cytokine in the innate immune response of invertebrates (malagoli et al., 2007, 2008). furthermore, using the anti-rdhf antibody, the macrophage-like drosophila embryonic hemocytes (sl2 cell line) have been found to promote the secretion of dhf following exposure to heat-inactivated bacteria and after the administration of the recombinant peptide rdhf (malagoli et al., submitted). although present data do not allow us to conclude that dhf is a homologue of vertebrate helical cytokines, the results do point to the first invertebrate candidate that could prove of some help in describing the evolution of one of the major classes of immune-related molecules. concluding remarks the major new insight into the invertebrate immunological system suggested by the above data is that, as in vertebrates, both lectins and cytokines are involved in the chemical communication among immunocompetent cells. the functions fit into the same framework, indeed these molecules share convergent and common functions, and one of the major roles of these messenger molecules is their participation in fighting against non-self. even though these conclusions may appear limited, this is a synopsis of what we know about lectins and cytokines in invertebrates. these two classes of molecules have been studied with a quite different attitude in the last 25 years. lectins have been essentially characterized from a functional and biochemical point of view. this has led to the identification of a plethora of factors, all indicated as lectins, among which it is quite difficult to find a starting point for an evolutionary analysis. conversely, considerable attention has been paid to cytokines for purposes of molecular characterization. the continuing search for elements of conservation between invertebrate and vertebrate mediators has moved towards sequence and domain analysis as a first step, maintaining the functional characterization as a necessary but not sufficient task. as such, we can say that currently only three cytokines, i.e., spätzle, upd-3 and astakine 1, are known in invertebrates. dhf is a likely further candidate, but it has still to be considered as a putative cytokine as a consequence of the sceptical attitude mentioned before. finally, we have not referred to other cytokine-like molecules that have been found in recent years in different invertebrate taxa. in absence of the required molecular, structural and functional characterization, the respective discoverers propose these as cytokine-like molecules. in conclusion, we would say that in the case of lectins, the adopted perspective has allowed the identification of an enormous number of family members, while the approach to cytokines has produced only three accepted members in the last 25 years. overall and for opposite reasons, present knowledge of both lectins and cytokines must be considered inadequate for evolutionary studies. acknowledgements we gratefully acknowledge prof l ballarin (university of padua, italy) for helpful discussion on this manuscript and an anonymous reviewer for valuable comments on an earlier version of the paper. this work was supported by far 2009 grants of the university of modena and reggio emilia (italy) to dm and eo. references agaisse h, perrimon, n. the roles of jak/stat signaling in drosophila immune responses. immunol. rev. 198: 72-82, 2004. agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev.cell 5: 441-450, 2003. angata t, brinkman-van der linden e. i-type lectins. biochim. biophys. acta 1572: 294-316, 2002. arimoto r, tripp mr. characterization of a bacterial agglutinin in the hemolymph in the hard clam, mercenaria mercenaria. j. invertebr. pathol. 30: 406-417, 1977. armstrong pb, swarnakar s, srimal s, misquith s, hahn ea, aimes rt, quigley jp. a cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins. j. biol. chem. 271: 14717-14721, 1996. azumi k, de santis r, de tomaso a, rigoutsos i, yoshizaki f, pinto mr, et al. genomic analysis of immunity in a urochordate and the emergence of the vertebrate immune system: waiting for godot. immunogenetics 55: 570-581, 2003. ballarin l, tonello c, guidolin l, sabbadin a. purification and characterization of a humoral opsonin, with specificity for d-galactose, in the colonial ascidian botryllus schlosseri. comp. biochem. physiol. 123b: 115-123, 1999. bianchet ma, odom ew, vasta gr, amzel lm. a novel fucose recognition fold involved in innate immunity. nat. struct. biol. 9: 628-634, 2002. barondes sh. bifunctional properties of lectins: lectins redefined. trends biochem. sci. 13: 480-482, 1988. barondes sh, cooper dn, gitt ma, leffler h. galectins. structure and function of a large family of animal lectins. j. biol. chem. 269: 20807-20810, 1994. beschin a, bilej m, brys l, torreele e, lucas r, magez s, de baetselier p convergent evolution of cytokines. nature 400: 627-628, 1999. beschin a, bilej m, magez s, lucas r, de baetselier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on a similar lectin-like activity. prog. mol. subcell. biol. 34: 145-163, 2004. beschin a, bilej m, torreele e, de baetselier p. on the existence of cytokines in invertebrates. cell. mol. life sci. 58: 801-814, 2001. blalock je. the syntax of immune-neuroendocrine communication. immunol. today 15: 504-511, 1994. 6 bloc a, lucas r, van dijck e, bilej m, dunant y, de baetselier p, beschin a. an invertebrate defense molecule activates membrane conductance in mammalian cells by means of its lectin-like domain. dev. comp. immunol. 26: 35-43, 2002. bolognani fantin am, ottaviani e. n-acetylmuramic acid in mollusca gastropoda: a histochemical and immunocytochemical study. histochem. j. 22: 475-479, 1990. bolognani l, masserini m, bodini pa, bolognani fantin am, ottaviani e. lipid composition in ganglia of mollusca. j. neurochem. 36: 821825, 1981. bonura a, vizzini a, salerno g, parrinello n, longo v, colombo p. isolation and expression of a novel mbl-like collectin cdna enhanced by lps injection in the body wall of the ascidian ciona intestinalis. mol. immunol. 46: 23892394, 2009. boswell ca, bayne cj. isolation, characterization and functional assessment of a hemagglutinin from the plasma of biomphalaria glabrata, intermediate host of schistosoma mansoni. dev. comp. immunol. 8: 559-568, 1984. bulgakov aa, nazarenko el, petrova iy, eliseikina mg, vakhrusheva nm, zubkov va. isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian cucumaria japonica. biochemistry (mosc) 65: 933-939, 2000. canicattì c, pagliara p, stabili l. sea urchin coelomic fluid agglutinin mediates coelomocyte adhesion. eur. j. cell biol. 58: 291-295, 1992. canicattí c, rizzo a. a 220 kda coelomocyte aggregating factor involved in holothuria polii cellular clotting. eur. j. cell biol. 56: 79-83, 1991. cebo c, vergoten g, zanetta jp. lectin activities of cytokines: functions and putative carbohydraterecognition domains. biochim. biophys. acta 1572: 422-434, 2002. conklin d. recognition of the helical cytokine fold. j. comput. biol. 11: 1189-1200, 2004. conklin d, haldeman b, gao z. gene finding for the helical cytokines. bioinformatics 21: 1776-1781, 2005. coombe dr, schluter sf, ey pl, jenkin cr. identification of the ha-2 agglutinin in the haemolymph of the ascidian botrylloides leachii as the factor promoting adhesion of sheep erythrocytes to mouse macrophages. dev. comp. immunol. 6: 65-73, 1982. cooper dn, barondes sh. god must love galectins; he made so many of them. glycobiology 9: 979-984, 1999. drago f, malagoli d, pezzino fm, d'urso v, sammartano f. presence of a low molecular weight lectin in the coelomic fluid of the sea urchin paracentrotus lividus inv. surv. j. 6:1520, 2009. drickamer k, taylor me. biology of animal lectins. annu. rev. cell biol. 9: 237-264, 1993. elola mt, vasta gr. lectins from the colonial tunicate clavelina picta are structurally related to acute-phase reactants from vertebrates. ann. ny acad. sci. 712: 321-323, 1994. eue i, kauschke e, mohrig w, cooper el. isolation and characterization of earthworm hemolysins and agglutinins. dev. comp. immunol. 22: 1325, 1998. ferrandon d, imler jl, hoffmann ja. sensing infection in drosophila: toll and beyond. semin. immunol. 16: 43-53, 2004. gabius hj. animal lectins. eur. j. biochem. 243: 543-576, 1997. gasparini f, franchi n, spolaore b, ballarin l. novel rhamnose-binding lectins from the colonial ascidian botryllus schlosseri. dev. comp. immunol. 32: 1177-1191, 2008. giga y, ikai a, takahashi k. the complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin anthocidaris crassispina. homologies with mammalian and insect lectins. j. biol. chem. 262: 6197-6203, 1987. giga y, sutoh k, ikai a. a new multimeric hemagglutinin from the coelomic fluid of the sea urchin anthocidaris crassispina. biochemistry 24: 4461-4467, 1985. gowda nm, goswami u, khan mi. purification and characterization of a t-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (holothuria scabra). fish shellfish immunol. 24: 450-458, 2008. hammarström s, kabat ea. purification and characterization of a blood-group a reactive hemagglutinin from the snail helix pomatia and a study of its combining site. biochemistry 8: 2696-2705, 1969. harrison da, mccoon pe, binari r, gilman m, perrimon n. drosophila unpaired encodes a secreted protein that activates the jak signaling pathway. genes dev. 12: 3252-3263, 1998. hatakeyama t, himeshima t, komatsu a, yamasaki n. purification and characterization of two lectins from the sea cucumber stichopus japonicus. biosci. biotechnol. biochem. 57: 1736-1739, 1993. hatakeyama t, kohzaki h, nagatomo h, yamasaki n. purification and characterization of four ca(2+)-dependent lectins from the marine invertebrate, cucumaria echinata. j. biochem. 116: 209-214, 1994. hibino t, loza-coll m, messier c, majeske aj, cohen ah, terwilliger dp, et al. the immune gene repertoire encoded in the purple sea urchin genome. dev. biol. 300: 349-365, 2006. himeshima t, hatakeyama t, yamasaki n. amino acid sequence of a lectin from the sea cucumber, stichopus japonicus, and its structural relationship to the c-type animal lectin family. j. biochem. 115: 689-692, 1994. hu j, yu x, fu w, zhang w. a helix pomatia lectin binding protein on the extraembryonic membrane of the polyembryonic wasp macrocentrus cingulum protects embryos from being encapsulated by hemocytes of host ostrinia furnaclis. dev. comp. immunol. 32: 356-364, 2008. huising mo, kruiswijk cp, flik g. phylogeny and evolution of class-i helical cytokines. j. endocrinol. 189: 1-25, 2006. 7 http://www.ncbi.nlm.nih.gov/pubmed/17027739?itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum&ordinalpos=4 http://www.ncbi.nlm.nih.gov/pubmed/17027739?itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum&ordinalpos=4 http://www.ncbi.nlm.nih.gov/pubmed/17027739?itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum&ordinalpos=4 ip wk, takahashi k, ezekowitz ra, stuart lm. mannose-binding lectin and innate immunity. immunol. rev. 230: 9-21, 2009. jimbo m, usui r, sakai r, muramoto k, kamiya h. purification, cloning and characterization of egg lectins from the teleost tribolodon brandti. comp. biochem. physiol. 147b: 164-171, 2007. jomori t, natori s. molecular cloning of cdna for lipopolysaccharide-binding protein from the hemolymph of the american cockroach, periplaneta americana. similarity of the protein with animal lectins and its acute phase expression. j. biol. chem. 266: 13318-13323, 1991. kamiya h, muramoto k, goto r, sakai m. lectins in the hemolymph of a starfish, asterina pectinifera: purification and characterization. dev. comp. immunol. 16: 243-250, 1992. kawabata s, iwanaga s. role of lectins in the innate immunity of horseshoe crab. dev. comp. immunol. 23: 391-400, 1999. kelly kl, cooper el, raftos da. purification and characterization of a humoral opsonin from the solitary urochordate styela clava. comp. biochem. physiol. 103b: 749-753, 1992. kishimoto t, taga t, akira s. cytokine signal transduction. cell 76: 253-262, 1994. komano h, mizuno d, natori s. purification of lectin induced in the hemolymph of sarcophaga peregrina larvae on injury. j. biol. chem. 255: 2919-2924, 1980. kopáček p, grubhoffer l, söderhäll k. isolation and characterization of a hemagglutinin with affinity for lipopolysaccharides from plasma of the crayfish pacifastacus leniusculus. dev. comp. immunol. 17: 407-418, 1993. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. lin x, kim y-a, lee bl, soderhall k, soderhall i. identification and properties of a receptor for the invertebrate cytokine astakine, involved in hematopoiesis. exp. cell res. 315: 1171-1180, 2009. lu j, teh c, kishore u, reid kb. collectins and ficolins: sugar pattern recognition molecules of the mammalian innate immune system. biochim. biophys. acta 1572: 387-400, 2002. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. malagoli d, mandrioli m, sacchi s, ottaviani e. drosophila helical factor (dhf) is an imdregulated cytokine that promotes the expression of defensin and drosomycin in sl2 cells. [submitted]. malagoli d, sacchi s, ottaviani e. unpaired (upd)-3 expression and other immune-related functions are stimulated by interleukin-8 in drosophila melanogaster sl2 cell line. cytokine 44: 269274, 2008. matsui t, ozeki y, suzuki m, hino a, titani k. purification and characterization of two ca(2+)dependent lectins from coelomic plasma of sea cucumber, stichopus japonicus. j. biochem. 116: 1127-1133, 1994. mckenzie an, preston tm. biological characteristics of the calliphora vomitoria agglutinin. dev. comp. immunol. 16: 85-93, 1992. mizuguchi k, parker js, blundell tl, gay nj. getting knotted: a model for the structure and activation of spätzle. trends biochem. sci. 23: 239-242, 1998. morisato d, anderson kv. the spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsalventral pattern of the drosophila embryo. cell 76: 677-688, 1994. multerer ka, smith lc. two cdnas from the purple sea urchin, strongylocentrotus purpuratus, encoding mosaic proteins with domains found in factor h, factor i, and complement components c6 and c7. immunogenetics 56: 89-106, 2004. muramoto k, kamiya h. the amino-acid sequence of multiple lectins of the acorn barnacle megabalanus rosa and its homology with animal lectins. biochim. biophys. acta 1039: 42-51, 1990. muta t, miyata t, misumi y, tokunaga f, nakamura t, toh y, ikehara y, iwanaga s. limulus factor c. an endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains. j. biol. chem. 266: 6554-6561, 1991. naganuma t, ogawa t, hirabayashi j, kasai k, kamiya h, muramoto k. isolation, characterization and molecular evolution of a novel pearl shell lectin from a marine bivalve, pteria penguin. mol. divers. 10: 607-618, 2006. nakatogawa s, oda y, kamiya m, kamijima t, aizawa t, clark kd, et al. a novel peptide mediates aggregation and migration of hemocytes from an insect. curr. biol. 19: 779785, 2009. nisticò g. communications among central nervous system, neuroendocrine and immune systems: interleukin-2. prog. neurobiol. 40: 463-475, 1993. ottaviani e. insect immunorecognition. inv. surv. j. 2: 142-151, 2005. ottaviani e. molluscan immunorecognition. inv. surv. j. 3: 50-63, 2006. ottaviani e, bolognani fantin am, bolognani l. muramic acid as a glycoconjugate component in mollusca gastropoda. comp. biochem. physiol. 96b: 627-632, 1990. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. ottaviani e, montagnani g. immunodetection of haemocyte subpopulations by n-acetylmuramic acid antibody in planorbarius corneus (l.) (gastropoda, pulmonata). histochem. j. 21: 675-678, 1989. ottaviani e, tarugi p. purification an induced bacterial agglutinin in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). boll. zool. 56: 313-315, 1989. 8 ottaviani e., tarugi p. isolation and charecterization of an agglutinin present in the haemolymph of the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). comp. biochem. physiol. 84b: 295-297. ozeki y, matsui t, suzuki m, titani k. amino acid sequence and molecular characterization of a d-galactoside-specific lectin purified from sea urchin (anthocidaris crassispina) eggs. biochemistry 30: 2391-2394, 1991. pace ke, baum lg. insect galectins: roles in immunity and development. glycoconj. j. 19: 607-614, 2004. pace ke, lebestky t, hummel t, arnoux p, kwan k, baum lg. characterization of a novel drosophila melanogaster galectin. expression in developing immune, neural, and muscle tissues. j. biol. chem. 277: 13091-13098, 2002. parodi aj. role of n-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. biochem. j. 348 pt 1: 1-13, 2000. parrinello n, arizza v, cammarata m, giaramita ft, pergolizzi m, vazzana m, et al. inducible lectins with galectin properties and human il1alpha epitopes opsonize yeast during the inflammatory response of the ascidian ciona intestinalis. cell tissue res. 329: 379-390, 2007. parrinello n, arizza v. sugar specific cellular lectins of phallusia mamillata hemocytes: purification, characterization and evidence for cell surface localization. dev. comp. immunol. 13: 113-121, 1989. paul we, seder ra. lymphocyte responses and cytokines. cell 76: 241-251, 1994. pauley gb, granger ga, krassner sm. characterization of a natural agglutinin present in the hemolymph of the california sea hare, aplysia californica. j. invertebr. pathol. 18: 207218, 1971. pearce s, newton ra, nair sv, raftos da. humoral opsonins of the tunicate, pyura stolonifera. dev. comp. immunol. 25: 377-385, 2001. pendland jc, boucias dg. characteristics of a galactose-binding hemagglutinin (lectin) from hemolymph of spodoptera exigua larvae. dev. comp. immunol. 10: 477-487, 1986. pereira me, andrade af, ribeiro jm. lectins of distinct specificity in rhodnius prolixus interact selectively with trypanosoma cruzi. science 211: 597-600, 1981. ravindranaths mh, paulson jc, irie rf. human melanoma antigen o-acetylated ganglioside gd3 is recognized by cancer antennarius lectin. j. biol. chem. 263: 2079-2086, 1988. renwrantz l, daniels j, hansen pd. lectin-binding to hemocytes of mytilus edulis. dev. comp. immunol. 9: 203-210, 1985. rögener w, renwrantz l, uhlenbruck g. isolation and characterization of a lectin from the hemolymph of the cephalopod octopus vulgaris (lam.) inhibited by alpha-d-lactose and nacetyl-lactosamine. dev. comp. immunol. 9: 605-616, 1985. rostam-abadi h, pistole tg. lipopolysaccharidebinding lectin from the horseshoe crab, limulus polyphemus, with specificity for 2-keto-3deoxyoctonate (kdo). dev. comp. immunol. 6: 209-2018, 1982. saito t, hatada m, iwanaga s, kawabata s. a newly identified horseshoe crab lectin with binding specificity to o-antigen of bacterial lipopolysaccharides. j. biol. chem. 272: 3070330708, 1997. schöbitz b, de kloet er, holsboer f. gene expression and function of interleukin 1, interleukin 6 and tumor necrosis factor in the brain. prog. neurobiol. 44: 397-432, 1994. schulenburg h, hoeppner mp, weiner j 3rd, bornberg-bauer e. specificity of the innate immune system and diversity of c-type lectin domain (ctld) proteins in the nematode caenorhabditis elegans. immunobiology 213: 237-250, 2008. sekine h, kenjo a, azumi k, ohi g, takahashi m, kasukawa r, et al. an ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, halocynthia roretzi. j. immunol. 167: 4504-4510, 2001. sharon n. lectin-carbohydrate complexes of plants and animals: an atomic view. trends biochem. sci. 18: 221-226, 1993. söderhäll i, kim ya, jiravanichpaisal p, lee sy, söderhäll k. an ancient role for a prokineticin domain in invertebrate hematopoiesis. j. immunol. 174: 6153-6160, 2005. stein ea, wojdani a, cooper el. agglutinins in the earthworm lumbricus terrestris: naturally occurring and induced. dev. comp. immunol. 6: 407-421, 1982. stein pc, basch pf. purification and binding properties of hemagglutinin from biomphalaria glabrata. j. invertebr. pathol. 33: 10-18, 1979. smith lc, chang l, britten rj, davidson eh. sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. j. immunol. 156: 593-602, 1996. suzuki t, takagi t, furukohri t, kawamura k, nakauchi m. a calcium-dependent galactosebinding lectin from the tunicate polyandrocarpa misakiensis. isolation, characterization, and amino acid sequence. j. biol. chem. 265: 1274-1281, 1990. takahashi h, komano h, kawaguchi n, kitamura n, nakanishi s, natori s. cloning and sequencing of cdna of sarcophaga peregrina humoral lectin induced on injury of the body wall. j. biol. chem. 260: 12228-12233, 1985. terada t, watanabe y, tateno h, naganuma t, ogawa t, muramoto k, et al. structural characterization of a rhamnose-binding glycoprotein (lectin) from spanish mackerel (scomberomorous niphonius) eggs. biochim. biophys. acta 1770: 617-629, 2007. terwilliger dp, clow la, gross ps, smith lc. constitutive expression and alternative splicing of the exons encoding scrs in sp152, the sea urchin homologue of 9 complement factor b. implications on the evolution of the bf/c2 gene family. immunogenetics 56: 531-543, 2004. trombetta es, helenius a. lectins as chaperones in glycoprotein folding. curr. opin. struct. biol. 8: 587-592, 1998. vasta gr, ahmed h, fink ne, elola mt, marsh ag, snowden a, et al. animal lectins as self/nonself recognition molecules. biochemical and genetic approaches to understanding their biological roles and evolution. ann. ny acad. sci. 712: 55-73, 1994. vasta gr, ahmed h, odom ew. structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates. curr. opin. struct. biol. 14: 617-30, 2004. vasta gr, cohen e. sialic acid binding lectins in the serum of american spiders of the genus aphonopelma. dev. comp. immunol. 8: 515522, 1984. vasta gr, hunt jc, marchalonis jj, fish ww. galactosyl-binding lectins from the tunicate didemnum candidum. purification and physicochemical characterization. j. biol. chem. 261: 9174-9181, 1986. vasta gr, quesenberry m, ahmed h, o'leary n. ctype lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. dev. comp. immunol. 23: 401-420, 1999. vijayan m, chandra n. lectins. curr. opin. struct. biol. 9: 707-714, 1999. yamaura k, takahashi kg, suzuki t. identification and tissue expression analysis of c-type lectin and galectin in the pacific oyster, crassostrea gigas. comp. biochem. physiol. 149b: 168175, 2008. yao jh, zhao xy, liao zh, lin j, chen zh, chen f, et al. cloning and molecular characterization of a novel lectin gene from pinellia ternata. cell res. 13: 301-308, 2003. yoshizaki n. functions and properties of animal lectins. zool. sci. 7: 581–591, 1990. yu xq, zhu yf, ma c, fabrick ja, kanost mr. pattern recognition proteins in manduca sexta plasma. insect biochem. mol. biol. 32: 12871293, 2002. 10 isj 5: 179-yyy, 2008 issn 1824-307x isj 5: 180-189, 2008 issn 1824-307x research report lipase and invertase activities in midgut and salivary glands of chilo suppressalis (walker) (lepidoptera, pyralidae), rice striped stem borer a zibaee, ar bandani, s ramzi plant protection department, faculty of agriculture, university of tehran, karaj 31584, iran accepted november 4, 2008 abstract the rice striped stem borer, chilo supprressalis, was introduced to iran in 1973 where it is now widely distributed and causes severe damages. lipases, which catalyses the hydrolysis of fatty acid ester bonds, are widely distributed among animals, plants and microorganisms. invertases (βfructofuranosidase) are glycosidehydrolases that catalyze the cleavage of sucrose (β-dglucopyranosyl-s-d-fructofuranoside) into the monosaccharides glucose and fructose. laboratoryreared 4th instar larvae were randomly selected, their midgut and salivary glands were removed by dissection under a light microscope and lipase and invertase activities were assayed. the activity of lipase/invertase in the midgut and salivary gland were 0.49/0.27 and 0.35/0.23 µmol/min/mg protein, respectively. the optimum ph and temperature for both the two enzymes were determined to be 1011 and 37-40 °c, which is consistent with ph and temperature values already observed in lepidoptera. the enzyme activity was reduced by addition of nacl, kcl, mgcl2, sds, urea and plant extracts from artemisia annua, but not by cacl2 which enhanced enzyme activity. pest control with usage of resistant varieties of plants is one of the most important practices that are dependant on inhibitors already present in nature. hence, characterization of insect digestive enzymes, especially examination of inhibition effects on enzyme activity, could be useful in developing new strategies for pest control. key words: α-amylase; rice striped stem borer; midgut; salivary glands introduction the rice striped stem borer (chilo supprressalis, walker) is a cosmopolitan and destructive pest in rice fields of the world (zibaee et al., 2008). this pest was introduced in iran in 1973 and since then has been widely distributed in the country rice fields. it causes severe damages in all rice fields and its present density is superior than the economic injury level (eil) (dezfoulian and moustofipoor, 1972). the chemical control by using organophosphorus compounds, has been a common control procedure, although other methods based on agricultural practices such as ploughing, usage of resistant varieties of plants, weed control as overwintering sites and biological control with trichogramma spp. have been incorporated. in recent ___________________________________________________________________________ corresponding author: ali reza bandani plant protection department college of agriculture and natural resources university of tehran, karaj, 31584 iran e-mail: abandani@ut.ac.ir years, resistant varieties and pheromones also have been added to control the diffusion of c. suppressalis like in other places of the world (muralidharan and pasaalu, 2006). a study on 78 different varieties of rice showed that binam with 15 % white head is the most resistant variety. germplasts studies showed that khazar variety is resistant to the first generation of rice striped stem borer. however, it is susceptible to the second generation. lipases (triacylglycerol–acyl-hydrolase ec 3.1.1.3), which catalyzes the hydrolysis of fatty acid ester bonds, are widely distributed among animals, plants and microorganisms (naumff, 2001). it has been showed that lipases can also hydrolyze a variety of esters in organic solvent systems and thus they can be widely used in many industrial areas, e.g., dairy, food, detergent and biofuel industries (ishaaya and swirski, 1970; henrissat and bairoch, 1993; grillo et al., 2007). the most characteristic property of lipases is that they act on substrate at the interface between the aqueous and the lipid phase (grillo et al., 2007). 180 to date, many research groups have carried out the isolation and purification of lipases from various sources, mainly microorganisms, fish, fungi, milk and plants (cherry and crandall, 1932; henrissat and bairoch, 1993; degerli and akpinar 2002; grillo et al., 2007). however, lipid biochemistry studies in insects is time-consuming and moved on very slowly due to high diversity of insects and changes in the lipid composition and lipophorin present in hemolymph during metamorphosis from larva to pupa (degerli and akpinar, 2002). recently, lipid mobilization and transport in insects is under investigation, especially lipases and lipophorin (a reusable lipoprotein particle in insect systems) because of their roles in energy production and transport of lipids at flying activity (ayre, 1967). although stored lipids in vertebrate adipose tissue are released as free fatty acids, in insects most fatty acids are released as 1,2-diacylglycerols and mobilization of lipid reserves from insect fat body is under the control of adipokinetic hormone (grillo et al., 2007). invertases (β-fructofuranosidase, ec3.2.1.26) also termed fructosidase, saccharase, or sucrase, are glycosidehydrolases (ec 3.2.1) that catalyze the cleavage of sucrose (β-d-glucopyranosyl-s-dfructofuranoside) into the monosaccharides, glucose and fructose (henrissat and bairoch, 1993; sturm and tang, 1999; naumoff, 2001). invertase, thus, appears to be a particularly important enzyme for plants and animals. given this general importance, a surprisingly limited number of studies have tried to quantify invertase activity in ants (ayre, 1963, 1967; ricks and vinson, 1972) or other animals (martinez del rio, 1990; zhang et al., 1993). this might be due to the particular methodological problems arising from the quantification of invertase in animals whose carbohydrate metabolism is highly active. digestion is a phase of insect physiology on which little research has been performed, despite the economic importance of the food of insects and the fact that the most important control measures involve the action of digestive juices on poisons taken into the digestive tract. a better understanding of enzyme catalysis is essential in order to develop methods of insect control (bandani et al., 2001; ghoshal et al., 2001; maqbool et al., 2001). the purpose of the present study is to identify and characterize the lipase and invertase activities from midgut and salivary glands (sg) of rice striped stem borer larvae to gain a better understanding of the digestive physiology. this understanding will hopefully lead to new management strategies for control of this pest. materials and methods insects to decrease the side effects of laboratory mass culture, 400 pupae were collected from fields and reared on the same variety seedling (taroum) as sampling sites. insects were reared based on the method mentioned by kammano and sato (1985) in 28 ± 1 °c, light cycle 16l:8d and rh > 80 %. when the larvae grow up to 4th instar larvae, 30 larvae were randomly selected for biochemical analysis. for 4th instar determination, dayer's formula was used which had been described by majidi et al. (2002). sample preparation and enzyme assays briefly, larvae were randomly selected and total midgut and sg were removed by dissection under a stereo microscope in ice-cold saline buffer (6 μm nacl). the midgut and sg were separated from the insect’s body, rinsed in ice-cold buffer, placed in a pre-cooled homogenizer and ground in 1 ml of universal buffer containing succinate, glycine and 2morpholinoethanesulfonic acid (ph 7.2) (hosseinkhani and nemat-gorgani, 2003). the homogenates from both preparations (midgut and sg) were separately transferred to 1.5 ml centrifuge tubes and centrifuged at 20,000 x g for 20 min at 4 °c. the supernatants were pooled and stored at -20 °c for subsequent analyses. lipase activity the enzyme assays were carried out as described by tsujita et al. (1989). thirty µl of gut and salivary glands tissue extracts and 100 µl of pnitrophenyl butyrate (50 mm), as substrate, were incorporated, mixed thoroughly and incubated at 37 °c. for negative control tubes, samples (midgut and salivary glands) were placed in a boiling water bath for 15 min to destroy the enzyme activity and then cooled. after 1 min, 100 µl distilled water were added to each tube (control and treatment) and absorbance was read at 405 nm. one unit of enzyme 0 0.1 0.2 0.3 0.4 0.5 0.6 midgut salivary glands a ct iv ity o f l ip as e (µ m ol /m in /m g pr ot ei n) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 midgut salivary glandsa ct iv ity o f i nv er ta se ( µm ol /m in /m g pr ot ei n) fig. 1 activity level of lipase (up) and invertase (down) in 4th instar larvae midgut and salivary glands of rice striped stem borer. 181 http://www.citeulike.org/user/biblio24/author/tsujita y = 0.0091x + 0.0556 r2 = 0.9693 0 0.1 0.2 0.3 0.4 0.5 0.6 0 10 20 30 40 50 concentration of p-nitrophenol a b so rb an ce a t 40 60 5 n m y = 0.2011x + 0.008 r2 = 0.9922 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 0.5 1 1.5 2 2.5 3 3. concentration of glucose a b so rb an ce a t 34 0 nm 5 fig. 2 standard calibration curve for the determination of p-nitrophenol and glucose released in the lipase (up) and invertase (down) assay. will release 1.0 nmol of p-nitrophenol per min at ph 7.2 at 37 °c using p-nitrophenyl butyrate as substrate. standard curve was used to calculate the specific activity of enzyme. invertase activity samples were transferred to 100 ml flasks and 1 ml toluene was added to arrest the enzyme activity. after 15 min, 6 ml of 0.2 m glycine buffer (ph 7.2) containing 18 mm sucrose was added to the samples and the flasks were closed with cotton plugs then held for 24 h at 30 °c. samples were passed through whatman filter paper and glucose in the filtrate was assayed at 340 nm (nelson, 1994). kinetic parameters measurements twenty µl of appropriately diluted enzyme preparation was used in each assay. final concentrations for substrate were 20, 30, 40, 50 and 60 mm for lipase and 0.09, 0.18, 0.36, 0.72 and 1.54 mm for invertase, respectively. the michaelis constant (km) and the maximum velocity (vmax) were estimated by sigmaplot software version 11 (systat software inc., chicago, il, usa) and the results of km and vmax were the means ± se of three replicates for every population. effect of ph and temperature on enzyme activity the effect of temperature and ph on lipase and invertase activity were examined using enzyme extractions from the larval midgut and sg. the effect of temperature on enzymes activity was determined by incubating the reaction mixture at 20, 25, 30, 35, 37, 40, 45, 50, 55, 60 and 70 °c for 24 h, followed by measurement of activity. optimal ph for their activities was determined using universal buffer with ph set at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. effect of activators and inhibitors on enzyme activity to test the effect of different ions on the enzymes, midguts and sg were dissected in distilled water. enzyme assays were performed in the presence of different concentrations of chloride salts of na+ (5, 10, 20 and 40 mm), k+ (5, 10, 20 and 40 mm), ca2+ (5, 10, 20 and 40 mm), mg2+ (5, 10, 20 and 40 mm), and ethylenediaminetetraacetic acid (edta; 0.5, 1, 2 and 4 mm), sodium dodecylsulfate (sds; 1, 2 and 4 mm), urea (0.5, 1, 2, 4, 6 and 8 mm) and artemisia annua extract (10, 15 and 25 % concentrations). these compounds were added to the assay mixture, and activity was measured after 30 min incubation. a control was also measured (no compounds added). effect of a. annua extract on enzymatic parameters of invertase and lipase for this experiment, 20 µl of appropriately diluted enzyme preparation was used in each assay. final concentrations for substrate were 20, 30, 40, 50 and 60 mm for lipase and 0.09, 0.18, 0.36, 0.72 and 1.54 mm for invertase, respectively. finally, 20 % of plant extract added to each well. km and vmax were estimated by sigmaplot software version 11 (systat software inc.) and the results of 0 0.2 0.4 0.6 0.8 1 3 4 5 6 7 8 9 10 11 12 13 ph a ct iv ity o f l ip as e (µ m ol /m in /m g pr ot ei n ) midgut salivary glands 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 3 4 5 6 7 8 9 10 11 12 ph in ve rt as e ac tiv ity (µ m ol /m in /m g pr ot ei n ) midgut salivary glands fig. 3 effect of ph on activity of lipase (up) and invertase (down) extracted from midgut and salivary gland of rice striped stem borer. 182 0 0.1 0.2 0.3 0.4 0.5 0.6 20 30 35 37 40 45 50 55 60 70 tem perature (°c) a ct iv it y o f l ip as e (µ m o l/m in /m g p ro te in ) midgut salivary glands results lipase and invertase activities studies showed that lipase and invertase are present in the midgut and salivary glands of adult c. suppressalis (fig. 1). the activity of lipase was 0.486 µmol/min/mg protein and 0.27 µmol/min/mg protein in midgut and sg, respectively. the invertase activity in midgut and sg was 0.35 and 0.23 µmol/min/mg protein, respectively. there was a significant difference in the degree of enzyme activity between midgut and salivary glands (figs 1, 2). effect of ph and temperature on enzyme activity the in vitro evaluation of c. suppressalis lipase and invertase from midgut and sg indicated that enzyme activity increased steadily from ph 3 to 11 and from 3 to 10, respectively. after reaching the threshold ph level, enzyme activity decreased with the increasing of ph and there were significant differences among measured values for each ph (fig. 3). both enzymes were considerably active over a broad range of temperatures. as the results show, the optimum temperature for lipase and invertase activities were 37 and 40 °c for midgut and sg, respectively (fig. 4). -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 20 30 35 37 40 45 50 55 60 70 tem perature (° c) a ct iv it y o f in ve rt as e (µ m o l/m in /m g p ro te in ) midgut salivary glands effect of activators and inhibitors on enzyme activity several molecules and chemical compunds affects the activity of lipase and invertase in midgut and sg of rice striped stem borer, although they had a similar effect on both enzymes (tables 1 and 2). activity level of enzymes in midgut and sg elevated due to increasing of cacl2 and edta concentrations for lipase and just cacl2 for invertase (table 1 and 2). activity level of enzyme decreased in presence of nacl, kcl, edta, mgcl2, sds, urea and a. annua extract in both midgut and sg (tables 1 and 2). fig. 4 effect of temperature on activity of lipase (up) and invertase (down) extracted from midgut and salivary gland of rice striped stem borer. km and vmax were the means ± se of three replicates for every population. for determination of a. annua extract effect on enzymatic parameters of lipase and invertase, 20 % of plant extract was added to each well. kinetic parameters as can be seen in table 3, lipase vmax of midgut and sg were 0.5 and 0.35 µmol/min/mg protein, respectively. lipase km of midgut and sg were 15 and 19 mm, respectively. invertase vmax was 0.9 and 0.5 µmol/min/mg protein in midgut and sg, respectively, while invertase km was 0.31 and 0.39 mm in midgut and sg (table 3, fig. 5). polyacrylamide gel electrophoresis (page) in order to determine the molecular mass of native lipase, native polyacrylamide disc-gel electrophoresis was carried out using the method of parish and marchalonis (1970) using 2.7 % and 7.7 % polyacrylamide for the stacking and resolving gels, respectively. the gel was stained with 1.5 % (w/v) coomassie brilliant blue g-250 and distained in glacial acetic acid-methanol-water 7.5: 5.0: 87.5. effect of a. annua extract on enzymatic parameters of invertase and lipase enzymes parameters changed due to using of a. annua extract. as it is shown in table 4, lipase kinetic parameter, vmax-km were 0.35 µmol/min/mg protein-37.5 mm in midgut and 2.12 µmol/min/mg protein-21 mm in sg. as well as invertase is concerned, vmax-km were 0.49 µmol/min/mg protein0.30 mm in midgut and 0.81 µmol/min/mg protein0.92 mm in sg (table 4, fig. 6). protein determination protein concentration was measured according to the method of bradford (1976), using bovine serum albumin (bio-rad, münchen, germany) as a standard. statistical analysis native page data were compared by one-way analysis of variance (anova) followed by tukey's studentized test when significant differences were found at p=0.05. enzyme kinetic parameters were analyzed by using the sigmaplot software version 11 (systat software inc.). analysis of midgut and sg lipase and invertase enzyme from homogenates of c. suppressalis by vertical slab electrophoresis on 8 % polyacrylamide gels indicated one band in all samples except for midgut's lipase (fig. 7). 183 table 1 relative activity of c. suppressalis lipase toward different compounds compounds concentration (mmol/l) relative activity (midgut) relative activity (salivary gland) control 100 100 nacl 5 102.77* 189.47* 10 77.77* 157.9* 20 33.33* 89.47* 40 6.38* 52.63* cacl2 5 50* 57.89* 10 77.77* 121.05* 20 88.88* 173.68* 40 119.44* 210.52* kcl 5 97.3* 100* 10 76.54* 87.36* 20 55.89* 68.66* 40 32.77* 45.25* mgcl2 5 247.22* 421.05* 10 208.33* 363.15* 20 91.66* 236.84* 40 44.44* 131.57* edta 0.5 38.88* 48.84* 1 72.22* 73.68* 2 105.55* 157.89* 4 231.55* 207.89* sds 2 113.88* 194.73* 4 80.55* 121.05* 6 44.44* 52.63* 8 26.66* 45.26* urea 1000 116.66* 205.23* 2000 102.77* 142.1* 4000 69.44* 115.78* 5000 27.77* 89.47* 6000 0.036* 31.57* plant extract 10 % 78* 86* 15 % 59* 63* 25% 34* 39* *p < 0.05 vs control 184 table 2 relative activity of c. suppressalis invertase in presence different compounds compounds concentration (mmol/l) relative activity (midgut) relative activity (salivary gland) control 100 100 nacl 5 102.77* 189.47* 10 77.77* 157.9* 20 33.33* 89.47* 40 6.38* 52.63* cacl2 5 50* 57.89* 10 77.77* 121.05* 20 88.88* 173.68* 40 119.44* 210.52* kcl 5 97.3* 100* 10 76.54* 87.36* 20 55.89* 68.66* 40 32.77* 45.25* mgcl2 5 247.22* 421.05* 10 208.33* 363.15* 20 91.66* 236.84* 40 44.44* 131.57* edta 0.5 38.88* 48.84* 1 72.22* 73.68* 2 105.55* 157.89* 4 231.55* 207.89* sds 2 113.88* 194.73* 4 80.55* 121.05* 6 44.44* 52.63* 8 26.66* 45.26* urea 1000 116.66* 205.23* 2000 102.77* 142.1* 4000 69.44* 115.78* 5000 27.77* 89.47* 6000 0.036* 31.57* plant extract 10 % 78* 86* 15 % 59* 63* 25% 34* 39* *p < 0.05 vs control 185 lipase-midgut 1/s -0.05 0.00 0.05 0.10 0.15 1/ v 0 1 2 3 4 5 6 lipase-salivary glands 1/s -0.05 0.00 0.05 0.10 0.15 1/ v 0 2 4 6 8 10 invertase-salivary glands 1/s -4 -2 0 2 4 6 8 10 12 1/ v 1 2 3 4 5 6 7 8 invertase-midgut 1/s -4 -2 0 2 4 6 8 10 12 1/ v 0 1 2 3 4 5 6 fig. 5 lineweaver-burk plot (vmax and km) of lipase and invertase extracted from 4th instar larvae of rice striped stem borer. discussion the present study shows that the larvae of c. suppressalis present lipase and invertase activities both in the midgut and in the sg. reports concerning lipase characterization have been obtained from several species of insects. metcalf (1945) found amylase, protease, and lipase to be absent from the sg of the mosquito anopheles quadrimaculatus while he observed that invertase is present in both midgut and crop homogenates. fisk and shambaugh (1954) found no activity of lipase in the sg of a. quadrimaculatus. a total body lipase was partially purified from abdomen homogenate of gryllus campestris l. (orthoptera, gryllidae) (orscelik et al., 2007). current study showed that both lipase and invertase are present in c. suppressalis and that the optimal ph for both enzymes are in alkaline condition (around ph 10). optimal temperatures for both enzymes are 37-40 °c in midgut and sg, respectively. ishaaya and swirski (1970) demonstrated that the optimum ph and temperature for invertase activity in the hemipteron chrysomphalus aonidum are 5.5 and 30 °c, respectively. degleri and anuipor (2002) showed that the optimal ph of lipases activity in the teleost fish cyprinion macrostomus is 7.5. studies on lipase properties of yeasts revealed that optimum ph and temperature for lipases activity are 7.5-8.2 and 3040 °c, respectively (vakhlu and kour, 2006). the utilization of dietary lipids was studied in adult females of the blood-sucking bug rhodnius prolixus with the use of radiolabeled triacylglycerol (grillo et al., 2007). these researches indicates that lipase activity is affected by ph and shows an optimal activity at a ph of 7.0-7.5. the optimal ph generally reflects the ph of the environment in which the enzyme normally functions. one way in which ph affects reactions rates is by altering the charge state of the substrate or of the active site of the enzyme. extreme phs can also disrupt the hydrogen bonds 186 lipase-midgut 1/s .08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 1/ v 0 2 4 6 8 10 12 14 16 invertase-midgut 1/s .0 -0.5 0.0 0.5 1.0 1.5 2.0 1/ v 0.0 0.2 0.4 0.6 0.8 1.0 invertase-salivary glands 1/s -4 -2 0 2 4 6 8 10 12 1/ v 0 2 4 6 8 10 12 14 16 fig. 6 lineweaver-burk plot (vmax and km) of lipase and invertase extracted from 4th instar larvae of rice striped stem borer due to using a. annua extract. that hold the enzyme in its three-dimensional structure, denaturing the protein (zeng et al., 2000). biological reactions occur faster with increasing temperature up to the point of enzyme denaturation, above which temperature enzyme activity and the rate of the reaction decreases sharply (applebaum 1985; agblor et al., 1994; zeng et al., 2002). current study results showed that ca2+ ions have activatory effects on the lipase and invertase activities of rice striped stem borer. podolor and applebaum (1971) reported that ca2+ ions have activatory effects on the lipase and invertase activities of the coleopteron callosobruchus chinensis. these results showed that these enzymes are metalloproteins which require calcium for maximum activity. researches have shown a significant correlation between the activity level of digestive enzymes in the hemolymph and the midgut. saleem and shakoori (1987) showed that sublethal concentrations of pyrethroids decrease amylase activity in larval gut of the beetle tribolium castaneum. lee et al. (1994) showed that some insect growth regulators decreased the activity level of alpha-amylase and esterase in treated larvae of c. suppressalis. ascher and ishaaya (2004) showed that the activity level of this enzyme increased 30 % in the noctuid moth spodoptera littoralis treated with phentine acetate compared with control. shekari et al. (2008) suggest that its activity level decreases 24 h after treatment and sharply increases at 48 h. digestive enzyme inhibitors occur naturally in many food plants and are particularly abundant in cereals and legumes (franco et al., 2002). insects gain access to food sources when they evolve enzymes that are not affected by inhibitors present in the food source, and plants become resistant when they evolve inhibitors effective against these 187 table 3 kinetic parameters of lipase and invertase enzymes extracted from midgut and sg of 4th instar larvae of c. suppressalis midgut* sg* enzymes vmax (µmol/min/mg protein) km (mm) vmax (µmol/min/mg protein) km (mm) lipase 0.5 ± 0.06 15 ± 3.74 0.35 ± 0.09 19 ± 6.45 invertase 0.9 ± 0.036 0.31 ± 0.047 0.5 ± 0.078 0.39 ± 0.77 *means ± se, n = 3 table 4 kinetic parameters of lipase and invertase enzymes extracted from midgut and sg of 4th instar larvae of c. suppressalis after exposure to a.annua extract midgut* sg* enzymes vmax (µmol/min/mg protein) km (mm) vmax (µmol/min/mg protein) km (mm) lipase 0.35 ± 0.087 37.5 ± 15.24 2.12 ± 0.68 21 ± 5.8 invertase 0.49 ± 0.087 0.30 ± 0.063 0.81 ± 0.021 0.92 ± 0.77 *means ± se, n = 3 insect enzymes. when the action of digestive enzymes is inhibited, insect’s nutrition is impaired, growth and development are retarded and eventually death occurred due to starvation. genes encoding for digestive enzyme inhibitors have been used to make transgenic crops by gene transfer technology. in transgenic pea expressing the αamylase inhibitor, the expression of digestive enzyme inhibitors makes plants harmful to target insects and pests, interfering with their digestive and fig. 7 native-page gel electrophoresis of midgut and sg from c. suppressalis. absorption processes, whereas neither antinutritional nor toxic effects were observed in rats (pusztai et al., 1999). the primary reason for producing insect-resistant transgenic crops is to reduce the use of chemical pesticides, which by one side lowers production costs and in the meantime reduces the insecticide loads in the environment. making insect-resistant plants requires the characterization of α-amylase and other digestive enzymes of the target insect and the identification of suitable inhibitors from plants or other sources. in our opinion, the purification and characterization of more insect digestive enzymes will greatly facilitate the understanding of the mechanisms responsible for this selectivity and will help to design new and more specific strategies for insect control. acknowledgment this research was supported by a university of tehran grant. we are really appreciated mr belbasi and ramadzan-khani for their assistances. we have special thanks to three anonymous reviewers for their helpful comments that resulted in a significant improvement of the article. references agblor a, henerson hm, madrid fj. characterization of alpha-amylase and polygalacturonase from lygus spp. (heteroptera: miridae). food res. inter. 27: 321-326, 1994. applebaum s w. biochemistry of digestion. in: ga kerkut, ll gilbert (eds.). comprehensive insect physiology, biochemistry and pharmacology. vol.4: regulation, digestion, excretion. pergamon press., pp. 279-307, 1985. 188 ascher krs, ishaaya i, antifeeding and protease and amylase inhibiting activity of phentin acetate in spodoptera littoralis larvae, pestic. biochem. physiol. 75: 326-336, 2004. ayre gl. feeding behaviour and digestion in camponotus herculeanus (l.) (hymenoptera: formicidae). entomol. exp. appl. 6: 165-170, 1963. ayre g l. the relationships between food and digestive enzymes in five species of ants (hymenoptera: formicidae). canad. entomol. 99: 408–411, 1967. baker je. interaction of partially-purified amylase with amylase inhibitors from wheat. comp. biochim. physiol. 93b: 239-246, 1989. bandani ar, amiri b, butt tm, gordon-weeks r. effects of efrapeptin and destruxin, metabolites of entomogenous fungi, on the hydrolytic activity of a vacuolar type atpase identified on the brush border membrane vesicles of galleria mellonella midgut and on plant membrane bound hydrolytic enzymes. biochim. biophys. acta 1510: 367-377, 2001. bradford ma. rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254, 1976. da silva mcm, mello lv, coutinho mv, rigden dj, neshich g, chrispeels m, et al. mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases. pesq. agropec. bras. 39: 201208, 2004. degerli n, akpinar ma. partial purification of intestinal triglycride lipase from cyprinion macrostomus and effect of ph on enzyme activity. turk. j. biol. 26: 133-143, 2002. dezfoulian a, moustofipoor p. rice striped stem borer. agri. org. iran. pub., pp 149, 1972. fisk fw, shambaugh gf. invertase activity in adult aedes aegypti mosquitoes. ohio. j. sci. 54: 237-240, 1954. franco ol, riggen dj, melo fr, bloch c, silva c, grossi de sa mf. activity of wheat α-amylase inhibitors towards bruchid α-amylases and structural explanation of observed specificities. eur. j. biochem. 267: 2166-2173, 2002. ghoshal d, sen s k, goyal a. introduction and expression of cowpea trypsin inhibitor (cpti) gene in transgenic tobacco. j. plant. biochem. biotech. 10: 19-25, 2001. grillo la, majerowicz d, gondim kc. lipid metabolism in rhodnius prolixus (hemiptera: reduviidae): role of a midgut triacylglycerollipase. insect biochem. mol. biol. 37: 579-588, 2007. henrissat b, bairoch a. new families in the classification of glycosyl hydrolases based on amino acid sequence similarities. biochem. j. 293: 781-788, 1993. hosseinkhani s, nemat-gorgani m. partial unfolding of carbonic anhydrase provides a method for its immobilization on hydrophobic adsorbents and protects it against irreversible thermoinactivation. enz. micro. tech. 33: 179184, 2003. ishaaya i, swirski e. invertase and amylase activity in the armoured scales chrysomphalus aonidum and aonidiella aurantii. j. insect. physiol. 16: 1599-1606, 1970. ishaaya i, moore i, joseph d. protease and amylase activity in larvae of the egyptian cotton worm, spodoptera littoralis. j. insect. physiol. 17: 945-953, 1971. lee hr, kim jw, lee i, studies on the toxicity of insect growth regulators against the fall webworm (hyphantaria cunea drury) and the rice stem borer (chilo suppressalis walker) ii. comparison in enzyme activities. korean, j. appl. entomol. 33: 88-95, 1994. maqbool sb, riazuddin s, loc nt, gatehouse amr, gatehouse ja, christou p. expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. mol. breeding 7: 85-93, 2001. martinez del rio c. dietary, phylogenetic, and ecological correlates of intestinal sucrase and maltase activity in birds. physiol. zool. 63: 9871011, 1990. mendiola-olaya e, valencia-jimenez a, valdesrodriguez s, delano-frier j, blanco-labra a. digestive amylase from the larger grain borer, prostephanus truncates horn. comp. biochem. physiol. 126b: 425-433, 2000. muralidharan k, pasalu ic. assessments of crop losses in rice ecosystems due to stem borer damage (lepidoptera: pyralidae). crop prot. 25: 409-417, 2006. naumff dg. β-fructosidase superfamily: homology with some α-l-arabinases and β-d-xylosidases. proteins: struct. funct. genet. 42: 66-76, 2001. nelson n . a photometric adapatation of somogyi method for determination of glucose. j. biol. chem. 153: 375-380, 1994. orscelk o, akpinar ma, gorgun s. partial purification of total body lipase from gryllus campestris l. (orthoptera: gryllidae). fen bilimleri dergisi. 27: 1-10, 2007. parish sh, marchalonis jj. polyacrylamide disc gel electrophoresis. anal. biochem. 34: 436. 1970. pusztai a, bardocz gg, alonso r, chrispeels mj, schroeder lm, tabe tj, et al. expression of the insecticidal bean alpha-amylase inhibitor transgene has minimal detrimental effect on the nutritional value of peas fed to rats at 30% of the diet. j. nutrition. 129: 1597-1603, 1999. ricks bl, vinson sb. digestive enzymes of imported fireant, solenopsis richteri (hymenoptera: formicidae). entomol. exp. appl. 15: 329-334, 1972. saleem ma, shakoori ar. point effects of dimilin and ambush on enzyme activies of tribolium castaneum larvae, pestic. biochem. physiol. 29: 127-137, 1987. shekari m, jalali sendi j, etebari k, shadparvar a. effects of artemisia annua l. (asteracea) on nutritional physiology and enzyme activities of elm leaf beetle, xanthogaleruca luteola mull. (coleoptera: chrysomellidae). pestic. biochem. physiol. 91: 66-74, 2008. sturm a, tang gq. the sucrose-cleaving enzymes of plants are crucial for development, growth 189 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t3f-49ngtv9-23m&_user=10&_coverdate=08%2f31%2f1970&_alid=774494413&_rdoc=9&_fmt=high&_orig=search&_cdi=4945&_sort=d&_docanchor=&view=c&_ct=11&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=012090ebbdc637c054faff0c696e76e6 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t3f-49ngtv9-23m&_user=10&_coverdate=08%2f31%2f1970&_alid=774494413&_rdoc=9&_fmt=high&_orig=search&_cdi=4945&_sort=d&_docanchor=&view=c&_ct=11&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=012090ebbdc637c054faff0c696e76e6 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t3f-49ngtv9-23m&_user=10&_coverdate=08%2f31%2f1970&_alid=774494413&_rdoc=9&_fmt=high&_orig=search&_cdi=4945&_sort=d&_docanchor=&view=c&_ct=11&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=012090ebbdc637c054faff0c696e76e6 and carbonpartitioning. trends plant sci. 4: 401-407, 1999. tsujita t, ninomiya, h, okuda, h. p-nitrophenyl butyrate hydrolyzing activity of hormonesensitive lipase from bovine adipose tissue. j lipid res. 30: 997-1004, 1989. vakhlu j, kour a. yeast lipases: enzyme purification. biochem. prop. gene. cloning. 9: 69-85, 2006. zeng f, cohen a c. partial characterization of αamylase in the salivary glands of lygus hesperus and l. lineolaris. comp. biochem. physiol. 126b: 9-16, 2000. zibaee a, jalali sendi j, alinia f, etebari k. a study on biochemical differences among five different groups of rice striped stem borer chilo suppressalis walker (lepidoptera: pyralidae). inv. surv. j. 5: 20-29, 2008. 190 http://www.citeulike.org/user/biblio24/author/tsujita http://www.citeulike.org/user/biblio24/author/ninomiya http://www.citeulike.org/user/biblio24/author/okuda introduction isj 6: 114-124, 2009 issn 1824-307x research report hemocytes in myriapoda (arthropoda): a review wer xylander senckenberg museum für naturkunde görlitz, görlitz, germany accepted august 5, 2009 abstract hemocyte types described in the different subtaxa of myriapoda are presented and characterised. they comprise (1) prohemocytes, (2) plasmatocytes and (3) two different forms of granular hemocytes; these three have been found in all taxa investigated. spherulocytes and coagulocytes were only found in some taxa. adipohemocytes occur quite infrequent, oenocytoids have not been detected but may be restricted to specific stages of the moulting cycle. cystocytes described earlier seem to be a preparation artefact. granular hemocytes are the most frequent hemocytes followed by the plasmatocytes. in general, diplopoda have fewer hemocytes than chilopoda. the differences in the hematogram and the functions of hemocytes are discussed. key words: arthropoda; myriapoda; chilopoda; diplopoda; hemocytes; immune reactions; plasmatocytes; granular hemocytes; phenoloxidase introduction in arthropoda, hemocytes have numerous functions: they are responsible for hemolymph clotting after cuticle and epidermis rupture, wound healing, self recognition, general and specific immune response and opsonisation (e.g., gupta, 1986; millar and ratcliffe, 1989; xylander, 1992, 1994). they phagocytise smaller pathogens like bacteria or fungal spores and encapsulate larger ones like metazoan parasites or xenografts. hemocytes, furthermore, produce and store substances which may be discharged after infections such as antibacterial substances and elements of the phenoloxidase system, as well as antibacterial substances, lectins and hemolysins. all these functions which were discovered in other groups of arthropoda have also been found to occur in the myriapoda (xylander and nevermann, 1990, 1993; xylander, 1992, in press; xylander and bogusch, 1992, 1997; nevermann, 1996). in some arthropods hemocytes are, furthermore, responsible for production and storage of the respiratory pigments. the number of hemocyte types investigated differs according to the subtaxon of arthropoda. additionally, the techniques and procedures of preparation, e.g., obtaining of hemolymph, in vitropreparation, fixation and staining techniques have ___________________________________________________________________________ corresponding author: willi xylander senckenberg museum für naturkunde görlitz postfach 300 154, 02806 görlitz, germany e-mail: willi.xylander@senckenberg.de impact on the reactions of hemocytes; as a consequence, the characteristics on which the nomenclature of hemocytes was based became highly diverse. as a consequence, the nomenclature of hemocytes in arthropods, especially in insects, became more and more confusing over the 100 years since the investigations started using more than 50 termini, often for the same cell types. with the aim to clarify at least in part this confusing situation, jones (1962), and ratcliffe and price (1974), presented a new nomenclature applicable more or less to hemocytes of all arthropod taxa. also in myriapoda the situation was quite complicated. early light microscopic investigations by gregoire (1955), ravindranath (1973, 1977, 1981) and rajulu (1971) resulted in the description of 7 different hemocyte types (prehemocytes, granular hemocytes, plasmatocytes, cystocytes, spherulocytes, adipohemocytes and oenocytoids). transmission electron microscopy (tem) investigations have described hemocyte types of chilopods and diplopods directly taken from the haemolymph, after in vitro preparation and partly during immune defense reactions (nevermann et al., 1991, 1996; nevermann, 1996; nevermann and xylander, 1996, 2006; hilken et al., 2003). by using light-microscopy, xylander (1992), xylander and nevermann (1993), and xylander and bogusch (1997), gave further characteristics applicable to differentiate the haematocyte types of myriapods. the intention of this paper is to review the recent results on myriapod hemocytes and their nomenclature. 114 mailto:willi.xylander@senckenberg.de fig. 1 (a-e) hemocytes of chicobolus spp., pure haemolymph, unfixed, spread on glass slides for different periods, observed by phase contrast microscopy and (f-j) r. virgator hemocytes, spread on glass slides for 10 min, fixed and stained. (a) various hemocytes (p= plasmatocytes, gl = granular hemocytes of type i, gii = granular hemocytes of type ii, the arrowhead points to cell proliferations). (b) plasmatocyte and prehemocyte (pr) (10 min). (c) two plasmatocytes with few grana spreading (“star-type”). (d) plasmatocytes representing various spreading-types (above: “star-type” with pseudopodia, below: “fried-egg-type” without pseudopodia, 20 min). (e) hemocyte with large grana in close vicinity to two different granular hemocytes (gi and gii, the arrowhead indicates cell proliferations of gii ,10 min). (f) may-grünwald and giemsa staining observed by phase contrast. (g) may-grünwald and giemsa staining observed in bright field. the prehemocytes are burgundy-coloured , the plasma of plasmatocytes is greyish and that of granular hemocytes (gi and gii) violet. (h) sudan-black-staining. several granular hemocytes are stained dark (bright field). (i) giemsa-staining. the more translucent plasma clearly differentiate plasmatocytes from granular hemocytes. (j) may-grünwald and giemsa staining. the plasma of granular hemocytes of type ii (gii) is bluish, their nuclei are blue. granular hemocytes of type i (gi) are faint purple , plasmocytes (p) are deep purple. (dark field). bars: 50 µm (a, c-e); 25 µm (b, f-j). 115 material and methods animals animals of different species were investigated. description of collecting sites, rearing conditions and other species specific informations have already been given in detail (xylander and nevermann, 1990; xylander, 1992; nevermann, 1996; nevermann and xylander, 1996). in vitro and staining techniques as well as hemocyte counts were described by xylander and nevermann (2006). electron microscopy preparations hemocytes were obtained by opening the cuticle of living animals. they spread and thus became attached to different substrates (e.g., cellophane) kept in cold ringer-solutions prior to fixation. such samples were fixed in the cold for at least 2 h in a modified karnoffsky´s fixative or in 2.5 % glutaraldehyde and postfixed in 2 % (w/v) oso4 and dehydrated in acetone [see xylander and nevermann 2006 for further details of the procedures]. after oso4-fixation samples were subdivided and (a) embedded for transmission electron microscopy samples in araldite or (b) critically point dried and sputtered with gold for scanning electron microscopy. tem-investigations were mainly done on a zeiss em 9a, sem investigations on a jeol or cambridge stereoscan s4 se-microscope. results hemocyte types six hemocyte types were found by light and electron microscopy, cytochemical and in vitro techniques in the two diplopod and three chilopod taxa. three of these occur in all taxa investigated: prehemocytes, plasmatocytes and granular hemocytes. in a limited number of chilopoda investigated, spherulocytes, coagulocytes and discoid cells were described (xylander and nevermann, 2006). prehemocytes three to 10 % of the hemocytes in the diplopods and chilopods investigated are prehemocytes. they are the smallest cells of all hemocytes and spherical in shape (figs 1b, f, g; 2c, g). their nucleus is large compared to the volume of cell plasma and located centrally (fig. 2g). in vitro, these cells spread later than other cell types keeping their shape for several minutes. only a few small grana may be found in the plasma of these prehemocytes and their phenoloxidase activity is weak (xylander and bogusch 1997). plasmatocytes plasmatocytes are significantly larger than the prehemocytes (fig. 1b) with a mean diameter of 30 to 70 µm. this cell type is characterized by intense spreading (figs 1, 2). furthermore, they contain no or just a few grana (figs 3a, c, d). their plasma is greyish in phase contrast (figs 1a-d). after activation of hemocyte cultures for phenoloxidase staining and incubation with dopa (xylander and bogusch 1997) there is no or just little reaction in the plasmatocytes. the nucleus of spread plasmatocytes is located eccentrically (figs 1b, d) and it occurs larger than in any other hemocyte type. directly after obtaining the haemolymph plasmatocytes are spherical to spindle-shaped but flatten and attach to glass slides within 8 to 10 min (in an undiluted haemolymph sample) forming pseudopodia (figs 2a, b, d; 3b, e). in the diplopoda, two types of spreading were observed in this cell type resulting in different hemocyte morphology (xylander and nevermann 2006): a) the “fried-egg-type”. in this type of spreading cells flatten more or less homogeneously not forming many pseudopodia; plasmatocytes showing such spreading behaviour contain no or just a few grana (fig. 1d) b) the “star-type”. here many thin pseudopodia grow centrifugally during spreading; in plasmatocytes of this type normally several small grana occur which are bluish in phase contrast (fig. 1d). tem observations show that in the plasmatocytes of chilopoda electron translucent vacuoles with moderately electron dense filamentous material occur (nevermann et al., 1991; hilken et al., 2003). in plasmatocytes of the spirobolid diplopod rhapidostreptus virgator comparable vacuoles were found containing (figs 3a, c, d) which, however contain flocculent rather than filamentous material; this material is discharged after some time during vitro-incubation. the material seems to attract other cell types thereby becoming involved in more complex aggregation (the immune reaction); such attraction of other cellular components of the immune system (against non-self material) is called opsonisation effect. granular hemocytes granular hemocytes are the most numerous hemocytes in myriapoda (xylander and nevermann 2006). they contain numerous electron dense grana of variable size and shape and some vacuoles (figs 1f-j; 2c, e; 4; 5). granular hemocytes are medium sized after spreading (larger than prohemocytes but smaller than plasmatocytes) (figs 1f-j). all cells of this type are phenoloxidase active (xylander 1996, xylander and bogusch 1997, xylander and nevermann 1993, 2006). briefly, after obtaining the haemolymph, granular hemocytes are more or less spherical (figs 1i, j). then they start to flatten in vitro and attach to the substrates within 15 min (figs 1f, g). they can, however, be distinguished from plasmatocytes for more than 1 h in light microscopy by their significantly lower spreading capabilities. during spreading, granular hemocytes often develop a unidirectional process (xylander and nevermann, 2006). intermediate forms of granular hemocytes and plasmatocytes have been found in a few cases (fig. 3d) indicating that granular hemocytes (at least of type i, see below) may constitute a transformation product of plasmatocytes. in the two species of diplopods investigated (chicobolus spp. and r. virgator) two types of granular hemocytes could be differentiated. granular 116 fig. 2 hemocytes of r. virgator. (a) hemolymph preparation with various hemocytes (pure haemolymph, 20 min after puncture, phase contrast. p= plasmatocyte, gi and gii= granular hemocyte type i and type ii.). (b) various hemocytes in a hemolymph smears (pure hemolymph, 30 min, phase contrast). (c) overview of various hemocytes observed by tem. (d) hemocyte aggregation in vitro observed by scanning electron microscopy (sem). the hemocytes with short extensions on the cell surface are plasmatocytes. (e) granular hemocytes of type i and type ii observed by tem. the grana of type ii are larger, more spherical and form fewer pseudopodia than type i. (f) tem observation of a granular hemocyte forming pseudopodia with contact to the substrate (g= granulum). (g) tem photograph of a prehemocyte. few small grana (g), high numbers of free ribosomes, rer and the nucleus (nu) are visible. bars: 50 µm (a, b); 5 μm (c, e); 10 µm (d); 0.2 µm (f); 1 µm (g). 117 hemocytes of type i show significantly higher spreading capabilities (fig. 1a). they contain fewer and smaller grana (figs 2c, i; 4a) which occur blue or green in phase contrast microscopy. in transmission electron microscopy their grana show to be irregular in shape (figs 2c; 4 a-c). after spreading the grana of type i granular hemocytes are predominantly located at the periphery of the nucleus; so mostly the nucleus is clearly visible. phenoloxidase activity is moderate in granular hemocytes of type i (and significantly lower than in type ii) (xylander and bogusch, 1997; fig. 3 plasmatocytes of r. virgator fixed after in vitro incubation on gelatine with i-ringer. (a) tem observation of a prehemocyte (with grana, left) and plasmatocyte (right). (b) two plasmatocytes observed by sem. (c) plasmatocyte. peripheral in the cytoplasm only few grana are visible. in the centre of the cell vesicles filled with flocculent material have gathered. (er= rough endoplasmic reticulum, m= mitochondrion, nu= nucleus, *: glycogen like inclusions). (d) presumed transitory stage between plasmatocyte and granular hemocyte type i. the number of grana increase, but they are less frequent and electron dense as in granular hemocytes. (e) plasmatocyte observed by sem. bars: 1 µm (a, c, d); 5 μm (b, e). 118 fig. 4 granular type i hemocytes of r. virgator under different types of fixation. hemocytes in a and c were fixed after in vitro incubation, 60 min in wenning-ringer on cellophane. hemocytes in b and d were fixed after in vitro incubation 60 min in i-ringer on gelatine. (a) for comparison: granular hemocytes of type i and type ii (nu= nucleus). (b) granular hemocyte of type i. at the periphery of the cell there are rer-cisterns and in the centre there are vacuoles. one of the vacuoles is filled with material resembling glycogen-rosettes (*). (c) in gr i there are fewer vacuoles after incubation in wenning-ringer on cellophane. (d) in unstained tem-sections (no double staining with uranyl acetate and lead citrate) the grana occur to be subdivided into a more electron dense and a more translucent part. bars: 1 µm. 119 xylander and nevermann 2006). in unstained temsections the grana of this type occur to be “subdivided” into a more electron dense and a more translucent part; the more electron dense part is smaller and enclosed eccentrically by the more translucent one (fig. 4d) (see also xylander and nevermann, 2006). granular hemocytes of type ii occur even smaller after spreading than plasmatocytes and granular hemocytes of type i and they spread less intensively (figs 1a, f, g). their volume, however, is about the same of the other two types. in many cases granular hemocytes of type ii also form a unidirectional process. in phase contrast they appear yellowish mostly due to the higher number of grana (fig. 1a). these grana are larger and spherical (figs 2c, e; 4a; 5a, c-e). when located peripherally in the granular hemocytes these grana may bulge their surface (figs 5a, b, f). granular hemocytes of type ii react stronger than the other hemocytes after prophenoloxidase activation and substrate incubation and stain dark brown (xylander and bogusch, 1997, xylander and nevermann, 2006). spherulocytes a fourth hemocyte type has been described for the chilopods lithobius forficatus and scutigera coleoptrata by nevermann et al. (1991) and hilken et al. (2003): the spherulocyte. it is characterised by regularly shaped spherical grana, significantly lower spreading capability and – as a major characteristic for differentiation from other hemocyte types the complete lack of phenoloxidase inactivity. these cells tend to form a single cytoplasmic protrusion which never bears grana. although spherulocytes may form such protrusions, contain grana, spread just moderately and therefore resemble the granular hemocytes intermediate forms between spherulocytes and granular hemocytes or plasmatocytes have never been detected. up to now, spherulocytes were only found in the two species of chilopoda. in diplopoda, as well as in scolopendra cingulata, this cell type does not occur (xylander 1992; nevermann, 1996; xylander and nevermann, 2006). in spite of the similarities, xylander and nevermann (2006) considered the spherulocytes to be a distinct hemocyte type possibly restricted to a subtaxon of chilopoda. coagulocytes in haemolymph preparations from lithobius forficatus, nevermann et al. (1991) described zones with typical plasma coagulations and postulated the occurrence of a fifth extremely fragile hemocyte type, which immediately disintegrates in vitro, the coagulocyte. nevermann (1996) described disintegrating hemocytes when dropping haemolymph directly into a fixative. these hemocytes resembled plasmatocytes containing only few electron dense grana and vacuoles but were characterised by large vacuoles containing flocculent material (indicating the disintegration process). nevermann (1996), therefore, considered the coagulocytes found just to represent “stressed plasmatocytes”. more recently, xylander and nevermann (2006), however, considered the coagulocytes to be one of the “valid” hemocyte types of myriapoda. which, due to their immediate disintegration during the procedures of obtaining the haemolymph, is just very rarely found in hemocyte preparation. naked nuclei and isolated membranes (most possibly remnants of hemocytes after disintegration) were also found in hemocyte aggregations (“capsules”) surrounding xenografts in the diplopod rhapidospreptus virgator (xylander, unpublished). they could also be remnants of coagulocytes indicating that this hemocyte type is more widely distributed throughout the myriapoda than considered earlier and just hard to detect with the methods usually applied. discoid hemocyte nevermann (1996) described a sixth hemocyte type by tem studies characterised by peripheral circular bundles of microtubules for s. cingulata. these microtubules disintegrate when the hemocytes attach to the substrate and start to spread. then they become invisible. bundles of microtubules equivalently arranged were also described from granular hemocytes of scutigera coleoptrata (hilken et al., 2003). so peripheral bundles of microtubules arranged in circles may represent a general characteristic of native plasmatocytes and granular hemocytes in vivo. cystocytes ravindranath (1981) in an early review of hemocytes in myriapoda described cystocytes for diplopoda. subsequently nevermann et al. (1991) showed, however, that this description was due to a preparation artefact: such hemocytes were exclusively found in cell preparations after mechanical stress (e.g., after sucking hemocytes into the narrow slit between a microscopic slide and cover slip). therefore, cystocytes are considered not to be a “valid hemocyte type” for the myriapoda. adipohemocytes in very few cases cells containing large lipid grana were found circulating in the hemolymph (fig. 1e). such numerous lipid grana (stainable with sudan black) are typical for so called “adipohemocytes”. but such observations were not reproducible with other specimens of the same species. therefore, it seems probable that such cells originated from the subepidermal fat body and are set free when obtaining the haemolymph by puncture. therefore, they represent rather an artifact originating from preparation and than a genuine hemocyte type in myriapoda. oenocytoids during all investigations performed in our working group with various diplopod and chilopod species, oenocytoids were never observed. however, oenocytoids are considered to be involved in the moulting process and their occurrence circulating free in the haemolymph may be restricted to a short period prior to moulting (which is a rare event in mature specimens of the species of diplopoda and chilopoda investigated during our 120 fig. 5 granular type ii hemocytes of r. virgator. hemocytes in a, d and e after in vitro incubation in wenningsaline for 60 min on cellophane; b, c and f, after in vitro incubation in i-ringer on gelatine for 60 min. (a) granular hemocyte of type ii. tem image displaying that grana are mostly spherical and bulge the plasma membrane at different sites (arrowheads); nu = nucleus. (b) grana responsible for extension of the plasma membrane are visible also by sem. (c) a few grana are surrounded by rer (arrowheads). (d) cisternae of rer could frequently been found at the cell periphery (er). free ribosomes are also widely distributed in the cytoplasm. note that mitochondria (mi) with electron dense matrix and translucent cristae are visible. (e) golgi complexes (di) with electron dense grana were occasionally observed in vicinity to the nucleus (nu). (f) sem micrograph of gii. bars: 1 µm (a, c); 5 μm (b, f); 0.2 µm (d, e). 121 studies). in fact, during our investigations specimens in the moulting process (which are extraordinary sensitive to cuticle rupture and may die briefly after injury) were excluded. this may be the reason for the absence of oenocytoids in our samples. therefore, definitive conclusions regarding the existence of oenocytoids in chilopods and diplopods are not possible. differential hemocyte counts for two diplopod species, r. virgator and chicobolus spp., the percentage of different hemocyte types were determined (xylander and nevermann, 2006). the granular hemocytes of type i was most frequent comprising 38 and 39 % of all hemocytes, respectively. granular hemocytes of type ii represented 30 % of all hemocytes in r. virgator and 17 % in chicobolus. in r. virgator 27 % of the hemocytes were plasmatocytes and 39 % in chicobolus. in both species prehemocytes represented about 5 %. total hemocyte counts the total number of hemocytes per hemolymph volume varies extremely between chilopoda and diplopoda: in the species investigated centipedes had a tenfold higher numbers of hemocyte than millipedes. in lithobius forficatus xylander and nevermann (2006) found 45,000 hemocytes µl-1 hemolymph, in scolopendra cingulata 31,500 and in s. oraniensis about 50.000. in contrast, there were only 2,450 and 6,500 hemocytes µl-1 hemolymph, respectively, in the diplopods r. virgator and chicobolus spp. discussion hemocyte types – a comparison within and outside the myriapoda in the arthropods at least 9 different hemocytes types can be differentiated (table 1). within the three major taxa of myriapods (chilopoda, diplopoda and symphyla) investigated with regard to their hemocytes until now prehemocytes, plasmatocytes and granular hemocytes occur in all three. other types described so far seem to be restricted to specific subtaxa or have to be considered as preparation artefacts. with regard of their microscopic and submicroscopic morphology the hemocytes of myriapod correspond to that of other arthropods (see for review jones, 1962; ravindranath, 1974; bauchau, 1981; sherman, 1981; xylander, 1992). this similarity of characters even on the electron microscopic level allows to use the generalised nomenclature for arthropod hemocytes not only for pragmatical reasons but also due to comparative morphology and function. however, the hemocyte types of decapod crustaceans which are separated into those bearing many granules (“granular cells”) and those bearing few (“semi-granular cells”) (bauchau, 1981; xylander et al., 2003) “resist” the inclusion into this system up to now. so further investigations are necessary to clarify the homology of the different hemocyte types. xylander and nevermann (2006) considered at least 4 hemocyte types to belong to the ground pattern of arthropods: the prohemocytes, the plasmatocytes, the granular hemocytes and the cyanocytes, the latter responsible for production and storage of the respiratory pigments. after the evolution of terrestrial arthropods with trachea, respiratory pigments and the hemocytes producing them were reduced. subsequently, immune defense, wound closure and hematopoiesis represented the major tasks of the remaining hemocytes. total hemocyte counts the range of hemocytes number per volume is extremely variable reaching from 500 µl-1 in decapod crustaceans to 60,000 µl-1 in cockroaches (overview in xylander and nevermann, 2006). diplopoda are among those taxa with a low number table 1 a comparison of the presence of different hemocyte types (listed in the results section) within the main taxa of arthropoda. myriapoda species are singularly reported. modified from xylander and nevermann (2006). taxon ph pl gr sph coa disc adi cyst oen cyan rhapidostreptus virgator x x x ? ? chicobolus spp. x x x ? lithobius forficatus x x x x (x) ? scolopendra cingulata x x x x ? scutigera coleoptrata x x x x insecta x x x x x x x crustacea x x x x x xiphosura ? x x x scorpiones x x x x x x ? aranea x x x ? x x 122 table 2 total hemocyte counts (hemocytes µl-1) [h (hc/µl)] for various arthropods. modified from xylander and nevermann (2006). myriapoda h (hc/µl) references rhapidostreptus virgator 2,500 xylander and nevermann, 2006 chicobolus spp. 6,500 xylander and nevermann, 2006 scolopendra cingulata 31,000 nevermann, 1996 scolopendra oraniensis ~50,000 xylander and nevermann, 2006 lithobius forficatus 45,000 nevermann, 1996 crustacea astacus leptodactylus ~500 ullrich, 1993; xilander et al., 1997 procambarus clarkii ~580 ullrich, 1993; xilander et al., 1997 crangon crangon 800-1,200 smith and johnston, 1992 insecta blatella germanica 23,000 gupta, 1986 periplaneta americana 60,000 crossley, 1975 chironomus thummi 1,000-3,000 götz and vey, 1974 drosophila melanogaster (2d) 2,000 rizki and rizki, 1992 drosophila melanogaster (4d) 23,000 rizki and rizki, 1992 manduca sexta (l5) 4,500 horohov and dunn, 1982 galleria melonella (l5) 25,000 chain and anderson, 1982 arachnida limulus polyphemus 30,000 sherman, 1981 arenea spp. 11,000 sherman, 1981 of hemocytes, whereas chilopoda range among those with quite high numbers. when looking at hemocyte numbers more generally, however, it should be taken into account that in those few species in which the topic was investigated, hemocyte numbers often differed significantly during ontogenetical stages. the variability of the hemocytes counts cannot be explained on the basis of the available data. but, as a tendency, those species with hard and strongly calcified cuticles or passive protection strategies against predators (such as glandular defense secretions) have fewer hemocytes (e.g., decapod crustaceans and diplopods). in these species the risk of injuries and infections and, therefore, the demand for wound closure and immune defense is most probably lower. in predatory groups with a thin cuticle (reducing body weight and enabling effective scavenging with lower energy demand than in those groups with calcified cuticles) or taxa with higher risk of injury due to their life style (e.g., chilopods, cockroaches, dipteran larvae) hemocyte numbers are often high. in this context, fründ (1992) showed that 28 to 60 % of the specimens of lithobius forficatus in his study had melanised scares or lost legs. thus the high total hemocyte count probably constitutes an adaptation to their predatory life style (xylander and nevermann, 2006). acknowledgements the author would like to thank dr l nevermann, dr h-u jahn and o bogusch for their support during the investigations on the immune system of myriapoda conducted at the justus-liebig university (germany). acknowledgments are also extended to prof dr g hilken and prof dr g seifert who contributed their part to this publication. references bauchau ag. crustaceans. in: ratcliffe na, rowley af (eds), invertebrate blood cells. vol. 2. academic press, london, pp 385-420, 1981. chain bm, anderson rs. selective depletion of plasmatocytes in galleria mellonella following injection of bacteria. j. insect physiol. 28: 377384, 1982. crossley ca. the cytophysiology of insects. adv. insect physiol. 11: 117-222, 1975. fründ h-c. the occurrence and frequency of scars in centipedes. ber. nat.-med. verein innsbruck, suppl. 10: 269-275, 1992. 123 götz p, vey a. humoral encapsulation in diptera (insecta): defence reactions of chironomus larvae against fungi. parasitology 68: 193-205, 1974. gregoire c. blood coagulation in arthropods. vi. a study by phase contrast microscopy of blood reactions in vitro in onychophora and various groups of arthropods. arch. biol. (liege) 66: 489-508, 1955. gupta ap. hemocytes of scutigerella immaculata and the ancestry of insecta. ann. entomol. soc. am. 61: 1028-1029, 1968. gupta ap. arthropod immunocytes. identification, structure, functions, and anologies to the functions of vertebrate band t-lymphocytes. in: gupta ap (ed), hemocytic and humoral immunity in arthropods. wiley and son, new york, chichester, brisbane, toronto, singapore, pp 3-59, 1986. hilken g, brockmann c, nevermann l. hemocytes of the centipede scutigera coleoptrata (chilopoda, notostigmophora) with notes on their interactions with the tracheae. j. morphol. 257: 181-189, 2003. horohov dw, dunn pe. phagocytosis and nodule formation by hemocytes of manduca sexta larvae following injection of pseudomonas aeruginosa. j. invert. pathol. 41, 203-213, 1982. jones jc. current concepts concerning insect hemocytes. amer. zool. 2: 209-246, 1962. millar da, ratcliffe na. the evolution of blood cells: facts and enigmas. endeavour 13: 72-77, 1989. nevermann l. untersuchungen an haemozyten von scolopendra cingulata und lithobius forficatus unter dem aspekt zellulärer abwehrreaktionen. doctoral thesis, university of giessen. www.nevermanns.de/hemocytes/, 1996 nevermann l, kaiser he, xylander wer. microbial induced haemocytic immune reactions in chilopods. in vivo 10: 161-168, 1996. nevermann l, xylander wer. in vitro cellular immune reactions of hemocytes against bacteria and their differential degradation in myriapods. geoffroy, jj, mauries l, nguyen duy-jacquemin m (eds), acta myriapodologica. mem. mus. natn. hist. natur. 169: 421-430, 1996. nevermann l, xylander wer, seifert g. the hemocytes of the centipede lithobius forficatus (chilopoda, lithobiomorpha): light and electron microscopic studies using in-vitro techniques. zoomorphology 110: 317-327, 1991. rajulu gs. a study of haemocytes in a centipede scolopendra morsitans (chilopoda: myriapoda). cytologia 36: 515-521, 1971. ratcliffe na, price cd. a reappraisal of insect haemocyte classification by the examination of blood from fifteen insect orders. z. zellforsch. 147: 537-549, 1974. ravindranath mh. the hemocytes of a millipede, thyropygus poseidon. j. morphol. 141: 257268, 1973. ravindranath mh. the hemocytes of a scorpion palamnaeus swammerdami. j. morpohol. 144: 1-10, 1974. ravindranath mh. a comparative study of the morphology and behaviour of granular haemocytes of arthropods. cytologia 42:743751, 1977. ravindranath mh. onychophorans and myriapods. in: ratcliffe na, rowley af (eds), invertebrate blood cells, vol. 2, academic press, london, pp 327-354, 1981. rizki tm, rizki rm. lamellocyte differentiation in drosophila larvae parasitized by leptopilina. dev. comp. immunol. 16: 103-110, 1992. sherman rg. chelicerates. na, rowley af. invertebrate blood cells. invertebrate blood cells, vol. 2, academic press, london, pp 335384, 1981. smith vj, johnston pa. differential haemotoxic effect of pcb congeners in the common shrimp, crangon crangon. comp. biochem. physiol. 101c: 641-949, 1992. ullrich g. antibakterielle substanzen bei astacus leptodactylus (crustacea, decapoda). 60 pp. thesis, university of giessen, 1993. xylander wer. immune defense reactions of myriapoda a brief presentation of recent results. in: thaler k, meyer e, schedl w (eds), advances in myriapodology (proceedings of the 8th international congress of myriapodology). ber. nat.-med. verein innsbruck, suppl. 10: 101-110, 1992. xylander wer. immunabwehr bei gliederfüßern wie sich spinnentiere, krebse, insekten und tausendfüßer gegen krankheitserreger schützen. spiegel der forschung 11: 27-30, 1994. xylander wer. antibacterial substances and characteristics from the haemolymph of chilopoda and diplopoda (myriapoda, arthropoda). soil organisms [in press]. xylander wer, bogusch o. investigations on the phenoloxidase of rhapidostreptus virgator (arthropoda, diplopoda). zool. jb. physiol. 96: 309-321, 1992. xylander wer, bogusch o. granular hemocytes as the main location of prophenoloxidase in the millipede rhapidostreptus virgator (diplopoda, spirostreptida, spirostreptidae). ent. scand. suppl. 51: 183-189, 1997. xylander wer, nevermann l. antibacterial activity in the hemolymph of myriapoda (arthropoda). j. inv. pathol. 56: 206-214, 1990. xylander wer, nevermann l. phenoloxidase-active hemocytes in lithobius forficatus, scolopendra cingulata (chilopoda) and chicobolus spec. (diplopoda). verh. dtsch. zool. ges. 86.1: 197, 1993. xylander wer, nevermann l. haemocytes in diplopoda and chilopoda (arthropoda, myriapoda) -types, structures, and numbers. scand. j. entomol. 53: 195-210, 2006. xylander wer, ullrich g, kaiser he. antibacterial immune response in astacus leptodactylus (decapoda, crustacea). in vivo 11: 195-200, 1997. xylander wer, ullrich g, nevermann l, eichelberg d. discrimination of haemocytes of astacus leptodactylus (decapoda: crustacea) by differential spreading and cytochemical staining. abh. ber. naturmuseum görlitz 75: 83-90, 2003. 124 http://www.nevermanns.de/hemocytes/ spherulocytes coagulocytes discoid hemocyte cystocytes adipohemocytes oenocytoids h (hc/µl) references crustacea insecta arachnida isj103.pdf 105 isj 2: 105-113, 2005 issn 1824-307x minireview toll-like receptors in invertebrate innate immunity l zheng1, l zhang1, 2, h lin1, 3, mt mcintosh1, ar malacrida4 1 yale university school of medicine, epidemiology and public health, 60 college street, new haven, ct 06520, usa 2 department of parasitology, medical college, jinan university, shipai, guangzhou 510632, guangdong, p. r. china 3 fujian provincial centers for disease control and prevention, 76 jintai avenue, fuzhou, fujian, 350001, p.r.china 4 dipartimento di biologia animale, università di pavia, piazza botta 9, 27100 pavia, italy accepted august 4, 2005 abstract among invertebrates, innate immunity is the only defense mechanism against harmful non-self agents. in response to recognition of microbial pattern molecules, drosophila melanogaster activates either the toll or imd pathway, leading to the translocation of nf-kb (or rel) transcription factors from the cytoplasm to the nucleus and the subsequent production of antimicrobial peptides, which provide systemic innate immunity. toll-like receptors (tlrs) are characterized by an extracellular leucine rich repeat (lrr) domain and an intracellular toll/interleukin-1 receptor (tir) domain. tlrs are found from cnidarians to mammals. here we argue that tlr mediated innate immunity developed during an early stage of evolution when organisms acquired a body cavity. this is supported by the distributions of tlr and rel genes in the animal kingdom. further, tlr mediated immunity appears to have developed independently in invertebrates and vertebrates. recent studies have shown that microbial molecules, with the potential to signal through tlr, can be beneficial to host survival. studies on this signaling pathway could open doors to a better understanding of the origins of innate immunity in invertebrates and potential transmission blocking strategies aimed at ameliorating vector-borne diseases. key words: toll; innate immunity; antimicrobial peptides; invertebrates; coelom introduction in invertebrates, innate immunity is the sole defense mechanism against infectious non-self agents. there are three manifestations of innate immunity: phagocytosis, activation of humoral responses leading to coagulation, melanization or opsonization, and the systemic production of antimicrobial peptides, or amps (hoffmann et al., 1999). production of amps may represent a more recent evolutionary adaptation of innate immunity, allowing for an effective and efficient means of protection against systemic infection. corresponding author: liangbiao zheng yale university school of medicine, epidemiology and public health, 60 college street, new haven, ct 06520, usa e-mail: liangbiao.zheng@yale.edu phagocytosis, being the most primitive feature, developed probably as a means of acquiring nutrients and was probably co-opted later as a defense mechanism. while origins of the other two forms of innate responses are not clear, it is likely they too developed quite early when life forms co-existed in primordial oceans and had to contend with a milieu of diverse microbial agents (friends and foes). that said, for primitive aquatic organisms lacking a real body cavity it would not have been efficient to produce and secrete amps onto the surfaces as these would be subjected to dilution by the environment and thus never reach the critical concentration required for effective clearance of microbes. it is therefore likely that systemic immunity in the form of production of amps mediated by toll-like receptors (tlrs) first developed in organisms with a body cavity. 106 systemic immunity in the model system drosophila melanogaster systemic production of amps is best characterized in the fruit fly, drosophila melanogaster (reviewed in hoffmann, 2003). generally speaking, immunity against fungi and gram(+) bacteria is mediated by the tolldif/dorsal pathway while anti gram(-) bacterial responses are mediated by the imd-relish pathway, leading to the activation and expression of a different set of antimicrobial peptide genes. imd was first identified genetically as a key factor for survival against bacterial challenge (lemaitre et al., 1995). how gram(-) bacteria are recognized remains to be determined; though it is known that the recognition eventually leads to the activation of a member of the peptidoglycan recognition protein (pgrp) family, pgrp-lc. pgrp-lc interacts directly with imd (choe et al., 2005), which then sends the signal through two separate channels. one leads to the activation of a signalsome consisting of ikkβ and ikkγ via tak1. the other channel leads to the activation of dredd, a member of the caspase family. both channels converge on the nf-kb factor, relish. ikkβ and ikkγ complex phosphorylate the i-kb domain of relish, while dredd presumably cleaves the i-kb domain (stoven et al., 2000, 2003). this allows the amino terminal portion of relish, carrying both dna binding and transcriptional activation domains, to enter into the nucleus and activate expression of antimicrobial peptide genes such as diptericin and cecropin. in d. melanogaster, fungal pathogens lead to an activation of an extracellular protease, persephone, while anti-gram(+) bacterial responses require the products of pgrp-sa and gnbp1, members pgrp and gram negative binding protein (gnbp) families, respectively. both fungal and gram(+) pathogens eventually converged on the cytokine-like molecule spaetzle, a protein with cysteine knots. proteolytic cleavage of pre-protein spaetzle produces the ligand for toll. toll then sends the signal through a multimeric protein complex, consisting of myd88, tube and pelle, leading to the phosphorylation of cactus, an i-kb like molecule, and subsequent activation and nuclear translocation of dif or dorsal, both belonging to the nfkb family of transcription factors. in adult flies, dif activates transcription of antimicrobial peptide genes such as drosomycin and metchnikowin (reviewed in hoffmann, 2003). toll was originally identified as a gene required for dorsal ventral patterning (nusslein-volhard et al., 1980) but was then found to be required for antifungal immunity in adults (lemaitre et al., 1996). toll is one of nine tlr genes in d. melanogaster that encode proteins with extracellular leucine rich repeat (lrr) arrays and an intracellular toll-interleukin-1 receptor (tir) domain (tauszig et al., 2000). thus far, genetic studies have shown that toll (or toll1) is the primary receptor in innate immunity. however, experiments in cell lines also suggest that toll5 (luo et al., 2001) and toll9 (ooi et al., 2002) can activate expression of antimicrobial peptide genes. toll1 and toll5 (also known as tehao) are similar in the tir domain, though toll1 carries a carboxyl terminal extension which toll5 lacks (luo and zheng, 2000). to date, screening by whole genome rna interference has not elucidated the functions of the other eight tlr genes in d. melanogaster. toll and imd signaling pathways in insects the origin of the innate immune systems will be better understood as more and more genomes of invertebrates are determined. the completion of the genome sequencing of anopheles gambiae, the main vector for human malaria (holt et al., 2002), in addition to the resolved genome of d. melanogaster, allows for comparative immunology to be examined between these two dipterans. although most of the toll and imd signaling components are conserved in a. gambiae, significant differences have been found (christophides et al., 2002). similar conclusions can be drawn from the ongoing genome sequencing of another major vector of human diseases, aedes aegypti, which transmits dengue and yellow fever viruses (unpublished observations). for the imd pathway, it was found that rel2, in both a. gambiae and a. aegypti, is both structurally and functionally different from its orthologue, relish, in d. melanogaster. rel2 in mosquitoes contains an additional death domain which is absent in the fly orthologue (shin et al., 2002). furthermore, the rel2 gene in mosquitoes produces different spliced variants. in a. aegypti, rel2 produces three spliced forms, yielding a full length protein, with both rel-homology domain (rhd) and i-kb ankyrin repeats, and partial length polypeptides, containing either the i-kb domain or the rhd domain, respectively (shin et al., 2002). in a. gambiae, two spliced variants have been found thus far, corresponding to the full length and rhd domain containing polypeptides from a. aegypti, respectively. more importantly, it has been shown in a. gambiae that the imd pathway functions in immune responses against both gram(+) and gram (-) bacteria (meister et al., 2005). components of the toll pathway are also found in a. gambiae, though once again significant differences have been found between it and d. melanogaster. the most conspicuous being the absence of a dif orthologue in both a. gambiae and a. aegypti. it was found recently that the dorsal homologue, rel1 from a. aegypti also produces spliced variants, rel1a and rel1b, differing at the carboxyl terminal domains (shin et al., 2005). similar spliced variants have been found for drosophila dorsal. concomitant over-expression of the two variants rel1a and rel1b from a. aegypti leads to the up-regulation of defa and ceca in a cell line (shin et al., 2005). alternative splicing also appears to occur in the a. gambiae dorsal homologue known as gambif (barillasmury et al., 1996) or rel1. one expressed sequence tag (ensangestt00000367694) of rel1 from a. gambiae represents a partial cdna clone which uses different splice sites than the full-length transcript rel1 transcript characterized previously (barillas-mury et al., 1996). at present, whether or not spliced forms similar to the 107 aedes rel1a and rel1b exist in a. gambiae remains to be determined. another major difference is the presence of four genes (toll1a, 1b, 5a and 5b) that share similarities in the tir domain with d. melanogaster toll1 and toll5. toll1a and 5a are arranged in tandem on the x chromosome, while toll1b and 5b are situated in tandem on the third chromosome (christophides et al., 2002). while it has been shown that over-expression of toll related genes from mosquitoes in d. melanogaster cell lines leads to elevated expression from the promoter of drosomycin (luna et al., 2002, 2003), there remains no additional functional evidence for the involvement of mosquito tolls in innate immunity. structure and functions of toll-like receptors (tlrs) as indicated above, toll receptors are transmembrane proteins with an extracellular lrr domain interspersed with cysteine knots. the intracellular domain shares sequence similarities with the interleukin-1 receptors from mammals, and is called the toll/interleukin-1 receptor (tir) domain (reviewed in imler and zheng, 2004). the tir domain is composed of approximately 150 amino acid residues. it is present not only in tlrs, but also in other intracellular signaling components, such as myd88. these intracellular tir-containing adaptor molecules, however, do not contain a leucine rich repeat. in addition, tir domains and leucine rich repeats are also present in some plant resistance (r) genes, typified by rpp5, which also has a nucleotide binding domain (nbd) (parker et al., 1997). in contrast to tlrs, the plant tir-nbd-lrr resistant proteins are intracellular. furthermore, their tir domains are phylogenetically distinct from tlrs. thus, it seems likely that the tir domain-containing adaptor and plant tir-nbd-lrr genes evolved independently from tlrs. the molecular structures of the tir domains of human tlr1 and tlr2 have been determined and show a five-stranded parallel β-sheet surrounded by five helices on both sides (xu et al., 2000). there is a conserved surface which primarily consists of a loop, known as the bb loop, which protrudes from the rest of the structure. mutations in the bb loop result in a loss or significant reduction in activity of tlrs from both man and drosophila. within this region are a few highly conserved charged amino acid residues, including an arginine at loop position 3 and an aspartic acid at loop position 4. a proline residue at loop position 7 is also known to be essential for lps signaling by human tlr4. as eluded to previously, this proline residue is not conserved in all tlrs (luna et al., 2003) and functional studies suggest that it is not a key structural determinant (xu et al., 2000). instead, substitution of this residue may disrupt interactions with other signaling components needed for activation of the pathway. mutation at a histidine residue in the bb loop position 1 in d. melanogaster, represented by allele tlrb1 or tlr6 in the flybase database (www.flybase.org), also results in a loss of function of toll in dorsal ventral formation in early embryogenesis (schneider et al., 1991). this histidine residue is highly conserved in insect tlrs, but not in vertebrate tlrs. loss of function mutations outside the bb loop yet still within the tir domain have also been documented in d. melanogaster. how these residues fully contribute to the function of toll signaling has yet to be determined. as tir domains of myd88 genes from different insects vary widely in sequence, variation on this interacting surface may contribute to species specific signaling in insects (unpublished observations). distribution of tlrs in the animal kingdom thus far, on the tree of life, tlrs have been found to be present in animals ranging from cnidarians to mammals, though they appear to be absent in platyhelminths (table 1). tlr genes have been found in all vertebrates where they also play an important role in bridging innate and adaptive immunity, reviewed in (pasare and medzhitov, 2004). for example, human and mouse genomes contain 10 and 12 tlr genes, respectively. these tlrs are activated, individually or in combinations, by different bacterial pattern molecules, such as lipopolysaccharide, peptidoglycan, or naked dna (for a recent review see kaisho and akira, 2004). tlrs are also found in invertebrates that have body cavities (table 2). d. melanogaster possesses 9 tlrs while a. gambiae encodes 10. thus far, at least one tlr gene has been found in all insect genomes. within the phylum arthropoda, one tlr gene has been found in the horseshoe crab tachypleus (inamori et al., 2000, 2004) and the lobster homarus americanus (accession number: cn852754, unpublished observation). limited genomic information has shown that at least one tlr gene is present in euprymna, the hawaiian squid which belongs to the phylum mollusca (hoa nt et al., unpublished observations). likewise, nematoda genome sequencing has revealed that caenorhbiditis elegans and caenorhbiditis briggsea each encode one tlr. interestingly, functional studies showed that this unique tlr gene (cetol-1) appears to have no function in innate immunity in c. elegans (pujol et al., 2001). a tlr gene has yet to be found in the phylum platyhelminth, though significant genomic information is available for schistosoma japonicum, schistosoma mansoni and schmidtea mediterranea. surprisingly though, on-going genome sequencing of a cnidarian recently showed that hydra magnipapillata has at least 3 tlr genes. these expressed sequence tags (accession number cb889349, cn630662 and cn630303) encode proteins with a tir domain and a transmembrane segment, thus establishing them as likely tlr genes. phylogenetic analysis provides further evidence that these tir domains cluster with animal tlrs, rather than with the intracellular tir domain containing genes. while it is conceivable that tlrs were lost during the evolutionary period leading to platyhelminths, as apparent in schistosoma and schmidtea, further genome 108 table 1. distribution of toll related receptors (tlr) and nf-kb genes in the animal kingdom phyla representative toll rel cnidaria hydra 3 -? platyhelminth schistosoma -? -? nematoda caenorhbiditis 1 annelida tubifex ? 1 mollusca euprymna/crassostrea 1? (+) arthropoda drosophila, 9 3 anopheles 10 2 tachypleus 1 ? homarus 1 ? echinodermata strongylocentrotus 2 1 chordata mus 12 5 homo 10 5 table 2. toll like receptor genes in arthropods† subclass infraclass order suborder # of genes representative neoptera orthopteroida orthoptera caelifera 2* schistocerca caelifera >1* locusta paraneoptera hemiptera 1* homalodisca endopterygota hymenoptera aculeata 5* apis coleoptera polyphaga 1* tribolium lepidoptera glossata 7* bombyx glossata 1* manduca diptera brachycera 9 drosophila brachycera 1* ceratitis brachycera 1* glossina nematocera 4* aedes nematocera 1* culex nematocera 3* clogmia nematocera 10 anopheles * indicates genome has not been fully sequenced or annotated. †not included here are one tlr gene each found in a lobster and a horseshoe crab, respectively. sequencing may yet reveal tlrs within the phylum platyhelminth. phylogeny of tlr genes the numbers of extracellular lrr arrays in different tlrs are different, making sequence alignment in this region highly subjective. therefore, we based the phylogenetic relationships among tlrs on the intracellular tir domain. the tir domain was identified using the smart algorithm (schultz et al., 2000; letunic et al., 2004) and aligned by clustal x (thompson et al., 1997). neighbor-joining showed that all tlrs from mollusca and arthropoda form a clade, except toll9s from drosophila and anopheles, which cluster with mammalian tlrs. tlrs from nematoda form a separate clade (fig. 1). this is consistent with the proposal that innate immunity developed independently in invertebrates and vertebrates (hughes, 1998; friedman and hughes, 2002). interestingly, the tlrs from cnidarian cluster with mammalian tlrs (fig. 1). distribution of rel transcription factors in the animal kingdom from studies in drosophila and mammals, toll signaling pathways in innate immunity require key transcriptional factors belonging to the nf-kb (or rel) family. these proteins are characterized by a rhd domain. thus far, nf-kb factors have been found in an 109 fig. 1 phylogenetic relationships among the tlrs. tir domains were identified using the smart algorithm and sequences before the conserved motif (f/y)da and after the motif fw(e/d) were removed. alignment was performed by clustal x, followed by visual inspection. edited alignment was used for tree generation using 1000 bootstrap iterations. nodes with bootstrap values >700/1000 are highlighted with filled circles. scale represents 0.1 amino acid change/residue. abbreviations are as follows: hu (human), dm (drosophila), aa (aedes), ag (anopheles), am (apis), at (arabidopsis), bf (branchiostoma), bm (bombyx), bom (boophilus), ca (clogmia), cc (ceratitis), ce (caenorhbiditis), ci (ciona), cp (culex), es (euprymna), ha (homarus), mx (manduca), sa (schistocerca), ss (strongyloides), tc (tribolium), tt (techypleus). due to the high throughput nature of the sequences analyzed, some insect tlr genes may be allelic. for simplicity, arthropod tlrs are indicated by a number after the species abbreviation, while the single tlr genes from caenorhbiditis, strongyloides and euprymna each are designated as tol-1. the alignment file is available upon request. 110 fig. 2 coelomata (panel a) versus ecdysozoa (panel b). if genes shared among organisms with a body cavity are absent in nematodes (black filled circle), they can be easily explained by the coelomata hypothesis (panel a). the ecdysozoa hypothesis would need to invoke a loss of genes in the nematodes (dashed open circle). genes shared among coelomates but absent in one of the two nematode species (filled rectangle) can be explained equally well by either the coelomata or ecdysozoa hypotheses (where gene losses are represented by dashed rectangles with different fill patterns for the two nematode species). the distribution of tlr and rel genes (last two columns in panel a) is consistent with the coelomata hypothesis. an asterisk indicates the absence of a corresponding gene, though it is conceivable that the gene could be found when the complete genome is sequenced. for simplicity, the rel gene from another coelomate annelid, tubifex is not shown here. 111 annelid, mollusks, arthropods and vertebrates, all of which possess real body cavities. none have been found in nematodes, platyhelminths or cnidarian (the former being pseudocoelomates while the later two acoelomates; see table 1 and fig. 2). this was based on a tblastn analysis (altschul et al., 1997) performed on april 7, 2005, using a. gambiae rel1 and d. melanogaster relish as queries against the updated non-redundant genbank database. tlr mediated systemic immunity developed in coelomates: a hypothesis among invertebrates, antimicrobial peptide production is the main form of systemic immunity. we argue that such immunity developed in coelomates (kanzok et al., 2004). before animals became terrestrial, it would not have been efficient to produce antimicrobial peptide on the surface, where the activity would have been diluted by sea water. when a coelom was developed in an animal, it provided a nutrient-rich and sterile environment that would have been envied by microbes. to protect against internal infection, the first coelomate patched together a signaling pathway that includes the existing tlr genes and may have acquired a novel transcription factor of the nf-kb family. this is consistent with the presence of both tlr and nf-kb genes in the phyla where sufficient genomic sequences are available: echinodermata, chordata, arthropoda and mollusca. further, antimicrobial peptides such as defensin have also been found in mammals, arthropods and mollusks. although one tlr gene each has been found in c. elegans and c. briggsae, no nf-kb-like transcription factor has been found in nematodes. further, genetic evidence with mutations in the tlr gene of c. elegans suggests that it is essential for development and is not involved in innate immunity (pujol et al., 2001). coelomata and ecdysozoa traditional classification of metazoans (or coelomata hypothesis) is based on the presence of a coelom and divides bilaterians into acoels, pseudocoels and coels (hyman, 1951). our hypothesis implies that classical division of the animal kingdom based on the presence or absence of a coelom (body cavity) is correct and promoted us to examine the relationships among taxa at the deep root of the evolutionary tree. both mollusca and arthropoda are protostomia coels and are considered more closely related to each other than they are to a pseudocoel phylum, nematoda. likewise, newer morphology-based classifications recognize protostomia and deuterostomia, with protostomia divided into spiralia and cycloneuralia, and these place mollusca and arthropoda as more closely related to each other than to nematoda (nielsen, 2001). a radical view proposed recently separates mollusca from arthropoda and groups arthropoda with nematoda and others, forming a clade named ecdysozoa. a separate clade, lophotrochozoa includes organisms such as annelids, mollusks and platyhelminths. this later view is primarily based on phylogenetic analysis of nuclear 18s ribosomal rna sequences (aguinaldo et al., 1997). evidence since has been accumulated in favor of (e.g. dopazo and dopazo, 2005; roy and gilbert, 2005) and against (e.g. hughes and friedman, 2004; steinauer et al., 2005) the ecdysozoa hypothesis. instead of examining sequence variations over time, which are subjected to varying evolutionary rates for different genes and homoplasy, recent studies show that large numbers of genes are shared between mollusks and arthropods yet are absent in the nematodes c. elegans and c. briggsae (hoa nt et al, unpublished observations). even taking into consideration gene loss that is known to occur in c. elegans, coelomata theory provides the most parsimonious explanation for the sharing of genes between mollusks and arthropods. if the coelomata theory is indeed correct, it would explain with the distributions of tlr and nf-kb genes in the animal kingdom. tlr genes first appeared in cnidarian and were subsequently lost in platyhelminths. the nf-kb transcription factor evolved once in coelomates and was retained in organisms such as annelids, mollusks, arthropods and vertebrates. the combination of tlr and nf-kb transcription factor genes provided the first coelomate a systemic immune response to internal infection by harmful bacteria. future perspectives we argue here that tlr mediated innate immunity developed after the appearance of coelomates. whether the imd pathway evolved in the same way remains to be examined. as more and more genome sequences become available, comparative immunology will shed further light on the origin of the systemic innate immune responses. interactions between microbes and their animal hosts range from pathogenic, to mutualistic, to symbiotic. thus far, we have examined host immune responses to microbes as non-self. in invertebrates, symbiotic microbes are essential to the survival and success of their hosts (rio et al., 2004). in a mutualistic interaction, the mollusk, euprymna scolopes or hawaiian squid, depends on its microbial partner, vibrio fisheri, to build a light-emitting organ. recent studies suggested that microbial pattern molecules are essential to the generation of this organ (koropatnick et al., 2004), though how these pattern molecules trigger organ development remains a mystery. whether toll or imd, both or additional pathways are involved in the formation of this unique organ or even other bacterial associated structures in invertebrates are questions which also remain unsolved. it is worth noting however that recent studies in mammals have shown that commensal microbes can activate host tlr receptors which in turn are required to maintain intestinal homeostasis (rakoffnahoum et al., 2004). in summary, understanding how microbial organisms interact with their hosts will provide 112 insights into the innate immunity of invertebrates and could lead to practical applications such as blocking the transmission of vector-borne pathogens. acknowledgements research in l zheng’s laboratory is supported by an nih grant (ai43053). l zhang and h lin received generous support from the chinese government. references aguinaldo am, et al. evidence for a clade of nematodes, arthropods and other moulting animals. nature 387: 489493, 1997. altschul sf, et al. gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids res. 25: 3389-3402, 1997. barillas-mury c, charlesworth a, gross i, richman a, hoffmann ja, kafatos fc. immune factor gambif1, a new rel family member from the human malaria vector, anopheles gambiae. embo j. 15: 4961-4701, 1996. choe km, lee h, anderson kv. drosophila peptidoglycan recognition protein lc (pgrp-lc) acts as a signaltransducing innate immune receptor. proc. natl. acad. sci. usa 102: 1122-1126, 2005. christophides gk, et al. immunity-related genes and gene families in anopheles gambiae. science 298: 159-165, 2002. dopazo h, dopazo j. genome-scale evidence of the nematode-arthropod clade. genome biol. 6: r41, 2005. friedman r, hughes al. molecular evolution of the nfkappab signaling system. immunogenetics 53: 964-974, 2002. hoffmann ja. the immune response of drosophila. nature: 426, 33-38, 2003. hoffmann ja, kafatos fc, janeway ca, ezekowitz ra. phylogenetic perspectives in innate immunity. science 284: 1313-1318, 1999. holt ra, et al. the genome sequence of the malaria mosquito anopheles gambiae. science 298: 129-149, 2002. hughes al. protein phylogenies provide evidence of a radical discontinuity between arthropod and vertebrate immune systems. immunogenetics 47: 283-296, 1998. hughes al, friedman r. differential loss of ancestral gene families as a source of genomic divergence in animals. proc. biol. sci. 271 (suppl. 3): s107-109, 2004. hyman lh. the invertebrates, vol 1 (new york, mcgraw-hill) 1951. imler jl, zheng l. biology of toll receptors: lessons from insects and mammals. j. leukoc. biol: 75, 18-26, 2004. inamori k, ariki s, kawabata s. a toll-like receptor in horseshoe crabs. immunol. rev. 198: 106-115, 2004. inamori k, koori k, mishima c, muta t, kawabata s. a horseshoe crab receptor structurally related to drosophila toll. j. endotoxin res. 6: 397-399, 2000. kaisho t, akira s. pleiotropic function of toll-like receptors. microbes infect. 6: 1388-1394, 2004. kanzok sm, hoa nt, bonizzoni m, luna c, huang y, malacrida ar, zheng l. origin of toll like receptor mediated innate immunity. j. mol. evol. 58: 442-448, 2004. koropatnick ta, engle jt, apicella ma, stabb ev, goldman we, mcfall-ngai mj. microbial factor-mediated development in a host-bacterial mutualism. science 306: 1186-1188, 2004. lemaitre b, et al. a recessive mutation, immune deficiency (imd), defines two distinct control pathways in the drosophila host defense. proc. natl. acad. sci. usa 92: 9465-9469, 1995. lemaitre b, nicolas e, michaut l, reichhart jm, hoffmann ja. the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults. cell 86: 973-983, 1996. letunic i, et al. smart 4.0: towards genomic data integration. nucleic acids res. 32: d142-144, 2004. luna c, et al. characterization of three toll-like genes from mosquito aedes aegypti. insect mol. biol. 12: 67-74, 2003. luna c, wang x, huang y, zhang j, zheng l. characterization of four toll related genes during development and immune responses in anopheles gambiae. insect biochem. mol. biol. 32: 1171-1179, 2002. luo c, shen b, manley jl, zheng l. tehao functions in the toll pathway in drosophila melanogaster: possible roles in development and innate immunity. insect mol. biol. 10: 457-461, 2001. luo c, zheng l. independent evolution of toll and related genes in insects and mammals. immunogenetics 51: 92-98, 2000. meister s, et al. functional characterization of rel2 from anopheles gambiae. proc. natl. acad. sci. usa 2005 (in press). nielsen c. animal evolution: interrelationships of the living phyla, 2nd, oxford university press, oxford, 2001. nusslein-volhard c, lohs-schardin m, sander k, cremer c. a dorso-ventral shift of embryonic primordia in a new maternal-effect mutant of drosophila. nature 283: 474-476, 1980. ooi jy, yagi y, hu x, ip yt. the drosophila toll-9 activates a constitutive antimicrobial defense. embo rep. 3: 82-87, 2002. parker je, et al. the arabidopsis downy mildew resistance gene rpp5 shares similarity to the toll and interleukin-1 receptors with n and l6. plant cell 9: 879-894, 1997. pasare c, medzhitov r. toll-like receptors: linking innate and adaptive immunity. microbes infect. 6: 1382-1387, 2004. pujol n, link em, liu lx, kurz cl, alloing g, tan mw, ray kp, solari r, johnson cd, ewbank jj. a reverse genetic analysis of components of the toll signaling pathway in caenorhabditis elegans. curr. biol. 11: 809-821, 2001. rakoff-nahoum s, paglino j, eslami-varzaneh f, edberg s, medzhitov r. recognition of commensal microflora by tolllike receptors is required for intestinal homeostasis. cell 118: 229-241, 2004. rio rv, hu y, aksoy s. strategies of the home-team: symbioses exploited for vector-borne disease control. trends microbiol. 12: 325-336, 2004. roy sw, gilbert w. resolution of a deep animal divergence by the pattern of intron conservation. proc. natl. acad. sci. usa 102: 4403-4408, 2005. schneider ds, hudson kl, lin ty, anderson kv. dominant and recessive mutations define functional domains of toll, a transmembrane protein required for dorsal-ventral polarity in the drosophila embryo. genes dev. 5: 797-807, 1991. schultz j, copley rr, doerks t, ponting cp, bork p. smart: a web-based tool for the study of genetically mobile domains. nucleic acids res. 28: 231-234, 2000. shin sw, kokoza v, ahmed a, raikhel as. characterization of three alternatively spliced isoforms of the rel/nf-kappa b transcription factor relish from the mosquito aedes aegypti. proc. natl. acad. sci. usa 99: 9978-9983, 2002. shin sw, kokoza v, bian g, cheon h-m, kim yj, raikhel as. rel1, a homologue of drosophila dorsal, regulates toll antifungal immune pathway in the female mosquito aedes aegypti. j. biol. chem. 280: 16499-16507, 2005. steinauer ml, nickol bb, broughton r, orti g. first sequenced mitochondrial genome from the phylum acanthocephala (leptorhynchoides thecatus) and its phylogenetic position within metazoa. j. mol. evol. 60: 706-715, 2005. stoven s, ando i, kadalayil l, engstrom y, hultmark d. activation of the drosophila nf-kappab factor relish by 113 rapid endoproteolytic cleavage. embo rep. 1: 347-352, 2000. stoven s, et al. caspase-mediated processing of the drosophila nf-kappa b factor relish. proc. natl. acad. sci. usa 100: 5991-5996, 2003. tauszig s, jouanguy e, hoffmann ja, imler j-l. toll related receptors and the control of antimicrobial peptide expression in drosophila. proc. natl. acad. sci. usa 97: 10520-10525, 2000. thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids res. 25: 4876-4882, 1997. xu y, et al. structural basis for signal transduction by the toll/interleukin-1 receptor domains. nature 408: 111-115, 2000. isj 3: 111-xx, 2006 isj 3: 137-145, 2006 issn 1824-307x research report brush border membrane vesicles from dipteran midgut: a tool for studies on nutrient absorption mg leonardi, s caccia, b giordana dipartimento di biologia, università degli studi di milano, milano, italy accepted december 20, 2006 abstract brush border membrane vesicles (bbmv) from insects midgut can be successfully used to study several membrane phenomena, including nutrient absorption, ions permeability and insecticides mode of action. midgut bbmv, purified from musca domestica whole larvae, were used for the functional characterization of leucine transport. the amino acid uptake was accelerated in the presence of sodium or potassium and increased significantly when the extravesicular ph was 5.0, in agreement with the luminal ph in vivo. radiolabelled leucine uptake was significantly reduced by an excess of cold leucine, histidine, serine and glycine, suggesting that the amino acid transporter is a broad scope carrier that does not recognize proline, glutamine and the dibasic amino acids lysine and arginine. midgut bbmv were also obtained from homogenization of m. domestica and bactrocera oleae adults. the final preparations showed a high enrichment in the specific activity of the bbm marker enzymes aminopeptidase n and γ-glutamyl transpeptidase, and were poorly contaminated by basolateral membranes, as indicated by the low specific activities of their marker enzyme na+/k+ atpase. electron microscopy of b. oleae bbm fraction showed the presence of closed vesicles. similar sds-page patterns, with numerous distinct bands, were detected for both b. oleae and m. domestica bbmv. key words: dipteran midgut; brush border membrane vesicles; amino acid absorption; bactrocera oleae; musca domestica _____________________________________________________________________________________________ introduction amino acids play a key role in several physiological processes and for this reason intestinal amino acid absorption, a crucial step in nitrogen metabolism, affects the biological development of the whole organism. the functional and regulative properties of the amino acid transport systems expressed in the insect midgut epithelium has been extensively studied in lepidopteran larvae (giordana et al., 1989, 1998, 2002; wolfersberger, 1996; leonardi et al., 1998a, 2001a, b; casartelli et al., 2001; ______________________________________________________________________________ corresponding author: m. giovanna leonardi dipartimento di biologia univerisity of milan via celoria 26, 20133 milano, italy e-mail: mgiovanna.leonardi@unimi.it parenti et al. 2002) while little is known on intestinal amino acid absorption in other insect orders (hong et al., 1995, 1997; neal, 1996; neal et al., 1996; parenti et al., 1986, 2001). brush border membrane fragments form sealed vesicles spontaneously maintaining their correct orientation (haase et al., 1978), that allow to define the internal and external environments according to the experimental needs. the brush border membrane vesicles (bbmv) isolated from lepidopteran midgut epithelium have been successfully used for the functional characterization of amino acid transport proteins (giordana et al., 1989, 1998; wolfersberger, 1996; leonardi et al., 1998a; casartelli et al., 2001), to analyze in vitro the activity of the insecticide fenoxycarb (leonardi et al. 1996, 1998b, 2001a) and, far more extensively, to study the mode of action of 137 whole dipteran adults homogenized in 1:10 w/v buffer a (300 mm mannitol, 2 mm tris-cl at ph 7.1) in polytron (2 periods lasting 1 min) and filtered through gauze homogenate centrifuged at 1000xg for 10 min s1 fig. 1 procedure for the preparation of bbmv from adults m. domestica and b. oleae (described in detail in materials and methods). p1 centrifuged at 20000xg for 15 min s2p2 resuspended in 30 ml of buffer a plus percoll 10.8%, with 3 strokes in a loose dounce and centrifuged at 40000xg for 35 min band collected, diluted to 30 ml with buffer a and centrifuged at 100000xg for 60 min p3b – fluffy pellet p3a – hard glassy percoll pellet s3 p4 s4 centrifuged at 48000xg for 20 min s5 p3b resuspended in 20 ml buffer a, mixed with cacl2 to10 mm final concentration and centrifuged at 1000xg for 15 min bbmv p5 138 cry1 toxins of bacillus thuringiensis (sacchi et al. 1986; gill et al., 1992; giordana et al., 1993; knowles, 1994; leonardi et al., 1997; bravo et al., 2002). the preparation of purified bbmv from the midgut of insects of interest is therefore fundamental to investigate several membrane phenomena. in the present paper we describe the preparation of purified midgut bbmv using whole musca domestica larvae as a starting material. the vesicles were then used to describe the functional properties of amino acid uptake in this dipteran larva. we also describe a method to obtain midgut bbmv from adults of m. domestica and bactrocera oleae, preparations that can be used to study the physiological processes active at the apical membrane of midgut cells or the effect on the same membrane of insecticides potentially active against the adults of these two dipteran pests. materials and methods materials l-[4,5-3h]leucine (71 ci/mmol) was purchased from amersham biosciences europe, italy. all the other reagents were supplied by sigma-aldrich s.r.l., italy. experimental animals m. domestica larvae, kindly provided by dr. marcello verdinelli (istituto di ricerca sul controllo biologico dell’ambiente (ircoba), cnr, sassari), were immediately frozen and preserved in liquid nitrogen for few weeks. pupae of m. domestica and b. oleae, also provided by ircoba, were maintained at 25 ± 1 °c, 65-70 % r.h. and 12l:12d photoperiod. immediately after eclosion, the adults were anaesthetized at 4 °c, then frozen in liquid nitrogen and there preserved for few weeks. bbmv preparation from larvae of m. domestica larvae of m. domestica, removed from liquid nitrogen, were placed in 1:10 w/v of an ice-cold buffer composed of 100 mm mannitol, 10 mm hepes-tris at ph 7.1, 1 mm pmsf and homogenized on ice with polytron (kinematica ch-6010 kriens-lu) for 3 periods lasting 30 s at velocity 5. the homogenate was filtered through 2 gauze layers and bbmv were prepared by ca++-precipitation and differential centrifugation as reported by giordana et al. (1982). the filtered homogenate (h) was added with cacl2 to a final concentration of 10 mm, the suspension was stirred on ice for 15 min and then centrifuged at 3000 x g for 15 min. the supernatant was decanted and kept on ice. the pellet was resuspended in half of the original volume of buffer (100 mm mannitol, 10 mm hepes-tris at ph 7.1) with the aid of a glass-teflon homogenizer (ika-labortechnick re 16) with five strokes at 1500 rpm. the suspension was mixed with cacl2 to a final concentration of 10 mm, blended on ice for 15 min and then centrifuged at 3000 x g for 15 min. the second pellet was discarded. the first and the second supernatants were pooled and centrifuged at 48000 x g for 20 min. the supernatant was discarded and the pellet resuspended in the original volume of buffer with five strokes at 1500 rpm in the glass-teflon homogenizer. the suspension was centrifuged at 48000 x g for 20 min and the final pellet, containing the brush border membranes, was resuspended in a suitable amount of buffer by 10 passes through a 22 gauge needle. protein concentration was assessed with the coomassie brilliant blue g-250 protein assay (pierce, rockford, il, usa) with bovine serum albumin as standard. bbmv preparation from adults of m. domestica and b. oleae the procedure is schematically presented in fig. 1. all the steps were performed at 4 °c. the frozen adults were placed in ice-cold buffer composed of 300 mm mannitol, 2 mm tris-cl at ph 7.1, in a 1:10 w/v proportion, and homogenized with a polytron (kinematica ch-6010 kriens-lu) for 2 periods lasting 1 min at velocity 5. the homogenate was filtered through 3 layers of gauze to remove large debris. the filtered homogenate (h) was centrifuged at 1000 x g for 10 min. the first pellet p1 was discarded and the supernatant s1 was centrifuged at 20000 x g for 15 min. the supernatant s2 was discarded and the pellet p2 resuspended in 30 ml of buffer containing 10.8 % of percoll with 3 strokes in a loose dounce homogenizer. the suspension was centrifuged at 40000 x g for 35 min with no brake. the plasma membranes were present as a band at the third upper portion of the continuous density gradient formed by percoll. the membrane fraction was carefully removed with a pasteur pipette, diluted to 30 ml with buffer and centrifuged at 100000 x g for 1 h. the supernatant s3 was carefully decanted and the fluffy membrane layer p3b, separated from the glassy percoll pellet p3a, was resuspended in 20 ml of buffer. cacl2 was added to the suspension to a final concentration of 10 mm, the mixture was blended on ice for 15 min and then centrifuged at 1000 x g for 10 min. the pellet p4 was discarded and the supernatant s4 centrifuged at 48000 x g for 20 min. the final pellet p5, containing the brush border membranes, was resuspended in a suitable volume of buffer by means of 10 passes through a 22 gauge needle. enzyme assays enzyme activities were measured in the filtered homogenate (h) and in bbmv. aminopeptidase n (ec 3.4.11.2) was determined by measuring the release of p-nitroaniline from l-leucine-p-nitroanilide in 40 mm trishcl at ph 7.5 and γ-glutamyl transferase (e.c. 2.3.2.2) by the release of 5-amino-2-nitrobenzoate from l-γ-glutamyl-glycilglycine in 100 mm trishcl at ph 8. incubations were carried out in a spectrophotometer (ultrospec 3000 pharmacia biotech, cambridge uk) with a thermostatic (25 °c) cuvette holder, in conditions in which activity was proportional to the protein concentration and time. 139 table 1 protein yield and activities of plasma membrane marker enzymes in homogenate and bbmv from m. domestica larvae protein yield (mg protein/g animals) bbmv 0.31 ± 0.09 (5) enzyme activity (mu/mg protein) aminopeptidase n γ-glutamyl transferase na+/k+ atpase homogenate 84.1 ± 3.0 (5) 2.0 ± 0.1 (3) 121 ± 21 (3) bbmv 642 ± 119 (5) 20.6 ± 0.6 (3) 189 ± 8 (3) enrichment factor* 7.5 ± 1.2 (5) 10.3 ± 0.2 (3) 1.7 ± 0.4 (3) values are means ± se in brackets are reported the number of independent preparations assayed *ratio of the enzyme specific activity in bbmv and in the homogenate one unit of enzyme activity corresponds to the hydrolysis of 1 μmol of substrate/min. na+/k+-atpase (e.c. 3.6.1.3.) activity was measured according to quigley and gotterer (1969). electrophoresis brush border membrane proteins were analyzed under denaturing conditions by sds-page (laemmli, 1970) in a mini protean ii bio-rad electrophoresis system. 4 % and 10 % acrylamide concentrations were used for stacking and running gels respectively. samples were prepared by mixing 1:1 (v/v) h or bbmv with the loading buffer 2x (4 % sds, 20 % glycerol, 10 % 2-mercaptoethanol, 0.004 % bromphenol blue, 0.125 m tris-hcl at ph 6.8) (sigmaaldrich s.r.l., italy). the mixture was incubated at 100 °c for 4 min before loading the samples. the bands were stained with coomassie blue r-250. transmission and scanning electron microscopy bbmv were fixed for 30 min in 0.1 m cacodylate buffer, ph 7.2, containing 2 % glutaraldehyde. samples were then washed in the same buffer and postfixed for 20 min with 1 % osmic acid in 0.1 m cacodylate buffer, ph 7.2. after a standard step of serial ethanol dehydratation, bbmv were pelletted and embedded in an epon-araldite 812 mixture. sections were obtained with a reichert ultracut s ultratome (leica, wien, austria). thin sections were stained by uranyl acetate and lead citrate and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). measurements of leucine uptake in bbmv from m. domestica larvae transport experiments were performed in triplicate at room temperature by rapid filtration (hanozet et al., 1980). to measure leucine uptake, 1 volume of the bbmv suspension was mixed to 4 volumes of the radiolabelled incubation medium, whose final concentration is reported in the legend of the figures. uptake was terminated by diluting the incubated mixture with 50 volumes of ice-cold stop solution (150 mm nacl, 10 mm hepes-tris at ph 7.2). the suspension was then filtered through a prewetted mixed cellulose ester filter (0.45 µm pore size, micro filtration systems, dublin, ca), then counted for radioactivity in a scintillation spectrometer (tri-carb, packard, model 1600 ca). results and discussion in the 1980’s two methods were published to prepare bbmv from the midgut of lepidopteran larvae (hanozet et al., 1980; giordana et al., 1982; wolfersberger et al., 1987). these procedures required that the midgut tissues were isolated from the larvae and were based on the calciumor magnesiumprecipitation of the membrane fragments obtained from the tissue homogenization, followed by differential centrifugations. later, modifications of the original protocols were introduced to prepare midgut bbmv sufficiently purified starting directly from the entire larva of the lepidoptera plutella xilostella (macintosh et al., 1994) or the diptera aedes aegypti (abdul-rauf and ellar, 1999) and chironomus riparius (parenti et al., 2000). the use as a starting material of the entire animal instead of the isolated midguts reduces enormously the time necessary to obtain bbmv. we applied the ca++-precipitation method (giordana et al., 1982), conveniently modified, to the larval stage of the important pest m. domestica. a satisfying homogenization of all the larval tissues could be obtained only by using a polytron homogenizer, and an adequately purified final preparation was achieved by adding a second cycle of ca++-precipitation and 140 fig. 2 initial rates (panel a) and time course (panel b) of leucine uptake in bbmv from m. domestica larvae. bbmv, resuspended in 100 mm mannitol, 10 mm hepes-tris at ph 7.2, were diluted 1:5 to obtain the following final composition: 0.1 mm l-[4,5-3h] leucine 30 μci/ml, 100 mm nascn (na+, ▼�) or kscn (k+, ○), or 200 mm sucrose (none, ●), 20 mm hepes-tris at ph 7.2 or 20 mm mes-tris at ph 5.0 (na+, �). values are means ± se of a typical experiment performed in triplicate. initial rates (panel a) and time course (panel b) of leucine uptake in bbmv from m. domestica larvae. bbmv, resuspended in 100 mm mannitol, 10 mm hepes-tris at ph 7.2, were diluted 1:5 to obtain the following final composition: 0.1 mm l-[4,5-3h] leucine 30 μci/ml, 100 mm nascn (na+, ▼�) or kscn (k+, ○), or 200 mm sucrose (none, ●), 20 mm hepes-tris at ph 7.2 or 20 mm mes-tris at ph 5.0 (na+, �). values are means ± se of a typical experiment performed in triplicate. differential centrifugation. the purity was verified by measuring the specific activity in the homogenate and in the final pellet of aminopeptidase n and γ-glutamyl transferase, two enzymes exclusively localized in the apical membrane of midgut cells, and of na+/k+ atpase, the marker enzyme of the basal plasma membrane. the enrichment factor was calculated as the ratio between the enzyme specific activity in the final preparation and in the homogenate. the brush border marker enzymes were enriched sevenfold and tenfold, respectively, with a negligible enrichment of na+/k+ atpase (table 1), so the final bbmv preparation was essentially free of contaminating basal membranes. altogether, the enrichment factors of this study are consistent with previous results, since a tenfold enrichment of the bbm marker enzymes were obtained for bbmv prepared from whole aedes aegypti larvae (abdul-rauf and ellar, 1999), and enrichment factors varying between 3 and 5 were calculated for bbm purified from the isolated larval midguts of m. domestica as a starting material (lemos and terra, 1992; jordao et al., 1995). we performed transport experiments in m. domestica bbmv to identify the main features of amino acid absorption in this insect. the time course of 0.1 mm leucine uptake into bbmv was measured in the absence of cations, or in the presence of an inwardly directed sodiumor potassium-gradient. if an amino acid is transported by a transport protein that also binds and translocates a cation, i.e. by a cotransporter, a transient accumulation of the amino acid in the intravesicular space should occur, due to the flow of the cation along its electrochemical gradient. otherwise, the uptake is merely equilibrative and the equilibrium will be attained at different times, according to the amino acid transport rate. when the extravesicular ph was 7, leucine initial uptake rate at 15 s increased fourfold in the presence of k+ and eightfold with na+ (fig. 2, panel a). the time course of leucine uptake in the absence of cations was very slow and the equilibrium was not yet reached after 120 min (fig. 2, panel b). in the presence of a k+or a na+-gradient, leucine uptake was accelerated but no transient accumulation of the amino acid was observed 141 fig. 3 inhibition of radiolabelled leucine uptake by a large excess of the indicated amino acids in bbmv from m. domestica larvae. bbmv, resuspended in 100 mm mannitol, 10 mm hepes-tris at ph 7.2, were diluted 1:5 to obtain the following final composition: 0.4 μm l-[4,5-3h]leucine 30 μci/ml, 100 mm nascn, 10 mm inhibitors or mannitol (control), 20 mm hepes-tris at ph 7.2. values are means ± se of a typical experiment performed in triplicate. the statistical analysis of the data was performed by student’s t-test. ** p < 0.01; *** p < 0.001. (panel b). therefore, na+ and k+ are able to activate leucine transporter in m. domestica midgut, but no evidence is given that the protein is a cotransporter. in coleopteran larval midgut, the uptake of tyrosine and methionine, but not that of leucine, was also stimulated by na+ and k+ (hong et al., 1995) and, in agreement with our results, no accumulation of the two amino acids was observed with a na+or a k+ gradient (neal, 1996). the na+-dependent leucine uptake increased markedly when the extravesicular ph was 5.0 (fig. 2, panels a and b), suggesting that leucine transporter is adapted to the physiological environment of m. domestica larval midgut, where the lumen contents is weakly acidic in the anterior and posterior regions and highly acidic in the middle region (terra, 1988; dubreuil, 2004). interestingly, leucine uptake in midgut bbmv of lepidopteran larvae is mediated by a transport system that is, in that case, strongly activated by an extravesicular alkaline ph, as tipically present in the luminal fluids of the larval midgut: the ph-gradient can be exploited to perform leucine intravesicular accumulation (giordana et al., 1998). therefore, the ability to operate more efficiently in the presence of the physiological ph is a functional property in common between dipteran and lepidopteran leucine transport systems. as expected for a carrier-mediated process, a 87 % reduction of radiolabelled leucine uptake was observed in the presence of a large excess of cold leucine (fig. 3). the transporter recognized other neutral amino acids, since a 56 %, 34 % and 37 % inhibition was observed with an excess of histidine, serine and glycine, respectively (fig. 3). the percent inhibitions were calculated with respect to the control value after subtraction of the residual uptake in the presence of an excess of leucine. the uptake was not affected by proline, glutamine or by the dibasic amino acids lysine and arginine, suggesting the presence of other transporters for the intestinal absorption of these amino acids. while protocols for the preparation of bbmv from whole larvae are now available (abdul-rauf and ellar, 1999; parenti et al., 2000; this paper), no procedures are actually known to obtain bbmv directly from adult diptera, a severe limit to the investigation of the physiological processes occurring at the apical membrane of midgut cells and to the detection of new drugs potentially noxious to the brush border, that may be used as pesticides. we have, therefore, developed a new procedure based on the fractionation of the homogenate on a continuous density gradient of percoll, followed by calcium-precipitation (fig. 1). a complete homogenization of b. oleae and m. domestica adults was obtained with the polytron homogenizer. the brown-black color developed during homogenization remained in the pellet of the first lowspeed centrifugation, that is discarded, so that no dark coloration was observed in the following steps of the procedure. the plasma membranes stratified as a distinct diffused band located at the third upper portion of the continuous density gradient formed by percoll. the residual percoll present in the collected fraction was later removed as a glassy pellet after the high speed centrifugation. 142 table 2 protein yield and activities of the plasmamembrane marker enzymes in homogenate and bbmv from m. domestica and b. oleae adults protein yield (mg protein/g insects) b. oleae m. domestica bbmv 0.03 ± 0.01 (3) 0.13 (2) enzyme activity (mu/mg protein) b. oleae aminopeptidase n γ-glutamyl transferase na+/k+ atpase homogenate 19.0 ± 0.04 (3) 2.0 ± 0.1 (3) 83 ± 7 (3) bbmv 531 ± 18.2 (3) 58.3 ± 6.8 (3) 275 ± 24 (3) enrichment factor* 31.9 ± 9.6 (3) 28.8 ± 3.2 (3) 3.3 ± 0.2 (3) m. domestica aminopeptidase n γ-glutamyl transferase na+/k+ atpase homogenate 17.2 (2) not detectable 380 ± 40 (2) bbmv 65.0 (2) 52.2 (2) not detectable enrichment factor* 3.8 (2) values are means ± se in brackets are reported the number of independent preparations assayed *ratio of enzyme specific activity in bbmv and in the homogenate as shown in table 2, the final pellet obtained from b. oleae was enriched 30-fold in aminopeptidase n and γ-glutamyl transferase activities with a very low enrichment for na+/k+ atpase, a reliable indication that the protocol leads to a highly purified brush border membrane preparation. however, bbmv protein yield was extremely low (table 2), a result more likely related to the fact that the intestinal epithelium represents a very small portion of the starting material rather than to the complexity of the procedure. the microphotograph of the final pellet (p5) indicated the presence of closed vesicles and of some amorphous material (fig. 4). the analysis of the purity of the bbmv from m. domestica (table 2) confirms that this final preparation is also fairly purified. the enrichment factor of aminopeptidase n was lower than that obtained for b. oleae but the final pellet was not contaminated by basal membranes, since no na+/k+ atpase activity was detectable. the enrichment in brush border membranes was also confirmed by the emergence in the bbmv pellet of a γ-glutamyl transferase activity, too diluted in the homogenate to be detected. a characterization of b. oleae and m. domestica bbmv was performed by sds-page (fig. 5). numerous distinct bands were resolved by the gel. the patterns produced by the bbmv of the two species were very similar (lanes 3 and 5), while they fig. 4 electron micrograph of the final membrane pellet from b. oleae whole adults. 143 fig. 5 comparison of sds-page profile of bbmv prepared from adults of b. oleae (lane c) and m. domestica (lane e). lane a) molecular weights (kda); lane b) bbmv prepared from bombyx mori larval midgut; lane d) homogenate from adults of m. domestica. were evidently different from that obtained with midgut bbmv of a lepidopteran larva (lane 1) here presented for comparison. the apparent molecular masses of some major bands, present in the two dipteran bbmv and lacking in the homogenate (lane 4), correspond to a number of proteins involved in the architecture of microvilli cytoskeleton, such as actin (43 kda), myosin i (110 kda) and fimbrin (68 kda). in conclusion, our experimentation provides a midgut preparation from larvae of a pest insect, that can be used to characterize fundamental physiological processes such as amino acid absorption. the in depth knowledge of these processes may provide targets for new pesticides. the novel preparation of midgut bbmv from dipteran whole adults, obtained for the first time, may contribute to solve the problem of the identification of the mechanism of action of insecticidal molecules acting on midgut brush border membrane in insects of small dimension. acknowledgements we are grateful to prof. m de eguileor (dipartimento di biologia strutturale e funzionale, università dell'insubria, italy) for the tem image of bbmv pellet. references abdul-rauf m, ellar dj. isolation and characterisation of brush border membrane vesicles from whole aedes aegypti larvae. j. invert. pathol. 73: 45-51, 1999. bravo a, miranda r, gómez i, soberón m. pore formation activity of cry1ab toxin from bacillus thuringiensis in an improved membrane vesicle preparation from manduca sexta midgut cell microvilli. biochim. biophys. acta 1562: 63-69, 2002. a c e d b 250 casartelli m, leonardi mg, fiandra l, parenti p, giordana b. multiple transport pathway for dibasic amino acid in the larval midgut of the silkworm bombyx mori. insect biochem. molec. biol. 31: 621-632, 2001. 116 dubreuil rr. copper cells and stomach acid secretion in the drosophila midgut. int. j. biochem. cell biol. 36: 745-752, 2004. gill ss, cowles fa, pietrantonio pv. the mode of action of bacillus thuringiensis endotoxins. annu. rev. entomol. 37: 615-636, 1992. giordana b, sacchi vf, hanozet gm. intestinal amino acid absorption in lepidopteran larvae. biochim. biophys. acta 692: 81-88, 1982. giordana b, sacchi vf, parenti p, hanozet gm. amino acid transport systems in intestinal brush-border membranes from lepidopteran larvae. am. j. physiol. 257: r494-r500, 1989. giordana b, tasca m, villa m, chiantore c, hanozet gm, parenti p. bacillus thuringiensis subsp. aizawai inhibits the k+/amino acid cotransporter of lepidopteran larval midgut. comp. biochem. physiol. 106c: 403-407, 1993. giordana b, leonardi mg, casartelli m, consonni p, parenti p. the k+/neutral amino acid symport of bombyx mori larval midgut operates in extreme environmental conditions. am. j. physiol. 274: r1361-r1371, 1998. giordana b, forcella m, leonardi mg, casartelli m, fiandra l, hanozet gm, et al. a novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut. j. insect physiol. 48: 585-592, 2002. haase w, schafer a, murer h, kinne r. studies on the orientation of brush-border membrane vesicles. biochem. j. 172: 57-62, 1978. hanozet gm, giordana b, sacchi vf. k+-dependent phenylalanine uptake in membrane vesicles isolated from the midgut of philosamia cynthia larvae. biochim. biophys. acta 596: 481-486, 1980. hong ys, reuveni m, neal jj. a sodium-stimulated and potassium-stimulated tyrosine transporter from leptinotarsa decemlineata midgut. j. insect physiol. 41: 527-533, 1995. hong ys, neal jj. tyrosine transporter in larval leptinotarsa decemlineata midgut brush border membrane. insect biochem. molec. biol. 27: 193200, 1997. jordao bp, terra wr, ferreira c. chemical determinations in microvillar membranes purified from brush-borders isolated from the larval midgut of coleoptera and two dipteran species. insect biochem. molec. biol. 25: 417-426, 1995. 66 45 29 20 144 knowles bh. mechanism of action of bacillus thuringiensis insecticidal delta-endotoxins. adv. insect physiol. 24: 275-308, 1994. laemmli uk. cleavage of structural proteins during the assembly of the head of bacteriofage t4. nature 227: 680-685, 1970. lemos fja, terra wr. a high yield preparation of musca domestica larval midgut microvilli and the subcellular distribution of amilase trypsin. insect biochem. molec. biol. 22: 433-438, 1992. leonardi mg, cappellozza s, ianne p, cappellozza l, parenti p, giordana b. effects of the topical application of an insect growth regulator (fenoxycarb) on some physiological parameters in the fifth instar larvae of the silkworm bombyx mori. comp. biochem physiol. 113b: 361-365, 1996. leonardi mg, parenti p, casartelli m, giordana b. bacillus thuringiensis cryiaa δ-endotoxin effects the k+/amino acid symport in bombyx mori larval midgut. j. membrane biol. 159: 209-217, 1997. leonardi mg, casartelli m, parenti p, giordana b. evidence for a low affinity high capacity uniport for amino acid in bombyx mori larval midgut. am. j. physiol. 274: r1372-r1375, 1998a. leonardi mg, cappellozza s, de eguileor m, grimaldi a, cappellozza l, casartelli m, et al. modification of the nutritional parameters and of midgut biochemical and absorptive functions induced by the igr fenoxycarb in bombyx mori larvae. arch. insect biochem. physiol. 39: 18-35, 1998b. leonardi mg, marciani p, montorfano g, cappellozza s, giordana b, monticelli g. effect of fenoxycarb on leucine uptake and lipid composition of midgut brush border membrane in the silkworm, bombyx mori (lepidoptera, bombycidae). pest. biochem. physiol. 70: 42-51, 2001a. leonardi mg, fiandra l, casartelli m, cappellozza s, giordana b. modulation of leucine absorption in the larval midgut of bombyx mori (lepidoptera, bombycidae). comp. biochem. physiol. 129: 665672, 2001b. macintosh sc, lidster bd, kirkham cl. isolation of brush border membrane vesicles from whole diamondback moth (lepidoptera: plutellidae) larvae. j. invert. phatol. 63: 97-98, 1994. neal jj. brush border membrane and amino acid transport. arch. insect biochem. physiol. 32: 5564, 1996. neal jj, wu d, hong ys. high affinity transport of histidine and metionine across leptinotarsa decemlineata midgut brush border membrane. j. insect physiol. 42: 329-335, 1996. parenti p, sacchi vf, hanozet gm, giordana b. nadependent uptake of phenylalanine in the midgut of a cockroach blabera gigantea. j. comp. physiol. 156b: 549-556, 1986. parenti p, forcella m, pugliese a, giacchini r, rossaro b, hanozet gm. leucine transport in membrane vesicles from chironomus riparius larvae display a mélange of crown-group features. arch. insect biochem. physiol. 48: 5162, 2001. parenti p, leonardi mg, forcella m, casartelli m, pugliese a, hanozet gm, et al. leucine methyl ester is a powerful allosteric activator of the neutral amino acid cotransport system in bombyx mori larval midgut. insect biochem. molec. 32: 719-727, 2002. quigley jp, gotterer gs. distribution of na+-k+ atpase activity in rat intestinal mucosa. biochim. biophys. acta 173: 456-468, 1969. sacchi vf, parenti p, hanozet gm, giordana b, luethy p, wolfersberger mg. bacillus thuringiensis toxin inhibits k+-gradient dependent amino acid transport across the brush border membrane of pieris brassicae midgut cells. febs lett. 204: 213-218, 1986. terra wr. physiology and biochemistry of insect digestion: an evolutionary perspective. brazilian j. med. biol. res. 21: 675-734, 1988. wolfersberger mg, lüthy p, maurer a, parenti p, sacchi vf, giordana b, et al. preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (pieris brassicae). comp. biochem. physiol. 86a: 301-308, 1987. wolfersberger mg. localization of amino acid absorption systems in the larval midgut of the tobacco hornworm manduca sexta. j. insect physiol. 42: 975-982,1996. 145 dipartimento di biologia, università degli studi di milano, milano, italy accepted december 20, 2006 transmission and scanning electron microscopy isj114.pdf 25 isj 3: 25-39, 2006 issn 1824-307x review the sea urchin immune system lc smith1, jp rast2, v brockton1, dp terwilliger1, sv nair3, km buckley1, aj majeske1 1department of biological sciences, george washington university, washington dc, usa 2division of molecular and cell biology, sunnybrook and women's research institute, department of medical biophysics, university of toronto, toronto, canada 3department of biological sciences, macquarie university, sydney, australia accepted may 10, 2006 abstract metchnikoff’s use of sea star larvae to observe encapsulation and phagocytosis, which was followed much later by allograft rejection kinetics, revealed that echinoderms had an innate immune system that was lacking of adaptive attributes. larval sea urchins mount defenses in response to contact with microbes, which are mediated by phagocytic blastocoelar cells and pigment cells. in the adult, the coelomocytes mediate immune responses through phagocytosis and encapsulation of foreign particles in addition to degranulation of antimicrobial molecules. molecular analysis of immune functions in the sea urchin has demonstrated a complement system that appears to have multiple alternative pathways and several activators of the lectin pathway, but may be missing the terminal pathway. other genes and proteins involved in the sea urchin immunity include expanded sets of lectins, proteins with scavenger receptor cysteine-rich repeats, toll-like receptors and associated signalling proteins. a vast array of proteins belonging to the 185/333 family are expressed in coelomocytes in response to lipopolysaccharide and show a surprising level of diversity. the sea urchin innate immune system has a number of large gene families with unexpected complexities and elevated levels of diversification. key words: evolution; deuterostome; echinoderm; coelomocyte; innate immunity; diversification; complement; 185/333 genes introduction for many years, it was thought that invertebrates did not possess a recognizable immune system, a viewpoint which ignored metchnikoff’s seminal research on the mechanisms of inflammation (metchnikoff, 1893). he was the first to demonstrate cellular encapsulation of a foreign body inserted into an invertebrate, and employed the larval sea star, astropecten pentacanthus. metchnikoff extended this observation to corresponding author: l. courtney smith department of biological sciences, george washington university, 2023 g st nw, washington dc 20052, usa email: csmith@gwu.edu a phylogenetically diverse array of animals and was able to establish that phagocytic cells are a basic characteristic of metazoan biology. these observations formed the basis of the emerging field of cellular immunity for which he was awarded the nobel prize in 1908. however, it was not until seventy years later that hildemann and colleagues demonstrated the ability of several echinoderm species to differentiate self from nonself tissues through allograft rejection studies (hildemann and dix, 1972; karp and hildemann, 1976). these initial observations were extended in studies of sea urchins, lytechinus pictus and strongylocentrotus purpuratus, in which the non-specific nature of innate immune responses of the sea urchins was defined (coffaro and hinegardner, 1977; coffaro, 1979, 1980; smith and davidson 1992). these and other studies identified the cells of the open circulatory system in adult echinoderms, the coelomocytes, as the main effectors of defense 26 responses and as the primary mediators of allograft rejection (hildemann and dix, 1972; coffaro and hinegardner, 1977), response to injury or infection, and the clearance of foreign substances (reinisch and bang, 1971; bertheussen, 1981; yui and bayne, 1983; plytycz and seljelid, 1993). of the echinoderms, the genus strongylocentrotus is one of the most comprehensively studied with respect to immunological defense capabilities, particularly in the adult (reviewed by gross et al., 1999). yet, the embryonic and larval forms also must contend with microbes in marine waters (reviewed by smith, 2005), and studies to understand the immune capabilities at those life stages are underway. larval immunity the life cycle of most sea urchin species begins with an egg in which fertilization and development are initiated after release into the water column. although there is significant variation among species, the resulting ciliated embryo typically hatches from the fertilization envelope just prior to gastrulation and develops into a simple feeding larva that lives in the plankton and bears little resemblance to the adult form (fig. 1). pluteus larvae are bilateral with an internal calcite skeleton that supports the overall structure of the larva including the gut, the blastocoelar spaces, and the “arms” that extend out from the oral side of the organism. cilia cover the animal but are concentrated in bands at the intersection of the oral and aboral ectoderm (strathmann, 1975). these ciliated bands enable larvae to swim and remain within the plankton near the surface, as well as to sweep food particles into the mouth and down the esophagus. planktonic larvae eat a variety of organisms including bacteria, single celled and multicellular eukaryotes. both the embryos and the larvae live in environments teeming with potential microbial pathogens, including those that are introduced into the gut. consequently, these animals must have mechanisms for protecting themselves against microbial colonization and invasion of the two major potential sites of pathogen entry; the ectodermal surfaces and along the internal surfaces of the gut (reviewed by smith, 2005). in a similar but more recent study than those by metchnikoff, silva (2000) demonstrated that embryonic mesenchyme cells were capable of phagocytosing yeast that had been injected into the blastocoelar cavity. although the identity of these cells was left unclear, possible candidates for embryonic phagocytes include the blastocoelar cells that populate the blastocoelar spaces of the larva and pigment cells that are found within and near the ectoderm (fig. 1) (gibson and burke, 1985, 1987). both of these cell types originate from the secondary mesenchyme cells that are specified from a mesodermal ring in the gastrulating embryo. during gastrulation ~30 pigment cell precursors migrate to positions in the aboral ectoderm (gibson and burke, 1985, 1987), and after differentiation, they are filled with granules containing red pigment and express a set of enzymes required to produce echinochrome (castellani et al., 2003). echinochrome a has been shown to have effective antibacterial properties (service and wardlaw, 1984; see below), suggesting that pigment cells may have protective functions within the larval ectoderm. the blastocoelar cells develop from the secondary mesenchyme cells that migrate from the tip of the extending archenteron at late gastrulation (tamboline and burke, 1992). approximately 20 of these cells take up residence in the blastocoel, form long pseudopodia, and are particularly concentrated around the gut (fig. 1). recently, both of these cell types have been shown to be capable of bacterial recognition and phagocytosis (hibino and rast, unpublished). the combination of these two cell types thus may ‘patrol and protect’ the larval ectoderm and gut from microbial colonization and invasion. commonly, phagocytosis either requires, or is significantly augmented by, molecular interactions between the phagocyte membrane and foreign particles. this interaction may be mediated by either an opsonin deposited on the foreign particle for which the phagocyte has a receptor, or by the presence of a receptor on the phagocyte that directly recognizes a microbial molecular pattern. no information is available as to whether opsonins or specific receptors are required for microbial phagocytosis by blastocoelar cells or degranulation of echinochrome by pigment cells. however, silva (2000) suggested that contact with blastocoelar fluid might be required for phagocytosis of yeast by cells in the embryos and larvae of the sea urchin, lytechinus variagatus. it is therefore interesting that two homologues of sea urchin complement c3 (spc3 and spc3-2; see below for more details) are expressed in the embryos and larvae. furthermore, the gene encoding spc3 shows low expression in embryos, but is elevated in response to contact with bacteria just prior to gastrulation (shah et al., 2003). spc3 is known to function as an opsonin in adult sea urchins (clow et al., 2004), and may have a similar function in the embryos and larvae. the second sea urchin c3 homologue, spc3-2, is not well characterized but is expressed at higher levels in the gastrulating embryo and larvae, whereas the transcripts are much less prevalent in adult coelomocytes (rast, unpublished). because spc3 is highly expressed in coelomocytes and spc3-2 is more highly expressed in embryos and larvae, these two c3 homologues may represent the core of two alternative complement pathways that operate at two different developmental stages of the sea urchin (see below). metamorphosis and adult coelomocytes the larvae of indirect-developing sea urchins feed in the plankton for weeks to months in a developmental process, which serves, disperse the larvae far distances from their benthic parents. after a period of feeding, the adult rudiment forms within left side of the larva (okazaki, 1975) and upon full development, the larvae leave the plankton and metamorphose if they settle on an appropriate substrate. the adult rudiment is everted into 27 fig. 1 cells with immune function in the sea urchin pluteus larva. blastocoelar cells with pseudopodia (some marked with white asterisks) occupy the blastocoel and surround the gut. pigment cells (some marked with red asterisks) are usually in close apposition to the aboral ectoderm. a single pigment cell with vesicles is shown among cells of the aboral ectoderm (left inset). the same pigment cell is shown in outline in the right inset. subdivisions of the gut are indicated for orientation. f, foregut; m midgut; h, hindgut. the spicular skeleton is birefringent in the preparation. a juvenile, leaving a “turban” of larval tissues on the dorsal side of a tiny sea urchin that has five tube feet and five spines. this process transforms the animal from a bilateral larval form to a pentamerous juvenile. the anatomy of the fully metamorphosed sea urchin is essentially the same as the adult with aristotle’s lantern (mouth parts), gut, and gonad being the major organs in the coelomic cavity. filling the spaces between the organs within the coelomic cavity is coelomic fluid that contains coelomocytes. there are a variety of coelomocyte types in s. purpuratus that can be differentiated based on their structural attributes (johnson, 1969; bertheussen and seljelid, 1978; edds, 1993; gross et al., 2000). four categories are currently recognized; phagocytes, red spherule cells, colorless spherule cells and vibratile cells (table 1; fig. 2). the proportions of each type of coelomocyte in the coelomic fluid vary considerably (table 1) at both the interand intra-individual level (smith, unpublished). this variability may reflect the nutritional, immunological and homeostatic status of the individual sea urchin from which the coelomocyte samples were taken (smith and davidson, 1994; reviewed by gross et al., 1999). the phagocytes constitute the majority of coelomocytes and have been reported to be involved in graft rejection, chemotaxis, phagocytosis, encapsulation, immune gene expression, agglutination (gross et al., 2000; clow et al., 2004; reviewed in gross et al., 1999) and clotting reactions (hillier and vacquier, 2003). phagocytes also appear to be the major cell type to express immune genes including complement homologues, a c-type lectin and a homologue of nfkb (fig. 3). phagocytes can be sub-categorized into three types based on morphology. type 1 phagocytes (fig. 2a, b), also known as discoidal cells, can be readily differentiated from type 2 phagocytes (fig. 2c, d), or polygonal cells, by their distinct cytoskeletal morphologies (edds, 1993; henson et al., 1992; 1999). discoidal cells have radially arranged actin filaments emanating from the central nuclear region of the cell, whereas polygonal cells have their actin filaments arranged laterally and lie along the length of the cell membrane forming irregular polygonal shapes (edds, 1993). the discoidal cells are stationary in vitro, while the polygonal cells show greater motility (henson et al., 1992) and can translocate across glass like fibroblasts when in contact with coelomic fluid proteins (edds et al., 1983). differences in these two cell types also include subcellular localization of kinesin, microtubules, and myosin ii, in addition to positioning of the mitochondria (henson et al., 1999). the possible developmental relationships between these two subtypes of phagocytes have not been described. small 28 phagocytes (fig. 2e), a third type of phagocyte identified by gross et al. (2000), are smaller with less cytoplasm than either of the other two cell types. they have not been reported previously, perhaps because of their small size and low numbers. to date, the function of these cells is unknown. there are two types of spherule cells, those with red spherules, also called morula cells (fig. 2f), and those with colorless spherules (fig. 2g). they are similar in size and are both amoeboid (matranga et al., 2006), however the red spherule cells are significantly more dense than the colorless type, enabling separation by density centrifugation (bertheussen and seljelid, 1978; gross et al., 2000). the red spherules contain echinochrome a, a naphthoquinone which gives the cells their characteristic red color. echinochrome a is degranulated in the presence of bacteria (johnson, 1969) and has antimicrobial properties against both gram positive and gram negative bacteria (service and wardlaw, 1984; gerardi et al., 1990; haug et al., 2002). red spherule cells accumulate around injuries and sites of infection (johnson, 1970; coffaro and hinegardner, 1977), suggesting that these cells and echinochrome a play a role in the immune response in adult sea urchins. the functions of the colorless spherule cells have yet to be identified. vibratile cells (fig. 2h) are spherical and show no amoeboid movement but have a single flagellum, which may propel them through the coelomic fluid. they have been associated with clotting reactions (bertheussen and seljelid, 1978), which is an important response to injury. upon immediate removal from a sea urchin, the phagocytes appear as bladder amoebocytes (bertheussen and seljelid, 1978) or petaloid phagocytes (edds, 1979; 1983), which is a description of lamellipodia that extend from the cell body in all dimensions. this may be the normal morphology of phagocytes while in the coelomic cavity, but upon settling on glass, the petaloid table 1 coelomocyte cell types and functions in the purple sea urchin strongylocentrotus purpuratus. cell type % in coelomic fluid morphology function type 1 discoidal cell dense nucleus with clear disc shaped cytoplasm, devoid of organelles with actin striations radiating from the center (fig. 2a, g) immune function: encapsulation, opsonisation (clow et al., 2004), chemotaxis and phagocytosis (reviewed by gross et al., 2000), graft rejection (reviewed by edds 1993) type 2 polygonal cell clear nucleolus (granular nucleus) and obvious organelles in the surrounding cytoplasm. forms large thin irregular shapes. actin bundles run parallel to straight edges of cell (fig. 2g, h) immune function: chemotaxis, opsonisation (clow et al., 2004), phagocytosis (reviewed by gross et al., 2000), antibacterial activity due to lysozyme (gerardi et al., 1990), clotting and encapsulation, graft rejection (reviewed by edds 1993) type 3 small phagocyte 40-80 % (total phagocytes) (edds 1993, gross et al., 2000 and smith l.c., unpublished) smaller than both type 1 and type 2 with little cytoplasm (fig. 2c) unknown red spherule 7-40 % (gross et al., 2000 and smith l.c., unpublished) amoeboid cells with red spherical inclusions, (fig. 2d) antibacterial activity not due to lysozyme (gerardi et al., 1990) contains echinochrome a (service and wardlaw 1984) colorless spherule 3.7-25 % (gross et al., 2000 and smith l.c., unpublished) colorless amoeboid cells with spherical inclusions; may be several types in other echinoderms (fig. 2e) unknown vibratile (boolootian and geise 1958, johnson 1969, karp and coffaro 1980, smith 1981) 11.9-20 % (gross et al., 2000 and smith l.c., unpublished) round colorless cells with a single flagellum (fig. 2f) movement or agitation of coelomic fluid? associated with clotting (bertheussen and seljelid, 1978) 29 fig. 2 coelomocyte types in the sea urchin, s. purpuratus. phase contrast images of live cells are shown for type 1 discoidal phagocyte (a), type 2 polygonal phagocyte (c), small phagocyte (e), red spherule cell (f), colorless spherule cell (g) and vibratile cell (h). fluorescent images of a type 1 discoidal cell (b) and a type 2 polygonal cell (d) were obtained after settling the cells on poly-l-lysine (0.1 �g/ml) coated coverslips for 5 min in calcium magnesium-free seawater with 30 mm edta, and then in coelomocyte culture media (henson et al., 1999) for 30 min. the cells were fixed and stained with a monoclonal anti-actin antibody (icn) followed by goat anti-mouse ig labeled with fluorescein and counterstained with hoescht 33258 (molecular probes) nuclear stain. all images were collected using a zeiss axioplan fluorescence microscope. scale bar in a and c is 15 µm. scale bar in e, f and g is 12 µm. scale bar in h is 10 µm. lamellipodia transform into discoidal or polygonal morphology and may show ruffling (henson et al., 1992; edds, 1993). this cellular behaviour has been considered akin to encapsulation, because the glass slide is perceived as foreign (edds, 1993). upon exposure to hypotonic media, lamellipodia of both discoidal and polygonal cell types transform into filopodia starting with the formation of serrations at the lamellipodial edge followed by cytoplasmic retraction and filopodial extension (edds, 1980). the filopodia have been described as forming physical links among several phagocytes, or filopodial intertwining, to result in a cellular clot, which is retracted through filopodial shortening (edds, 1977; smith, 1981). a complement system in sea urchins coelomocytes are the central mediators of immune responses in sea urchins. besides graft rejection, encapsulation, and cellular clot formation (smith and davidson, 1994; reviewed by gross et al., 1999), coelomocyte activities include the efficient clearance of a broad array of foreign cells or particles injected into the coelomic cavity, including bacteria (bertheussen, 1981; yui and bayne, 1983; plytycz and seljelid, 1993), xenogeneic cells (reinisch and bang, 1971), latex beads and yeast (bertheussen, 1981), bacteriophage (coffaro, 1979), and red blood cells (kaplan and bertheussen, 1977; bertheussen, 1981; for reviews see smith and davidson, 1994; gross et al., 1999). fig. 3 phagocytes are the major expressors of immune response genes. expression of several immune genes were analyzed in fractions of coelomocytes by reverse transcriptase – pcr. the genes analyzed encode complement proteins spc3 (clow et al., 2000) and spbf (smith et al., 1998; terwilliger et al., 2004), a c-type lectin, sp056, called spechinoidin (genbank accession number; aar02404) and the transcription factor spnfkb (pancer et al., 1999). actin expression was used as the control. phagocytes show the strongest expression of all genes while the red spherule cells also express spnfkb. total rna was isolated from density separated coelomocytes (see gross et al., 2000), reverse transcribed with a random hexamer and employed in pcr with primers specific for the genes listed to the right of the image. lane 1, phagocytes; lane 2, mix of vitratile cells and colorless spherule cells; lane 3, red spherule cells. lanes 2 & 3 contain < 2.1% phagocytes, and may account for the minor bands in those lanes. 30 phagocytosis of sheep rbcs (srbcs) by coelomocytes was not efficient until the srbcs were opsonized with human igm and c5-depleted serum (which blocked the lytic pathway) (kaplan and bertheussen, 1977; bertheussen, 1981) and led to further investigations. using isolated human complement components for controlled srbc opsonization also augmented phagocytosis of srbcs and suggested that both c3b and the inactivated form, c3bi, were involved in phagocytosis (bertheussen, 1982). these results inferred that type 3 complement receptors were present on the phagocytes and lead to the speculation that sea urchins might have a complement system (bertheussen, 1983). molecular identification of sea urchin complement components an early molecular investigation of genes expressed in sea urchin coelomocytes showed that profilin transcripts were up-regulated in response to injections of lipopolysaccharide (lps) into the coelomic cavity (smith et al., 1992, 1995). the implication was that because profilin functions in cytoskeletal modifications (paavilainen et al., 2004), its elevated expression indicated immune activation in coelomocytes as reflected by increased amoeboid movement, phagocytosis and secretion, all of which requires changes in cell shape. consequently profilin was used as a marker for cell activation, and pools of coelomocytes were employed in an expressed sequence tag (est) study to generate a snapshot of gene expression in the coelomocytes responding to lps and to provide a basic understanding of the molecular immunological functions of these cells (smith et al., 1996). this study yielded 307 ests, of which 89 matched to known sequences derived from 55 distinct genes. of those ests, two matched to mammalian complement proteins as had been predicted by bertheussen (1982). spc3 identification est064 was a new member of the thioestercontaining complement protein family that includes c3, c4, and c5 (smith et al., 1996). alignments of the full length sequences showed that the deduced sea urchin protein matched most closely to other c3 proteins and was therefore called spc3 (al-sharif et al., 1998). the spc3 protein contained a conserved α/β cleavage site to produce a mature protein with two chains and a conserved thioester site in the α chain. sequence analysis of spc3 identified n-linked glycosylation sites, cleavage sites for factor i and binding sites for factor h and factor b, leading to speculation that spc3 functioned similarly to other c3 homologues (al-sharif et al., 1998; smith et al., 1999, 2001; smith, 2001). immunoquiescent sea urchins have either undetectable or very low levels of spc3 in the coelomic fluid (gross et al., 1999). upon immune challenge with lps, however, spc3 protein appears in the coelomic fluid within 15 minutes (clow et al., 2000). furthermore, spc3 is produced by a subset of phagocytes that appear to maintain the protein within small cytoplasmic vesicles (fig. 4). in addition to the spc3 secretion response, there is also an increase in number of phagocytes containing spc3 after challenge (gross et al., 2000). spc3 was the first complement component identified in an invertebrate, which galvanized others to search for and to find complement systems in a wide variety of animals. phylogenetic analysis of the thioester-containing protein family placed spc3 basal to the chordate complement proteins and therefore identified it as a homologue of the common ancestor of the c3, c4 and c5 family in deuterostomes (al-sharif et al., 1998). c3 homologues have also been identified in the tunicates, halocynthia roretzi, styela plicata and ciona intestinalis (nonaka and azumi, 1999; marino et al., 2002; raftos et al., 2002) and until recently, only deuterostomes were thought to contain complement-like proteins. however, c3 homologues have been found in the horseshoe crab, carcinoscorpius rotundicauda (zhu et al., 2005) and in the gorgonian coral, swiftia exerta (dishaw et al., 2005), indicating that c3 appeared prior to the protostomedeuterostome split. thioester-containing proteins have also been identified in both drosophila (lagueux et al., 2000) and the mosquito, anopheles gambiae (levashina et al., 2001), but phylogenetic analysis of these proteins indicates that they fall into a different clade than either the complement clade or the α2-macroglobulin clade proteins (blandin and levashina, 2004). fig 4. spc3 is localized in small vesicles in phagocytes. confocal fluorescent micrographs of spc3 localization in the type 1 and type 2 phagocytes of s. purpuratus. density separated phagocytes were centrifuged onto slides, fixed and stained with spc3-anti-peptide antiserum followed by goat anti rabbit ig conjugated to alexa (gárig-a, pierce). images were captured using a bio rad mrc 1024 confocal laser scanning system attached to an olympus imt2-rfc inverted microscope (for details, see gross et al., 2000). scale bar is 15 µm. 31 the identification of thioester containing proteins, which appear to be present throughout the animal kingdom, suggests that this mechanism for recognizing, binding to, and eliminating pathogens is very ancient. spc3 function sequence analysis had indicated that spc3 was a c3 homologue, but it was important to determine whether the function of spc3 was conserved. based on the structure of c3 and c4, the active thioester site is recessed in a pocket and partially protected from deactivation (reviewed in sim and sim, 1983). however, when activated c3 proteins are denatured and heated under alkaline conditions, thioester sites undergo autolysis causing α chain cleavage (sim and sim, 1981, 1983). autolysis does not occur when the thioester has either not been activated or has been deactivated, and therefore the chemical reaction can be used to assess the fraction of proteins containing active thioester sites. alternatively, thioester function can also be demonstrated by binding small nucleophiles such as methyl amine or hydroxylamine. when these approaches were used to analyze spc3, autolysis was found to occur in a fraction of the proteins that varied among individual sea urchins (smith, 2002). furthermore, spc3 bound both methylamine and yeast, which blocked autolysis, and suggested that spc3 could function as an opsonin. consequently, when yeast were opsonized using coelomic fluid from immune activated sea urchins, phagocytosis was augmented (clow et al., 2004). overall, these data indicate that the thioester site is functional on spc3 and that it is a major opsonin in sea urchin coelomic fluid (smith, 2001, 2002). spbf identification factor b (bf), which has been found throughout the deuterostomes (ishiguro et al., 1992; nakao et al., 1998; smith et al., 1998; nonaka and azumi, 1999; azumi et al., 2003), is the second component of the alternative pathway, and the second complement homologue identified in the sea urchin (smith et al., 1996, 1998). bf is a mosaic protein containing several short consensus repeats (scrs), a von willebrand factor (vwf) domain and a serine protease domain. most bf proteins have three scrs, however, exceptions include the carp bf homologue with four scrs (nakao et al., 1998), and the sea urchin bf protein (spbf) with five scrs (smith et al., 1998). the bf protein in the tunicate, c. intestinalis, is quite different and has three scrs and two cub (c1r, u-epidermal growth factor, bone morphogenic protein) domains (azumi et al., 2003). expression of the gene encoding spbf (sp152) was constitutive and not affected by challenge with lps (terwilliger et al., 2004). along with the full-length sp152 transcript containing five scrs, some sp152 transcripts were alternatively spliced into remove the first and/or the fourth scr (terwilliger et al., 2004). however, only those messages that were missing the fourth scr encoded an in-frame protein. messages in which the first scr was deleted had a frame shift which resulted in an early stop codon and a truncated protein. therefore, it appears that in most higher deuterostomes, bf proteins have three scrs, while in lower deuterostomes, including s. purpuratus and the tunicate c. intestinalis, bf proteins have four or five scrs (azumi et al., 2003; terwilliger et al., 2004). it is not clear from the phylogenetic analyses of bf proteins whether the evolutionary process has selected for the loss of scrs in higher deuterostomes from an ancestral protein structure containing five scrs, or if the lower deuterostomes have undergone domain duplication more recently to result in bf proteins with five scrs (terwilliger et al., 2004). complement systems in lower deuterostomes in higher vertebrates, the complement system is composed of about 35 serum and cell surface proteins (volanakis, 1998) and is categorized into three activating pathways, called classical, alternative and lectin, which unite to trigger the terminal pathway (dodds and law, 1998) (fig. 5). in addition, there are a number of regulatory proteins located in both the serum and on cell surfaces, and complement receptors located on many cell types including phagocytes. analysis of the c. intestinalis genome has shown preliminary identification of many complement components in this invertebrate including duplications of proteins in the alternative and lectin pathways, and several matches to c6 in the terminal pathway (azumi et al., 2003; reviewed in nonaka and yoshizaki, 2004). although cloning and sequencing have identified c3 and bf homologues in the sea urchin, searches of the first build of the sea urchin genome (7/18/05 assembly) have uncovered additional gene models that match to complement homologues. these include four members of the c3/4/5 family, three members of the bf/c2 family, mannose binding lectin (mbl), and several matches to c1q (fig. 5) (unpublished). in addition, a number of matches were identified to variant forms of mbl-associated serine proteases (masps) and to proteins with a perforinmembrane attack complex (macpf) domain. with three gene models that encode c3 homologues and three that encode bf homologues, the sea urchin complement system appears as an expansion of the alternative pathway, perhaps with activation through an expanded lectin pathway using mbl, c1q and masps, plus a possible terminal pathway. however, without clear identification of members of the terminal pathway, the major function of this system may be opsonization. a complement system consisting of the alternative and lectin pathways and functioning in opsonization can be of significant value in combating microbial invasion. the alternative pathway in higher vertebrates has been considered to be the core of the complement system, and the current characterization of the sea urchin system emphasizes this notion. the positive feedback loop significantly augments the rate at which foreign particles can be opsonized and is therefore more effective and efficient than individual opsonins such as simple lectins that rely on diffusion to bind to the target. the presence of a c3-convertase in the sea urchin, composed of a complex of spc3 and spbf, and the presence of a positive feedback loop has been suggested previously 32 fig. 5 the complement cascade. the mammalian complement cascade is shown with the known (green circles) and predicted (green striped circles) sea urchin complement proteins mapped onto it. complement proteins known in ciona intestinalis are circled with a bold line. the dotted line represents the positive feedback loop in the alternative pathway. the dashed line suggests the possible activation of masps by c1q homologues in both sea urchins and tunicates. and might be of significant benefit to the species (fig. 5) (smith et al., 1999, 2001; smith, 2001). the importance of the complement system within the immune response of the sea urchin is reflected by multiple alternative pathways that may be activated through mbl or c1q. these results infer the importance of opsonization followed by phagocytosis by coelomocytes for removing and destroying invading microbes. regulatory proteins and receptors in the complement system the amount of functional c3 in body fluids depends on the rate of thioester activation vs. deactivation either by the formation of covalent bonds with a target or by hydrolysis. the thioester site is an intra-chain bond between the side groups of cysteine and a nearby glutamic acid (dodds and law, 1998) and when activated, becomes available for ester or amide bond formation with nearby substrate molecules. normally, if covalent bonds are not formed, the reactive thioester undergoes hydrolysis due to the abundance of surrounding water, thereby limiting the spread of reactive thioester-containing molecules (dodds and law, 1998). the short time frame of thioester reactivity helps to ensure that covalent bond formation occurs near where complement activation was initiated, usually on the surface of a pathogen rather than in the plasma or on host cell surfaces. however, autoactivation of c3 and augmented c3 activation by c3-convertase functioning within the positive feedback loop can lead to large amounts of activated c3 and inappropriate deposition of c3 fragments on host cells. consequently, to protect self tissues from autologous complement attack, a number of regulatory proteins are required both in body fluids and on cell surfaces. many of the regulatory proteins and complement receptors in higher vertebrates are constructed of short consensus repeats (scrs) or complement control protein (ccp) modules. searches of the sea urchin genome reveal 247 proteins with scr domains (7/18/05 assembly). it is feasible that some of these gene models will be characterized in the future to encode complement receptors and regulatory proteins that function to protect self. two examples of expressed genes have been identified that encode proteins with putative complement regulatory function or may be members of a primitive terminal pathway (multerer and smith, 2004). they are spcrl (s. purpuratus complement related protein, long form) and spcrs (short form) and both have multiple scrs and a fimac (factor i-membrane attack complex) domain. both share domains with factor h and factor i, which have regulatory functions, and c6 and c7, which are members of the terminal pathway. other genes expressed in coelomocytes lectins in addition to the complement system, c-type lectins may also be of significant importance in immune defense p c3 c3b c5 c6 c7 c8 c9 c3a c5a c4 masps microbes terminal pathway lectin pathway alternative pathway d c1q c1r, c1s c4b2bc4b c2 c2a c4a c4b2b3b classical pathway antigen antibody c3 c3b c3bbb c3bbb3b c3a bf ba mbl ficolin ? p c3 c3b c5 c6 c7 c8 c9 c3a c5a c4 masps microbes terminal pathway lectin pathway alternative pathway d c1q c1r, c1s c4b2bc4b c2 c2a c4a c4b2b3b classical pathway antigen antibody c3 c3b c3bbb c3bbb3b c3a bf ba mblmbl ficolinficolin ? 33 in the sea urchin. c-type lectins are carbohydrate binding proteins that require ca2+ for proper conformation and function of the carbohydrate recognition domain (crd). an est that matched to a c-type lectin, shows significant sequence similarity to echinoidin (smith et al., 1998), which was previously characterized in the sea urchin, anthocidaris crassispina (giga et al., 1987). spechinoidin (echinoidin from s. purpuratus; genbank accession ay336600) has six cysteines in conserved positions and a seventh that suggests that it may form a homodimer (smith et al., unpublished). the carbohydrate binding motif composed of three amino acids within the crd, predicts that spechinoidin may bind galactose or its derivatives. the gene encoding spechinoidin, sp056, is expressed exclusively in the phagocyte class of coelomocytes and only after immune challenge (multerer and smith, 2004; terwilliger et al., 2004; smith et al., unpublished). analyses of small lectins in the coelomic fluid of s. purpuratus suggest that there is vast array that have differing carbohydrate binding specificities and are expressed in response to immune challenge (smith et al., unpublished). this is in agreement with searches of the sea urchin genome (7/18/05 assembly) in which a large number of gene models matched to single domain c-type lectins and have motifs for binding an array of carbohydrates. srcrs coelomocytes express a large, complex family of transcripts that contain domains called scavenger receptor cysteine-rich (srcr) repeats (pancer et al., 1999; pancer, 2000) and comprise a protein superfamily in metazoans. srcr domains are 110 amino acids in length and exhibit a conserved spacing of six to eight cysteine residues, which is important for intradomain disulfide bonds (freeman et al., 1990). srcr-containing proteins have been identified in a wide variety of animals (sarrias et al., 2004) and in invertebrates, have been shown to function as an aggregation receptor in a marine sponge (blumbach et al., 1998), or a sperm activation receptor in a sea urchin (dangott et al., 1989). in vertebrates, however, srcrcontaining proteins play a critical role in the regulation and development of the immune response. srcrs are expressed in both hematopoietic and non-hematopoietic vertebrate immune cells (sarrias et al., 2004), and down-regulate antigen receptor signalling on t and b cells (perez-villar et al., 1999), down-regulate host response to endotoxin (trahey and weissman, 1999), regulate apoptosis (miyazaki et al., 1999), inhibit endocytosis (takito et al., 1996), and bind pulmonary lectins (holmskov et al., 1999). relevant functions of srcr proteins within innate immunity include binding repetitive polyanionic structures such as modified lipoproteins, bacterial lipids and certain nucleotide aggregates (sarrias et al., 2004). in the sea urchin, a diverse set of srcr-containing transcripts was isolated from coelomocytes, revealing six types of srcr molecules (pancer et al., 1999; pancer, 2000). the proteins have between 2 and 20 srcr domains plus a variety of other domains, including a von willebrand factor (vwf) domain, epidermal growth factor (egf) domains, scrs, and an extracellular-matrix-like domain (ecm). the srcr transcripts are derived from a large gene family in the sea urchin and genome blots revealed complex hybridization patterns suggesting that srcr genes may exist in multiple forms and may be clustered. furthermore, the diversity of srcr genes is high both within individuals and within the population, which is supported by a preliminary analysis of the sea urchin genome (7/18/05 assembly), which indicates that there are 228 srcr gene models present. the srcr transcript repertoire of individual sea urchins is highly transient in unchallenged animals and is greatly altered after injury and bacterial or fungal challenge (pancer, 2000). unchallenged animals display changes in srcr expression of 20to 30-fold over a period of three months. animals challenged with pathogens also display variations in transcript expression of this magnitude, although there is no specific expression pattern observed among the animals. although the regulation of these genes is unknown, a high degree of conservation in the 5’ flanking region suggested possible coordinated regulation of srcr gene transcription. many of the srcr-containing genes in the sea urchin may have immunological relevance, although the specific functions of the proteins encoded by the individual genes are unknown. toll receptors and transcription factors animal toll receptors are characterized by extracellular leucine rich repeats (lrr), which function to recognize molecular pathogen signatures (pasare and medzhitov, 2005), and cytoplasmic tir (toll-il1 receptor) domains, which are important for signalling. drosophila has eight toll genes and humans have eleven, however, plant genomes have hundreds of r genes which have lrrs. similar to the plant system, the purple sea urchin has a very large array of predicted toll genes (pancer and cooper, 2006). although expression of most of these genes is currently unknown, a few are expressed in coelomocytes (see genbank accessions aak25761, aak25762). in correlation with the expansion of the toll genes in the sea urchin genome, there is a moderate expansion of genes encoding proteins that initiate the signalling pathways that are activated by toll (rast, unpublished). these include myd88 homologues, which are cytoplasmic proteins that interact with the tir domains. the end of the toll signalling pathway results in the release of rel transcription factors from their cytoplasmic inhibitors, followed by their translocation into the nucleus. spnfkb (s. purpuratus nuclear factor kappa b) was the first rel protein described in the sea urchin and is homologous to drosophila relish and vertebrate p105 and p65 proteins (pancer et al., 1999). other rel proteins have been identified in the sea urchin genome including a second nfkb homologue and one nfat homologue (unpublished). spnfkb and sprunt expression in non-activated coelomocytes is undetectable, but both transcription factors are highly expressed 6 to 12 hrs after bacterial challenge or injury (pancer et al., 1999). on the other hand, spgatac has 34 the opposite expression pattern in coelomocytes. its expression in non-activated coelomocytes is downregulated in response to bacteria and injury. based on the expression patterns in coelomocytes, spnfkb may be transcription activator for immune genes while spgatac may be a repressor (pancer et al., 1999). in embryos, sprunt expression is associated with proliferating cells (robertson et al., 2002) and consequently in coelomocytes it may be involved in proliferative responses to immune challenge. other lps-responsive ests in an effort to identify transcripts that appear in coelomocytes in response to lps challenge, an est study employed probes produced from subtractive suppressive hybridization using coelomocyte mrna from immunoquiescent sea urchins and the same sea urchins after lps challenge (nair et al., 2005). screens of arrayed coelomocyte cdna libraries identified ~6000 clones (of 92,160 clones in a library) representing putative lps-responsive genes (nair et al., 2005). est analysis of 1247 clones led to the identification of numerous novel genes expressed in s. purpuratus coelomocytes during lps challenge and included proteins that function in host defense, as cell surface receptors, signalling molecules, cytoskeletal and cytoskeleton modifying molecules, proteases, rna splicing, protein synthesis, protein processing, protein degradation, cell proliferation, and apoptosis. one of the largest categories of ests identified from both of the est studies (smith et al., 1996; nair et al., 2005) matched to cytoskeletal proteins including α and β-tubulin, dynein heavy chain, kinesin light chain, actin, gelsolin, fascin, and thymosin-β. additional matches include the mena neural variant protein, a receptor for activated protein kinase c (rack), integrin βc, protein tyrosine phosphatase receptor type f, protein tyrosine kinase 9-like protein, rho, rho-gdp dissociation inhibitor, cofilin, avena, and microtubule associated protein. this set of genes, which are upregulated in response to lps and encode both cytoskeletal proteins and cytoskeleton-regulating proteins, suggest that active and extensive remodelling of the cytoskeleton is a direct response of coelomocytes to immune challenge. this is in agreement with the initial identification of profilin up-regulation in coelomocytes responding to lps (smith et al., 1992). changes in nuclear activities were inferred from matches to proteins involved in dna transcription and mrna splicing. alternative splicing of defense-related transcripts has been observed for three complement components, spbf (terwilliger et al., 2004), spcrl and spcrs (multerer and smith, 2004), and the number of est matches with putative splicing function suggests that alternative splicing may be common. increases in messages encoding proteins involved in the synthesis, processing and degradation of proteins corresponds with coelomocyte responses to immune challenge. furthermore, matches to proteins that function in sorting (e.g. a p24 homologue which regulates vesicular traffic between the er and the golgi apparatus) and vesicular trafficking within the endosomal system (e.g. rab5interacting protein and the mannose-6-phosphate receptor homologues). indeed, previous studies have inferred that spc3 is synthesized and transported in cytoplasmic vesicles prior to secretion (clow et al., 2000; gross et al., 2000; smith, 2001). these results suggest that coelomocytes are involved in a significant level of protein production, transport and secretion beyond what is known about the complement components. est matches also indicate that coelomocytes express genes encoding proteins involved in stimulating cell proliferation (e.g. a polo-like kinase and allograft inflammatory factor), while other genes encode proteins that inhibit apoptosis (e.g. bax inhibitor-1). the activities of both these groups of proteins (i.e. proliferative and antiapoptotic) may serve to enhance coelomocyte numbers during immune challenges. such changes have been noted as increases in the numbers of cells expressing spc3 that appear in the coelomic fluid after immune challenge (clow et al., 2000). this suggests changes in cell numbers may be due to proliferation or reduced turnover rather than the release of emarginated coelomocytes into the coelomic fluid. aggressive clotting reactions are essential for protecting sea urchins from injuries and damage to the body wall because these animals have a ridged skeleton, or test, that cannot be contracted to close wounds. clotting of the coelomic fluid is a response to injuries and is a complex reaction involving both coelomocytes and coelomic fluid components. a recent study characterizing the clotting reaction in s. purpuratus showed that the coelomic fluid protein amassin is a prime mediator of the clotting reaction (hillier and vacquier, 2003) by employing disulfide bond formation; bertheussen and seljelid, 1978). this protein, which was also found to be expressed by lps-activated coelomocytes (nair et al., 2005), contains an olfactomedin domain and mediates the intercellular adhesion of coelomocytes during cellular clot formation. current analysis of the sea urchin genome (7/18/05 assembly) reveals four additional gene models with olfactomedin domains, although their functions are unknown (unpublished). although the cell surface receptor for amassin has not been identified, it may function in clotting by binding to integrins. it is noteworthy that a match to integrin βc was identified (nair et al., 2005), which was reported previously from sea urchin embryos (murray et al., 2000; burke et al., 2004). both the βc and βl integrins can be detected serologically on the surface of coelomocytes (r burke, personal communication), and a number of integrins have been identified in the sea urchin genome (unpublished). the 185/333 gene family message structure screens of the bacterially activated arrayed cdna library using subtracted probes showed that about 60 % of the positive clones matched to an unknown sequence that encoded a family of proteins (nair et al., 2005). re-screens showed that approximately 6.5 % of 35 fig. 6 full-length sequences from 81 cdna clones from the arrayed library were manually aligned and gaps (represented by horizontal lines) were inserted to optimize the alignment. the gaps separated blocks of sequence or elements, which are represented by colored boxes (numbered at the top). the figure shows a representative set of element patterns, which were established on the presence and type of element 15 (shown as varying sizes). an example of each group of cdna is represented here. group 1 is defined by element 15a (a2), group 2 by element 15b (b4), group 3 by element 15c (c1), group 4 by element 15d (d1), group 5 by element 15e (e2), and groups 6 (01) and 7 (not shown) do not have element 15. element 25 was divided into three sub-elements, 25a, b and c, based on the location of the stop codon (black vertical lines). the deduced protein shows little secondary structure. it is separated into a glycine-rich region (dotted horizontal line) and histidine-rich region (solid horizontal line). symbols indicate the presence of an rgd motif in element 7 (black star); conserved n-linked glycosylation sites (red circles) and o-link glycosylation sites (black circle); five types of repeats shown as numbered colored ovals (type 1 = red; type 2 = blue, type 3 = green; type 4 = purple; type 5 = yellow); secondary structure predictions (either á-helices or âstrands); short stretches of acidic amino acids (red vertical bars); histidine patches (purple vertical bars); and the five elements which are surrounded by putative cryptic splice signals. the scale bar is located at the lower right. (modified from terwilliger et al., 2006). the clones in the bacterially activated coelomocyte library were positive, compared to only 0.0087 % of the clones in the non-activated coelomocyte cdna library. this was in agreement with northern blots that revealed significant up-regulation in expression of this message in response to bacterial challenge (rast et al., 2000). originally identified as est333 (smith et al., 1996), later called dd185 (rast et al., 2000), and now referred to as 185/333 (nair et al., 2005), these transcripts are unusual because they display an elevated level of sequence diversity that is unexpected for an invertebrate. comparisons among 81 cdnas identified 67 different sequences encoding 64 different proteins (terwilliger et al., 2006) (fig. 6). the variability in the messages is based on the presence or absence of 25 blocks of sequence called elements, which resulted in 22 different element patterns (some of those patterns are shown in fig. 6) (terwilliger et al., 2006). besides the variations in element patterns, the 185/333 cdna sequences also show significant sequence diversity within the elements resulting from non-random single nucleotide polymorphisms (snps) and small insertions and deletions (indels). one effect of the extensive number of snps is to alter the position of the stop codon in element 25 (fig. 6). an initial analysis of sequence diversity for 42 unique ests and subsequent analysis of 81 full length cdnas found that synonymous/non-synonymous (dn/ds) ratios of the leader and first element are under positive selection for diversification, although most of the diversity is located within the first element and not the leader (nair et al., 2005; terwilliger et al., 2006). the 185/333 cdnas can be categorized into seven groups based on the presence or absence and length of element 15 (fig. 6), which is shared by most of the sequences, and has a large number of indels. notably, within these groups, the presence or absence of elements 13-25a (histidine-rich region and part of the terminal region) is identical, suggesting that certain suites of elements tend to appear together (terwilliger et al., 2006). analysis of the messages reveals elevated dn/ds ratios which suggest that the genes are undergoing selection for diversification, possibly as a result of pathogen pressure. initial analyses of 185/333 expression in individual sea urchins responding to bacterial and fungal molecular patterns show that the element patterns in messages present prior to challenge are generally different from those present after challenge (terwilliger et al., unpublished). furthermore, the sequence diversity of the messages present in coelomocytes after challenge is elevated compared to messages isolated prior to challenge. changes in the expression patterns of the various 185/333 transcripts in response to immune challenges suggests that expression 36 may be under some type of transcriptional or posttranscriptional control. the 185/333 proteins the 185/333 proteins are not similar to any known protein and showed little discernible secondary structure. all have a leader, all lacked cysteines, and have three distinct regions; a glycine-rich region, a histidine-rich region and a terminal region (fig. 6) (terwilliger et al., 2006). five types of repeats, 11 histidine patches, and six acidic patches are also present. a number of conserved sites for posttranslational modification are predicted throughout the deduced proteins, the most interesting of which is an rgd motif in element 7 suggesting that these proteins may bind to integrins. recent cytological analyses has shown that the 185/333 proteins are expressed by a subset of phagocytes and that they appear as diverse bands on western blots (brockton, et al.; unpublished). although the functions of the 185/333 proteins are not known, based on the timing of expression of the 185/333 genes in response to immune challenge (rast et al., 2000; nair et al., 2005), they may have an important immunological function. 185/333 gene structure and diversity the numerous element patterns initially identified from the transcripts suggested a significant level of alternative splicing (nair et al., 2005). this was thought to imply long primary transcripts from a very large gene that were spliced to produce all possible mature mrnas, a diversification mechanism similar to that found for dscam transcripts in drosophila (schmucker et al., 2000; watson et al., 2005). however, the two 185/333 genes identified in an early version of the sea urchin genome are not as predicted, and instead have two exons and a single, small intron (terwilliger et al., 2006). the first exon encodes the leader, while the second exon encodes the rest of the open reading frame, i.e., elements 1 through 25 (fig. 6). analysis of genomic dna by quantitative pcr suggests that the 185/333 gene family consisted of ~100 alleles, or 50 loci per genome (terwilliger et al., 2006). analysis of a later sea urchin genome assembly (7/18/05) shows two contigs with two or three 185/333 genes, which are spaced approximately 3 kb apart. based on the estimated number of genes per individual and the diversity levels of these genes, it is reasonable to conclude that the observed 185/333 transcript diversity may result from many small, closely linked genes that are members of a large family. this characteristic of clustered immune genes has been observed repeatedly in both animals and plants, and may be integral to gene diversification. sequences of genes cloned from two individuals show that element patterns mimic those found in the transcripts. the diversity observed from analyses of gene sequences corresponds to transcript diversity with respect to percent of unique nucleotide sequences, single nucleotide and amino acid polymorphisms, and diversity of sequences within sets of genes that have the same element pattern. these data suggest that the immune system of the sea urchin has an unknown mechanism of generating immune diversity in the 185/333 gene family. this is a shift from current paradigms, which assume that innate immunity is mediated by germline encoded proteins that have broad recognition specificities for identifying conserved molecular patterns in large classes of microbes. conclusions as we currently understand the sea urchin immune system, its most important functions appear to be opsonization and phagocytosis. the immune cells in the embryo, larva, and adult that mediate immune functions are amoeboid phagocytes. the adult coelomocytes are found in high concentrations in the coelomic fluid and are present in or on all tissues and organs in the animal. their mobility, phagocytosis and secretion functions are reflected in the large numbers of ests that match to cytoskeletal proteins and proteins involved in cytoskeletal modifications. opsonization to augment phagocytosis (or encapsulation) may be the general molecular response of sea urchins to foreign pathogens. a variety of types of opsonins are present in the sea urchin, including lectins, which bind oligosaccharides, srcrs, which bind many types of molecules including modified lipids, and the thioester-containing proteins of the complement system, which covalently bind to hydroxyl and amine groups. although the functions of the 185/333 proteins are not known, the expression of the genes in response to challenge from bacteria or lps plus their diversity supports the hypothesis that these proteins are somehow involved in coelomocyte interactions with microbes. the arrays of lectins, srcr proteins, 185/333 proteins, in addition to the expanded complement activation pathways that lead to opsonization, demonstrate the molecular diversity that is part of the sea urchin immune system. furthermore, the putative recognition system, as exemplified by the large numbers of tlrs and other llrcontaining proteins in the sea urchin genome, illustrates that both the detection and effector systems appear to be expanded and diverse. it is likely that diversification of innate immune systems in both invertebrates and plants may be driven by pathogen pressure. diversification of innate immune systems is a recent change in the previously accepted paradigm that innate immune systems are undiversified and static. the new paradigm results from recent data on sea urchins reviewed here and from analyses of other invertebrates. the importance of understanding the sea urchin system is based on the phylogenetic position of the echinoderms at the base of the deuterostome lineage and as a sister phylum to the chordates. determining how the sea urchin immune system functions and defining how it might be similar to other invertebrate and vertebrate systems will be of interest with respect to understanding the evolution of immune functions within the deuterostomes. the on-going analysis of the sea urchin genome is expected to provide additional surprises and insights into this invertebrate immune system and to accelerate the rate at which experimental data is 37 generated to test predictions from the genome. initial efforts will be centered on understanding how large families of duplicated genes are generated and maintained, and understanding the underlying mechanisms of innate immune diversification. acknowledgements writing this review was supported by the national science foundation (lcs), the canadian institutes of health research and the national sciences and engineering research council of canada (jpr), and the australian research council (svn). references al-sharif wz, sunyer jo, lambris jd, smith lc. sea urchin coelomocytes specifically express a homologue of the complement component c3. j. immunol. 160: 29832997, 1998. azumi k, de santis r, de tomaso a, rigoutsos i, yoshizaki f, pinto mr et al. genomic analysis of immunity in a urochordate and the emergence of the vertebrate immune system: "waiting for godot". immunogenetics 55: 570-581, 2003. bertheussen k, seljelid r. echinoid phagocytes in vitro. exp. cell res. 111: 401-412, 1978. bertheussen k. endocytosis by echinoid phagocytes in vitro. ii. mechanisms of endocytosis. dev. comp. immunol. 5: 557-564, 1981. bertheussen k. receptors for complement on echinoid phagocytes. ii. purified human complement mediates echinoid phagocytosis. dev. comp. immunol. 6: 635-642, 1982. bertheussen k. complement-like activity in sea urchin coelomic fluid. dev. comp. immunol. 7: 21-31, 1983. blandin s, levashina ea. thioester-containing proteins and insect immunity. mol. immunol. 40: 903-908, 2004. blumbach b, pancer z, diehl-seifert b, steffen r, munkner j, muller i et al. the putative sponge aggregation receptor. isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats. j. cell sci. 111: 2635-2644, 1998. boolootian ra, geise ca. coelomic corpuscles of echinoderms. biol. bull. 15: 53-56, 1958. burke rd, murray g, rise m, wang d. integrins on eggs: the beta c subunit is essential for formation of the cortical actin cytoskeleton in sea urchin eggs. dev. biol. 265: 5360, 2004. castellani c, rast jp, davidson eh. isolation of pigment cell specific genes in the sea urchin embryo by differential macroarray screening. development 130: 4587-4596, 2003. clow la, gross ps, shih cs, smith lc. expression of spc3, the sea urchin complement component, in response to lipopolysaccharide. immunogenetics 51: 1021-1033, 2000. clow la, raftos da, gross ps, smith lc. the sea urchin complement homologue, spc3, functions as an opsonin. j. exp. biol. 207: 2147-2155, 2004. coffaro ka. memory and specificity in the sea urchin lytechinus pictus. doctoral dissertation, university of california, 1979. coffaro ka. memory and specificity in the sea urchin lytechinus pictus. elsevier/north holland biomedical press, new york, usa, 1980. coffaro ka, hinegardner rt. immune response in the sea urchin lytechinus pictus. science 197: 1389-1390, 1977. dangott lj, jordan je, bellet ra, garbers dl. cloning of the mrna for the protein that crosslinks to the egg peptide speract. proc. natl. acad. sci. usa 86: 2128-2132, 1989. dishaw lj, smith sl, bigger ch. characterization of a c3-like cdna in a coral: phylogenetic implications. immunogenetics 57: 535-548, 2005. dodds aw, law sk. the phylogeny and evolution of the thioester bond-containing proteins c3, c4 and alpha 2macroglobulin. immunol. rev. 166: 15-26, 1998. edds kt. dynamic aspects of filopodial formation by reorganization of microfilaments. j. cell. biol. 73: 479-491, 1977. edds kt. isolation and characterization of two forms of a cytoskeleton. j. cell. biol. 83: 109-15, 1979 edds kt. coelomocyte cytoskeletons. interaction with cytochalasin b. exp. cell. res. 130: 371-6, 1980. edds kt. cell biology of echinoid coelomocytes. i. diversity and characterization of cell types. j. invert. biol. 61: 173-178, 1993. freeman m, ashkenas j, rees dj, kingsley dm, copeland ng, jenkins na, krieger m. an ancient, highly conserved family of cysteine-rich protein domains revealed by cloning type i and type ii murine macrophage scavenger receptors. proc. natl. acad. sci. usa 87: 8810-8814, 1990. gerardi p, lassegues m, canicatti c. cellular-distribution of seaurchin antibacterial activity. biol. cell 70: 153-157, 1990. gibson aw, burke rd. the origin of pigment cells in embryos of the sea urchin strongylocentrotus purpuratus. dev. biol. 107: 414-419, 1985. gibson aw, burke rd. migratory and invasive behaviour of pigment cells in normal and animalized sea urchin embryos. exp. cell res. 173: 546-557, 1987. giga y, ikai a, takahashi k. the complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin anthocidaris crassispina. homologies with mammalian and insect lectins. j. biol. chem. 262: 61976203, 1987. gross ps, al-sharif wz, clow la, smith lc. echinoderm immunity and the evolution of the complement system. dev. comp. immunol. 23: 429-442, 1999. gross ps, clow la, smith lc. spc3, the complement homologue from the purple sea urchin, strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes. immunogenetics 51: 1034-1044, 2000. haug t, kjuul ak, styrvold ob, sandsdalen e, olsen om, stensvag k. antibacterial activity in strongylocentrotus droebachiensis (echinoidea), cucumaria frondosa (holothuroidea), and asterias rubens (asteroidea). j. invert. path. 81: 94-102, 2002. henson jh, nesbitt d, wright bd, sholey js. immunolocalization of kinesin in sea urchin coelomocytes: association of kinesin with intracellular organelles. j. cell. sci. 103: 309-320, 1992. henson jh, svitkina tm, burns ar, hughes he, macpartland kj, nazarian r, borisy gg. two components of actinbased retrograde flow in sea urchin coelomocytes. mol. biol. cell 10: 4075-4090, 1999. hildemann wh, dix tg. transplantation reactions of tropical australian echinoderms. transplantation 14: 624-633, 1972. hillier bj, vacquier vd. amassin, an olfactomedin protein, mediates the massive intercellular adhesion of sea urchin coelomocytes. j. cell biol. 160: 597-604, 2003. holmskov u, mollenhauer j, madsen j, vitved l, gronlund j, tornoe i, kliem a, reid kb, poustka a, skjodt k. cloning of gp-340, a putative opsonin receptor for lung surfactant 38 protein d. proc. natl. acad. sci. usa 96: 10794-10799, 1999. ishiguro h, kobayashi k, suzuki m, titani k, tomonaga s, kurosawa y. isolation of a hagfish gene that encodes a complement component. embo j. 11: 829-837, 1992. johnson pt. the coelomic elements of the sea urchins (strongulocentratus) iii. in vitro reaction to bacteria. j. invert. pathol. 13: 42-62, 1969. johnson pt. infection with diatoms and other microorganisms in the sea urchin spines (strongylocentrotus fanciscanus). j. invert. pathol. 16: 268-276, 1970. kaplan g, bertheussen k. the morphology of echinoid phagocytes and mouse peritoneal macrophages during phagocytosis in vitro. scand. j. immunol. 6: 1289-1296, 1977. karp rd, coffaro ka. cellular defense systems of the echinodermata. in: manning ed (ed), phylogeny of immunological memory, elsevier/north holland, amsterdam, the netherlands, pp 257-282, 1980. karp rd, hildemann wh. specific allograft reactivity in the sea star dermasterias imbricata. transplantation 22: 434439, 1976. lagueux m, perrodou e, levashina ea, capovilla m, hoffmann ja. constitutive expression of a complement-like protein in toll and jak gain-of-function mutants of drosophila. proc. natl. acad. sci. usa 97: 11427-11432, 2000. levashina ea, moita lf, blandin s, vriend g, lagueux m, kafatos fc. conserved role of a complement-like protein in phagocytosis revealed by dsrna knockout in cultured cells of the mosquito, anopheles gambiae. cell 104: 709718, 2001. marino r, kimura y, de santis r, lambris jd, pinto mr. complement in urochordates: cloning and characterization of two c3-like genes in the ascidian ciona intestinalis. immunogenetics 53: 1055-1064, 2002. matranga v, pinsino a, celi m, di bella g, natoli a. impacts of uv-b radiation on short-term cultures of sea urchin coelomocytes. mar. biol. 149: 25-34, 2006. metchnikoff e. lectures on the comparative pathology of inflamation: delivered at the pasteur institute in 1891. kegan paul, trench, trubner & co. ltd.,1893. miyazaki t, hirokami y, matsuhashi n, takatsuka h, naito m. increased susceptibility of thymocytes to apoptosis in mice lacking aim, a novel murine macrophage-derived soluble factor belonging to the scavenger receptor cysteine-rich domain superfamily. j. exp. med. 189: 413422, 1999. multerer ka, smith lc. two cdnas from the purple sea urchin, strongylocentrotus purpuratus, encoding mosaic proteins with domains found in factor h, factor i, and complement components c6 and c7. immunogenetics 56: 89-106, 2004. murray g, reed c, marsden m, rise m, wang d, burke rd. the alphabbetac integrin is expressed on the surface of the sea urchin egg and removed at fertilization. dev. biol. 227: 633-647, 2000. nair sv, del valle h, gross ps, terwilliger dp, smith lc. macroarray analysis of coelomocyte gene expression in response to lps in the sea urchin. identification of unexpected immune diversity in an invertebrate. physiol. genomics 22: 33-47, 2005. nakao m, fushitani y, fujiki k, nonaka m, yano t. two diverged complement factor b/c2-like cdna sequences from a teleost, the common carp (cyprinus carpio). j. immunol. 161: 4811-4818, 1998. nonaka m, azumi k. opsonic complement system of the solitary ascidian, halocynthia roretzi. dev. comp. immunol. 23: 421-427, 1999. nonaka m, yoshizaki f. primitive complement system of invertebrates. immunol. rev. 198: 203-215, 2004. okazaki k. normal development to metamorphosis. springer verlag, new york, 1975. paavilainen vo, bertling e, falck s, lappalainen p. regulation of cytoskeletal dynamics by actin-monomer-binding proteins. trend. cell biol. 14: 386-394, 2004. pancer z, rast jp, davidson eh. origins of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes. immunogenetics 49: 773-786, 1999. pancer z. dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin. proc. natl. acad. sci. usa 97: 13156-13161, 2000. pancer z, cooper md. the evolution of adaptive immunity. annu. rev. immunol. 24: 497-518, 2006. pasare c, medzhitov r. toll-like receptors: linking innate and adaptive immunity. adv. exp. med. biol. 560: 11-18, 2005. perez-villar jj, whitney gs, bowen ma, hewgill dh, aruffo aa, kanner sb. cd5 negatively regulates the t-cell antigen receptor signal transduction pathway: involvement of sh2containing phosphotyrosine phosphatase shp-1. mol. cell. biol. 19: 2903-2912, 1999. plytycz b, seljelid r. bacterial clearance by the sea urchin, strongylocentrotus droebachiensis. dev. comp. immunol. 17: 283-289, 1993. raftos da, nair sv, robbins j, newton ra, peters r. a complement component c3-like protein from the tunicate, styela plicata. dev. comp. immunol. 26: 307-312, 2002. rast jp, pancer z, davidson eh. new approaches towards an understanding of deuterostome immunity. curr. top. microbiol. immunol. 248: 3-16, 2000. reinisch cl, bang fb. cell recognition: reactions of the sea star (asterias vulgaris) to the injection of amebocytes of sea urchin (arbacia punctulata). cell. immunol. 2: 496-503, 1971. robertson aj, dickey ce, mccarthy jj, coffman ja. the expression of sprunt during sea urchin embryogenesis. mech. dev. 117: 327-330, 2002. sarrias mr, gronlund j, padilla o, madsen j, holmskov u, lozano f. the scavenger receptor cysteine-rich (srcr) domain: an ancient and highly conserved protein module of the innate immune system. crit. rev. immunol. 24: 1-37, 2004. schmucker d, clemens jc, shu h, worby ca, xiao j, muda m, dixon je, zipursky sl. drosophila dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. cell 101: 671-684, 2000. service m, wardlaw ac. echinochrome-a as a bactericidal substance in the coelomic fluid of echinus esculentus (l). comp. biochem. physiol. 79b: 161-165, 1984. shah m, brown km, smith lc. the gene encoding the sea urchin complement protein, spc3, is expressed in embryos and can be upregulated by bacteria. dev. comp. immunol. 27: 529-538, 2003. silva jr. the onset of phagocytosis and identity in the embryo of lytechinus variegatus. dev. comp. immunol. 24: 733-739, 2000. sim rb, sim e. autolytic fragmentation of complement components c3 and c4 under denaturing conditions, a property shared with alpha 2-macroglobulin. biochem. j. 193: 129-141, 1981. sim rb, sim e. autolytic fragmentation of complement components c3 and c4 and its relationship to covalent binding activity. ann. ny acad. sci. 421: 259-276, 1983. smith vj. the echinoiderms. in: ratcliffe na, rowley af (eds), invertebrate blood cells, academic press, new york, ny, pp 513-562, 1981. smith lc, britten rj, davidson eh. spcoel1: a sea urchin profilin gene expressed specifically in coelomocytes in response to injury. mol. biol. cell 3: 403-414, 1992. 39 smith lc, davidson eh. the echinoid immune system and the phylogenetic occurrence of immune mechanisms in deuterostomes. immunol. today 13: 356-362, 1992. smith lc, davidson eh. the echinoderm immune system. characters shared with vertebrate immune systems and characters arising later in deuterostome phylogeny. ann. ny acad. sci. 712: 213-226, 1994. smith lc, britten rj, davidson eh. lipopolysaccharide activates the sea urchin immune system. dev. comp. immunol. 19: 217-224, 1995. smith lc, chang l, britten rj, davidson eh. sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. j. immunol. 156: 593-602, 1996. smith lc, shih cs, dachenhausen sg. coelomocytes express spbf, a homologue of factor b, the second component in the sea urchin complement system. j. immunol. 161: 6784-6793, 1998. smith lc, azumi k, nonaka m. complement systems in invertebrates. the ancient alternative and lectin pathways. immunopharmacology 42: 107-120, 1999. smith lc. the complement system in sea urchins. adv. exp. med. biol. 484: 363-372, 2001. smith lc, clow la, terwilliger dp. the ancestral complement system in sea urchins. immunol. rev. 180: 16-34, 2001. smith lc. thioester function is conserved in spc3, the sea urchin homologue of the complement component c3. dev. comp. immunol. 26: 603-614, 2002. smith lc. host responses to bacteria: innate immunity in invertebrates, cambridge university press, 2005. strathmann rr. larval feeding in echinoderms. amer. zool. 15: 717-730, 1975. takito j, hikita c, al-awqati q. hensin, a new collecting duct protein involved in the in vitro plasticity of intercalated cell polarity. j. clin. invest. 98: 2324-2331, 1996. tamboline cr, burke rd. secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells. j. exp. zool. 262: 51-60, 1992. terwilliger dp, buckley km, mehta d, moorjani pg, smith lc. unexpected diversity displayed in cdnas expressed by the immune cells of the purple sea urchin, strongylocentrotus purpuratus. physiol. genomics, 2006, in press. terwilliger dp, clow la, gross ps, smith lc. constitutive expression and alternative splicing of the exons encoding scrs in sp152, the sea urchin homologue of complement factor b. implications on the evolution of the bf/c2 gene family. immunogenetics 56: 531-543, 2004. trahey m, weissman il. cyclophilin c-associated protein: a normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo. proc. natl. acad. sci. usa 96: 3006-3011, 1999. volanakis je. overview of the complement system. immunol. med. 20: 9-32, 1998. watson fl, puttmann-holgado r, thomas f, lamar dl, hughes m, kondo m, rebel vi, schmucker d. extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 1874-1878, 2005. yui m, bayne c. echinoderm immunity :bacterial clearance by the sea urchin strongylocentrotus purpuratus. biol. bull. 165: 473-485, 1983. zhu y, thangamani s, ho b, ding jl. the ancient origin of the complement system. embo j. 24: 382-394, 2005. isj099.pdf 75 isj 2: 75-81, 2005 issn 1824-307x short communication sensitivity of the silkworm, bombyx mori l. (lepidoptera: bombycidae) larvae to uvirradiation si faruki, pk kundu department of zoology, university of rajshahi, rajshahi, bangladesh accepted may 31, 2005 abstract the effects of uv-radiation on some commercially relevant traits of three instars viz. 1st, 2nd and 3rd of the two multivoltine strains, nistari-m and urboshi-1 of the silkworm, bombyx mori l. have been investigated. uv-rays reduced the weight of larvae, pupae and adults of both the strains and sexes of b. mori independently of the instar that has been treated. the cocoon weight, shell weight and shell ratios were also reduced due to uv-irradiation. increased larval mortality was recorded at all the doses of uvrays. key words: uv-radiation; bombyx mori l. growth; cocoon characters; larval mortality introduction in sericulture, the growth of various developmental stages of the mulberry silkworm, bombyx mori l. is of paramount importance because the quality of successful cocoon crop depends mostly on a healthy larval growth. radiation studies have been extensively carried out in different insects (calderon et al., 1985; mehta et al., 1990, 1991; islam et al., 1992; faruki and khan, 1993; sharma and dwivedi, 1997; hasan et al., 1998). in silkworms, few attempts have been made to find out the radiation sensitivity on different developmental stages through the use of chemical agents and ionizing radiations (mallik et al., 1968; park and hyun, 1968; subramany and reddy, 1982; tazima, 1983, 1984; ali and ali, 1998). it has been observed that radiosensitivity varies according to species, strain, individual and even at different developmental stages of the individual (tazima, 1978). dose dependent sensitivity of silkworm growth to different forms of ionizing radiations have also been reported by molnar et al. (1964), corresponding author: saiful islam faruki department of zoology, rajshahi university, rajshahi, 6205, bangladesh e-mail: faruki64@yahoo.com murakami and kondo (1964), shankarnarayanan (1982) and singh et al. (1990). moreover, gamma radiation has been used to identify the resistant and less resistant strains of silkworm (hirobe, 1974). the ultraviolet (uv) portion of the spectrum is widely used as germicide (bruce, 1975), in embryological-physiological studies (bodenstein, 1953) and for the surface disinfection of insect eggs from pathogens (guerra, et al. 1968). in bangladesh, some works have been conducted on the effect of gammarays on the eri-silkworm, samia cynthia ricini (rahman et al., 1982; khan and khan, 1991) and b. mori (rahman et al., 1983a, b). unfortunately, no investigation was done on the effect of ultraviolet radiation on the mulberry silkworm, b. mori. keeping in view the importance and feasibility of the use of uv-rays the present investigation was undertaken to evaluate the effect of uv-irradiation on commercially relevant aspects of b. mori. materials and methods the multivoltine strains of the silkworm, b. mori used in the present investigation were nistari-m and urboshi-1. newly-hatched larvae were brushed to wooden rearing trays (40 x 29 x 7.5 cm) and were reared on finely-chopped fresh, tender mulberry (morus 76 alba l.) leaves to get the 2nd and 3rd instar larvae . first instar larvae were exposed to uv-rays just after hatching from eggs, and the 2nd and 3rd instar larvae were irradiated just after completion of their moulting, i.e. no feeding was provided before irradiation. the larvae of all instars were irradiated with 254 nm wavelength of uv-rays at different durations (doses), e.g. 2, 4 and 8 min. a 15w germicidal lamp, ge15t8 measuring 20 x 4 cm was the source of uv radiation, emitting at a wavelength of 254 nm. for irradiation the test insects were kept in 15 cm diameter petri dishes placed on the surface apart 12 cm from the lamp. irradiated larvae were then reared on mulberry leaves in rearing trays up to pupation. a single batch of nonirradiated worms was simultaneously reared as controls on fresh mulberry leaves up to spinning. from the fourth instar onwards entire mulberry leaves were supplied to both irradiated and control groups. food was provided four times a day. three replications, each with 50 larvae, were made for each uv treatment and for controls. the rearing trays were kept in fine-netted cabinets. the weight of larvae was determined at maturity, i. e. one day before spinning. thirty larvae were taken randomly from each treatment and were individually weighed on an electric balance. mature larvae were transferred to bamboo-made mountages for spinning cocoons. after spinning and pupation the cocoons were harvested and stored according to their sexes. the sex was determined by cutting cocoons with a sharp blade and observing the external genitalia. cocoons were then retained for adult emergence. pupal and adult weight were individually recorded. the adult weight was determined after emergence but before coupling. the cocoon characters, i. e. whole cocoon and shell weight, and shell-ratio (%) were also noted. for each character and each treatment 30 males and 30 females were randomly selected. data of all the characters were subjected to analyses of variance. here, the variance ratio f was calculated from the ratio between treatment mean square and residual mean square and the value was compared with the tabulated value for significance. the differences between means were determined by the “student-newman-keuls (snk) test”. the mortality of b. mori larvae was observed up to pupation and data were corrected by abbott’s (1925) formula. all the experiments were conducted at a mean room temperature of 24 ± 2 °c. results and discussion the results on the effect of uv-rays on the weight of mature larvae, pupae and adults are shown in tables 1, 2 and 3. it was found that the weight of larvae decreased with increased radiation doses at all the instars of both the strains of b. mori (p < 0.001 for nistari and p < 0.05 for urboshi) (table 1). it was also observed that the effect of uv-rays was more pronounced at an early stage than an advanced stage. table 1 effect of uv-radiation on the weight (mg) of mature b. mori larvae (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 1926.93 ± 15.03a 1926.93 ± 15.03a 1926.93 ± 15.03a 2 1530.86 ± 19.68b 1599.33 ± 21.74b 1670.00 ± 17.75b 4 1472.10 ± 15.63b 1703.16 ± 20.26b 1656.26 ± 24.75b nistari-m 8 1447.53 ± 20.97b 1584.43 ± 20.68b 1653.86 ± 20.57b (a) 23.91*** (b) 5.55* 0 (control) 1929.36 ± 20.92a 1929.36 ± 20.92a 1929.36 ± 20.92a 2 1735.73 ± 27.16b 1824.53 ± 50.29b 1917.30 ± 18.20a 4 1735.20 ± 45.49b 1726.30 ± 38.23bc 1916.70 ± 26.32a urboshi-1 8 1710.80 ± 37.26b 1693.96 ± 31.01c 1822.76 ± 18.67a (a) 6.92* (b) 6.25* (a) = between doses, (b) = between instars; * p < 0.05, *** p < 0.001 f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). 77 table 2. effect of uv-radiation on the weight (mg) of b. mori pupae (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 808.26 ± 10.59a (914.60 ± 7.57k) 808.26 ± 10.59a (914.60 ± 7.57k) 808.26 ± 10.59a (914.60 ± 7.57k) 2 763.06 ± 9.28b (891.80 ± 6.40k) 745.66 ± 10.28b (895.86 ± 5.44k) 767.30 ± 8.56b (892.10 ± 6.33k) 4 690.73 ± 7.92c (799.26 ± 2.81l) 680.40 ± 6.48c (893.63 ± 4.46k) 732.50 ± 7.44bc (853.26 ± 7.39k) nistari-m 8 645.50 ± 11.33d (744.66 ± 8.17l) 607.23 ± 4.07d (901.10 ± 5.03k) 694.86 ± 6.87c (851.63 ± 8.65k) (a) 36.46*** (2.92ns) (b) 4.19ns (2.78ns) 0 (control) 810.13 ± 10.04a (875.26 ± 9.60k) 810.13 ± 10.04a (875.26 ± 9.60k) 810.13 ± 10.04a (875.26 ± 9.60k) 2 748.10 ± 11.30b (857.20 ±12.12k) 794.90 ± 8.77a (870.26 ± 10.78k) 800.86 ± 8.12a (858.60 ± 11.46k) 4 743.63 ± 10.35ab (849.06 ± 17.60k) 694.20 ± 5.61b (865.96 ± 8.05k) 794.33 ± 8.39a (863.50 ± 13.42k) urboshi-1 8 731.73 ± 8.17ab (783.80 ± 14.52l) 692.70 ± 4.36b (843.66 ± 8.25k) 722.90 ± 4.92b (847.86 ± 9.47k) (a) 6.07* (5.07*) (b) 1.44ns (2.24ns) (a) = between doses, (b) = between instars; * p < 0.05, *** p < 0.001 ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). there was a significant weight difference between the instars of both the strains (p < 0.05). in all the instars of nistari, uv-rays deleteriously reduced the weight of male pupae (p < 0.001) in comparison to controls but produced no effect on the weight of female pupae (table 2). the weight of male and female pupae of urboshi was significantly reduced (p < 0.05). similarly, the adult weight of both the strains and sexes were significantly reduced due to uv-irradiation (p < 0.01 for male and female of nistari, and p < 0.001 and p < 0.01 respectively for male and female of urboshi)(table 3). there was no significant difference regarding weight between the instars of both the sexes of pupae and adults of the two strains. lassota (1966), shigematsu and takeshita (1968) working with gamma-ray and coulon (1969) working with x-ray on b. mori reported that higher doses either on the eggs or the larvae decreased the body weight that corroborates with the present findings. similarly, khan and khan (1991) stated that the growth of the eri-silkworm, s. cynthia ricini was adversely affected when the eggs were irradiated with gamma rays. in the present investigation, significantly increased larval mortality was also recorded at all the instars and strains of b. mori due to uvirradiation (table 4). the cocoon weight of the two strains of b. mori was adversely affected when larvae of different instars were irradiated with uv-rays. the lighter cocoons were recorded at all the doses of uv-rays in both the strains and instars in comparison to controls (table 5). in nistari male cocoon weight was significantly (p < 0.001) reduced whereas both male and female cocoons of urboshi were severely affected (p < 0.05 and p < 0.01 respectively for male and female). the uv-rays produced no adverse effects on the shell weight of b. mori but except the male shells in 3rd instar of the strain nistari-m, the weight was reduced at all the doses of uv-rays in comparison to controls, which was not statistically significant (table 6). similarly, the shell ratios (%) was not affected except in the males of nistari where the shell ratios were significantly reduced (p < 0.001) due to uv-radiation (table 6). singh et al. (1990) also observed reduced cocoon and shell weight in b. mori due to gamma irradiation. similar result was observed by khan and khan (1991) using gamma irradiation against the eggs of s. cynthia ricini. 78 table 3. effect of uv-radiation on the weight (mg) of b. mori adults (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 342.73 ± 4.20a (541.80 ± 5.77k) 342.73 ± 4.20a (541.80 ± 5.77k) 342.73 ± 4.20a (541.80 ± 5.77k) 2 326.80 ± 3.88a (522.30 ± 4.75k) 317.36 ± 4.82ab (557.66 ± 4.38k) 314.33 ± 3.20b (566.73 ± 3.01k) 4 290.83 ± 4.30b (495.53 ± 4.21l) 303.70 ± 2.54b (514.96 ± 3.13l) 308.93 ± 2.66b (551.66 ± 4.67k) nistari-m 8 263.93 ± 3.56c (480.60 ± 4.13l) 265.30 ± 4.91c (490.86 ± 4.77l) 303.63 ± 2.52b (512.93 ± 3.77l) (a) 14.24** (9.99**) (b) 0.97ns (6.22*) 0 (control) 343.50 ± 3.83a (700.06 ± 6.63k) 343.50 ± 3.83a (700.06 ± 6.63k) 343.50 ± 3.83a (700.06 ± 6.63k) 2 329.93 ± 4.72b (639.80 ± 3.93l) 331.36 ± 3.78b (634.16 ± 4.67l) 338.00 ± 3.08ab (640.16 ± 9.13l) 4 321.93 ± 3.09bc (546.76 ± 6.00m) 320.83 ± 3.03c (614.70 ± 4.50l) 330.96 ± 2.98b (602.40 ± 3.17lm) urboshi-1 8 318.53 ± 3.32c (489.10 ± 4.93m) 314.10 ± 2.99c (580.10 ± 5.95l) 310.83 ± 4.08c (574.03 ± 5.78m) (a) 26.18*** (18.56**) (b) 0.69ns (2.59ns) (a) = between doses, (b) = between instars; * p < 0.05, ** p < 0.01, *** p < 0.001 ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). table 4. effect of uv-radiation on the mortality of b. mori larvae corrected (%) mortality / instars f-ratio strains doses (min.) 1st 2nd 3rd 2 2.04 2.04 0.67 4 5.44 4.08 4.75 nistari-m 8 6.12 6.80 6.12 (a) 78.50*** (b) 0.85ns 2 2.71 8.16 2.71 4 3.39 3.39 4.75 urboshi-1 8 6.80 12.24 8.16 (a) 11.84** (b) 2.30ns control mortality of both the strains and all the instars = 2.00%, (a) = between doses, (b) =between instars, ** p < 0.01, *** p < 0.001; ns = not significant. f = variance ratio. 79 table 5. effect of uv-radiation on the cocoon weight (mg) of b. mori (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 886.62 ± 10.73a (1004.93 ± 7.52k) 886.62 ± 10.73a (1004.93 ± 7.52k) 886.62 ± 10.73a (1004.93 ± 7.52k) 2 842.06 ± 9.59b (982.86 ± 6.54k) 823.26 ± 10.34b (982.36 ± 5.72k) 851.96 ± 8.48b (980.26 ± 6.52k) 4 766.49 ± 8.13c (879.86 ± 2.96m) 751.70 ± 7.46c (983.20 ± 4.40k) 815.53 ± 7.62c (941.20 ± 7.28k) nistari-m 8 718.56 ± 11.62d (820.30 ± 8.38m) 679.63 ± 4.12d (978.00 ± 4.96k) 744.02 ± 6.74d (937.80 ± 8.67k) (a) 60.19*** (3.25ns) (b) 5.52* (2.65ns) 0 (control) 955.40 ± 11.32a (1039.40 ± 10.41k) 955.40 ± 11.32a (1039.40 ± 10.41k) 955.40 ± 11.32a (1039.40 ± 10.41k) 2 871.36 ± 11.41a (1001.36 ± 12.62l) 930.63 ± 7.91a (1018.06 ± 12.81k) 931.86 ± 12.80a (1019.60 ± 9.01k) 4 881.56 ± 12.06a (1015.83 ± 17.02kl) 820.00 ± 5.55b (1007.42 ± 8.25kl) 807.26 ± 12.96b (1011.46 ± 13.50k) urboshi-1 8 867.03 ± 8.67a (938.36 ± 14.45m) 812.50 ± 4.28b (983.66 ± 6.04l) 836.40 ± 5.38b (984.36 ± 8.35l) (a) 9.25* (14.12**) (b) 0.20ns (1.49ns) (a) = between doses, (b) = between instars, * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). 80 table 6. effect of uv-radiation on the shells of b. mori (n = 30) (a) = between doses, (b) = between instars, * p < 0.05, *** p < 0.001, ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. shell weight / instars shell ratios (%) / instars 1st 2nd 3rd 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio mean ± se mean ± se mean ± se f-ratio 0 (control) 78.36 ± 1.37 (90.33 ± 1.01) 78.36 ± 1.37 (90.33 ± 1.01) 78.36 ± 1.37 (90.33 ± 1.01) 8.84 ± 0.18 (8.99 ± 0.12) 8.84 ± 0.18 (8.99 ± 0.12) 8.84 ± 0.18 (8.99 ± 0.12) 2 79.00 ± 1.22 (91.06 ± 1.13) 77.60 ± 0.61 (86.56 ± 1.08) 84.66 ± 1.44 (88.16 ± 1.41) 9.38 ± 0.16 (9.26 ± 0.12) 9.43 ± 0.14 (8.81 ± 0.10) 9.94 ± 0.19 (8.99 ± 0.19) 4 75.76 ± 0.81 (80.60 ± 0.78) 71.30 ± 0.65 (89.56 ± 0.42) 83.10 ± 1.11 (87.83 ± 1.07) 9.88 ± 0.12 (9.16 ± 0.08) 9.49 ± 0.11 (9.11 ± 0.06) 10.19 ± 0.14 (9.33 ± 0.13) nistarim 8 73.06 ± 1.42 (75.63 ± 1.46) 72.40 ± 0.53 (76.90 ± 0.61) 79.16 ± 1.18 (86.26 ± 1.29) (a) 2.64ns (4.22ns) (b) 6.98* (0.90ns) 10.18 ± 0.22 (9.22 ± 0.18) 10.65 ± 0.09 (7.86 ± 0.08) 10.64 ± 0.16 (9.20 ± 0.15) (a) 26.80*** (0.73 ns) (b) 2.57ns (2.03ns) 0 (control) 144.90 ± 2.67 (164.13 ± 3.12) 144.90 ± 2.67 (164.13 ± 3.12) 144.90 ± 2.67 (164.13 ± 3.12) 15.16 ± 0.23 (15.79 ± 0.30) 15.17 ± 0.23 (15.79 ± 0.30) 15.17 ± 0.23 (15.79 ± 0.30) 2 123.26 ± 2.61 (144.16 ± 2.50) 135.73 ± 0.69 (147.80 ± 1.52) 131.00 ± 3.81 (161.00 ± 2.60) 14.15 ± 0.33 (14.40 ± 0.26) 14.58 ± 0.13 (14.52 ± 0.22) 14.06 ± 0.32 (15.79 ± 0.22) 4 137.93 ± 3.37 (166.76 ± 2.58) 125.66 ± 1.46 (141.46 ± 1.03) 122.93 ± 3.10 (147.96 ± 2.77) 15.65 ± 0.33 (16.42 ± 0.37) 15.32 ± 0.20 (14.04 ± 0.15) 15.23 ± 0.27 (14.63 ± 0.28) urboshi1 8 135.30 ± 2.79 (154.60 ± 3.02) 119.86 ± 0.62 (140.00 ± 1.94) 113.50 ± 2.41 (136.50 ± 2.97) (a) 4.47ns (2.47ns) (b) 0.90ns (0.95ns) 15.60 ± 0.30 (16.48 ± 0.37) 14.75 ± 0.11 (14.23 ± 0.19) 13.57 ± 0.26 (13.87 ± 0.27) (a) 3.19ns (0.61ns) (b) 1.70ns (1.41ns) 81 hirobe (1974) stated that in silkworms, growth and other quantitative characters are changed by gammairradiation depending upon dose rate, total dosage, developmental stage, temperature, moisture and other environmental conditions. it has been demonstrated that the developmental stages of insects renew their cells and tissues, and a particular stage of these animals determine their radio-sensitivity to ionizing radiation (allotey, 1985). in the present investigation dose dependent sensitivity was observed in the strains of nistari-m and urboshi-1 and in different instars of b. mori. moreover, uv-radiation reduced the relevance of some commercial traits e.g. larval, pupal, adult, cocoon and shell weight, and increased larval mortality in b. mori, which are very much undesirable from the economic point of view. future experiments with an array of doses on various developmental stages of b. mori and ecological factors are greatly to be desired. acknowledgements the authors remain grateful to the chairman, department of zoology, rajshahi university, for providing necessary laboratory facilities. references abbott ws. a method for computing the effectiveness of an insecticide. j. econ. entomol. 18: 265-267, 1925. ali ai, ali a. seasonal effect in nistari (m) silkworm, bombyx mori l. under gamma irradiation of eggs. bull. sericult. res. 9: 43-45, 1998. allotey j. study of radiosensitivity of the immature stages of corcyra cephalonica (stainton)(lepidoptera: galleriidae). insect sci. applic. 6: 621-625, 1985. bodenstein d. embryonic development. in: roder kd (ed), insect physiology, john wiley, new york, pp. 780-822, 1953. bruce wa. effect of uv-radiation on egg hatch of plodia interpunctella (lepidoptera: pyralidae). j. stored prod. res. 11: 243-244, 1975. calderon m, bruce wa, leesch lg. effect of uv-radiation on eggs of tribolium castaneum. phytoparasitica 13: 179244, 1985. coulon m. studies on x-ray damage in the early larvae of bombyx mori l. compt. rend. ser. d. 268: 959-962, 1969. faruki si, khan ar. potency of uv-radiation on carda cautella (walker) (lepidoptera: phycitidae) larvae treated with bacillus thuringiensis var. kurstaki. univ. j. zool., rajshahi univ. 12: 73-79, 1993. guerra aa, ouye mt, bullock hr. effect of ultraviolet irradiation on egg hatch, subsequent larval development, and adult longevity of the tobacco budworm and the bollworm. j. econ. entomol. 61: 541-542, 1968. hasan m, jahan ms, khan ar. effect of uv-radiation on the uzi-fly, exorista sorbillans widemann, an endoparasitoid of the silkworm, bombyx mori l. insect sci. appl. 18: 8791, 1998. hirobe t. utilization of gamma-ray effects in the field of silkworm breeding. indian j. genet. 34a. proc. 2nd general congress sabrao, new delhi, 1973, 224-228, 1974. islam s, mannan ma, begum m, afreen ks, saha ak. a preliminary report on the effects of ultra-violet radiation on fecundity and fertility of culex pipiens fatigans wiedemann (diptera: culicidae). j. asiatic soc. bangladesh (sci.) 18: 57-63, 1992. khan ar, khan sh. growth and development of the erisilkworm, samia cynthia ricini (boisd.) (lepidoptera: saturnidae) irradiated on the eggs with gamma rays. bull. sericult. res. 2: 91-94, 1991. lassota z. intestinal damage and water imbalance in gamma irradiation larvae of bombyx mori l. bull. acad. pol. sci. biol. 14: 293-296, 1966. mallik mu, hossain mm, mollah sa. preliminary study of the stimulating effect of low dose gamma-radiation on the larvae of silkworm, bombyx mori l. nucl. sci. appl. 4: 710, 1968. mehta va, sethi gr, garg ak. development of tribolium castaneum (herbst) larvae after gamma irradiation of eggs. j. nucl. agric. biol. 19: 54-57, 1990. mehta va, sethi gr, garg ak. gamma irradiation of pupae and adults of tribolium castaneum (herbst). j. nucl. agric. biol. 19: 184-188, 1991. molnar a, gubicza a, babos l. a study of silkworms from the eggs of bombyx mori l. irradiated with gamma rays. ann. biol. tihany 31: 50-54, 1964. murakami a, kondo. relative biological effectiveness of 14 mc v-neutrons to gamma rays for inducting mutations in silkworm. gonia japan. j. genet. 39: 102-114, 1964. park kw, hyun js. preliminary study on the biological effects of gamma rays on the silkworms, bombyx mori l. j. nucl. sci. 8: 19-25, 1968. rahman s, khan ar, hoque a. effect of gamma radiation on the oviposition of the silkworm, bombyx mori l. strain – nistari (non-spotted). j. asiatic soc. bangladesh (sci.) 9: 129-130, 1983a. rahman s, khan ar, hoque a. effect of gamma radiation on the filament length of the cocoons of the silkworm, bombyx mori l. bangladesh j. zool. 11: 42-44, 1983b. rahman s, khan ar, jalil a. effect of radiation on the erisilkworm, philosamia ricini boisd. (lepidoptera: saturnidae). j. asiatic soc. bangladesh (sci.) 8: 59-62, 1982. shankarnarayanan k. genetic effects of ionizing radiation in multicellular eukaryotes and the assessment of genetic variation hazards in man. elsevier biomedical press, amsterdam, pp. 83-85, 1982. sharma mk, dwivedi sc. investigation on the effects of ultraviolet and infra-red light on the life cycle of callosobruchus chinensis linn. j. advan. zool. 18: 27-31, 1997. shigematsu h. takeshita h. formation of silk protein by he silkworm, bombyx mori l. after gamma-ray irradiation in the embryonic stage. j. insect physiol. 14: 1013-1024, 1968. singh r, nagaraju j, vijayaraghavan k, premalatha v. radiation sensitivity of the silkworm bombyx mori. indian j. seric. 29: 1-7, 1990. subramany g, reddy sg. isolation of a mutant line with shorter larval duration by induction of mutation in the silkworm, bombyx mori l. indian j. exp. biol. 20: 139-141, 1982. tazima y. radiation mutagenesis of the silkworm. in: tazima y (ed), the silkworm – an important laboratory tool, kodensha ltd., tokyo, japan, pp 213-245, 1978. tazima y. environmental mutagenesis: a view from the study of the silkworm. in: proc. xv int. cong. genet., new delhi, pp. 43-52, 1983. tazima y. effect of dose rate and fractionated delivery of ionizing radiation on mutation induction in silkworm spermatogenesis. in: tazima et al. (eds), problems of threshold in chemical mutagenesis, the environmental mutagen society of japan, pp. 169-173, 1984 technical report isj 5: 192-215, 2008 issn 1824-307x technical report immune-neuroendocrine biology of invertebrates: a collection of methods l ballarin1*, m cammarata2*, f cima1*, a grimaldi3*, s lorenzon4*, d malagoli5*, e ottaviani5* 1department of biology, university of padova, padova, italy 2marine immunobiology laboratory, department of animal biology, university of palermo, italy 3department of structural and functional biology, university of insubria, varese, italy 4department of biological oceanography, national institute of oceanography and experimental geophysics, trieste, italy 5department of animal biology, university of modena and reggio emilia, modena, italy *the authors are inserted in alphabetical order accepted december 5, 2008 abstract in the last decade there has been a considerable increase of interest towards the elucidation of several aspects of invertebrate biology, including immunity and neuroendocrinology. however, due to the difficulties connected to the great variety of morphology and adaptations displayed by invertebrates, and also in consideration of the number of techniques that are applied in the various laboratories, research on invertebrates still suffers from hampering that have been substantially overcome in vertebrate models, especially in mammals. the aim of this technical report is to provide the reader a useful list of well-established morphological and morpho-functional protocols in order to facilitate the design and make more homogeneous the realization of experiments in the field of invertebrate immune-neuroendocrinology. key words: morphology; laboratory techniques; immunity; neuroendocrinology contents • introduction • collection of the hemolymph/immunocytes • morphological methods • morpho-functional methods • quantitative methods • concluding remarks • references introduction in the last decade the literature in the field of the invertebrate immune-neuroendocrine biology has been widely increasing and important contributions have allowed a better understanding of the mechanisms and the strategies of defenses set up from the species of different taxa. however, many ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it problems remain unsolved in the field of comparative invertebrate immunology, such as the classification of circulating cells (ribeiro and bréhelin, 2006), the evolution of soluble mediators as the cytokines (gerber et al., 2007; malagoli and ottaviani, 2007), the presence of memory (bréhelin and roch, 2008) and the characterization of immune-related stem cells (bachère et al., 2004; söderhall et al., 2005) among others. in the insect drosophila melanogaster (lemaitre and hoffmann, 2007) and the nematode caenorhabditis elegans (alper et al., 2008) several steps have been taken towards a better comprehension of the molecular mechanisms of immune response, but comparative immunologists well know that two organisms, even if carefully and deeply characterized, cannot be considered as the paradigmatic representation of the million of species widespread along the protostomian and deuterostomian lineages of invertebrates. among comparative immunologists, it is commonly accepted that the difficulties in extending the results obtained in a given invertebrate species to others invertebrate taxa are not only the direct 192 consequence of the intrinsic difficulties connected to the great variety of morphology and adaptations displayed, but also to the variety of techniques that are applied and that often lead to different results in the various laboratories (hooper et al., 2007). indeed, the majority of the utilized techniques derive from those developed for investigations in vertebrate models and laboratories moving their first steps in the field of comparative immunology may encounter problems in adapting available protocols to their models. remarkably, molecular biology approaches are giving an important contribution in supporting/clarifying the data collected by mean of morphological and functional investigations. notwithstanding that, morphological and functional assays obviously remain the firstchoice approaches to collect those evidences that could eventually direct future molecular biologybased experiments. in this technical report we collected several well-established morphological and morphofunctional protocols used in the field of invertebrate immune-neuroendocrinology. this report has not the ambition to represent a complete guide for all the possible experiments that can be performed with invertebrate models. our aim is rather to provide the reader a useful guide in order to facilitate and unify the design of future studies. besides this, we also hope to offer a brief collection of methods that should be applied when studying invertebrate immune-neuroendocrinology. in particular, we will refer to protostomian and deuterostomian invertebrates, such as mollusca (planorbarius corneus, viviparus ater, lymnaea stagnalis, mytilus galloprovincialis and mytilus edulis), annelida (eisenia foetida and hirudo medicinalis), crustacea (astacus leptodactylus, homarus americanus, neprops norvegicus, munida rugosa, paguristes oculatus, palaemon elegans and squilla mantis), insecta (calliphora vomitoria and galleria mellonella, cell lines from estigmene acraea, lymantria dispar and mamestra brassicae) and tunicata (botryllus schlosseri, ciona intestinalis, phallusia mamillata, styela plicata). collection of the hemolymph mollusca in gastropods the hemolymph is obtained by exerting slight pressure on the animal’s foot and collected by a pasteur pipette. in bivalves, e.g., mytilus spp. the classical protocols indicate that hemolymph can be taken from the posterior adductor muscle using a 2 ml syringe (ottaviani, 1983; ottaviani et al., 1997b). recently, we have also proposed a protocol for m. galloprovincialis in which the hemolymph is collected from mussels directly by inserting a sterile syringe between the valves, just beneath the exit-point of the byssus. after insertion, hemolymph can be obtained by exerting a gentle aspiration. this method is useful when large amount of fluid is needed, but it is of extreme importance to check for the quality of the hemolymph immediately after the withdrawal, in order to ensure that neither the gonads nor the digestive gland have been touched or damaged by the needle (malagoli et al., 2007). annelida h. medicinalis immune cells, which are entrapped in a thick connective tissue, can be isolated and cultured utilizing the injection of matrigel matrix gel (mg) supplemented with different growth factor/cytokines selected among those that play a major role in invertebrate/vertebrate wound healing (grimaldi et al., 2008). mg is an extract of the murine engelbreth-holm-swarm (ehs) tumor grown in c57/b16 mice and produced as described by kleinman (1986). it is rich in basement membrane components (laminin, collagen iv, nidogen and perlecan) and it is a thermo-sensitive material liquid at 4 °c which polymerizes when warmed to room temperature (rt). leeches are injected with 300 μl of mg added either with 50 ng of monocyte chemoactractant protein-1 (mcp-1/ccl2, pepro tech, london, uk), with a pivotal role in the recruitment of monocytes and macrophages, or with 50 ng of vascular endothelial growth factor (vegf, pepro tech), playing a pivotal role in the recruitment of hematopoietic and endothelial precursor cells. for in vivo analysis, anesthetized leeches are dissected and polymerized matrigel pellets are removed and histologically, ultrastructurally examined (grimaldi et al., 2008). for in vitro analysis, each matrigel pellet is minced in small pieces using sterilized razor blades and plated in wells of 60 μm in diameter in dulbecco's modified eagle's medium (dmem, celbio, milan, italy) modified by dilution (1:4) to reach iso-osmolarity and supplemented with 1 % glutamine and 10 % fetal bovine serum (grimaldi et al., 2008). the coelomocyte collection from the earthworm e. foetida is performed putting the animals in a sterile plastic petri dish in cold phosphate buffered saline (pbs-1) (137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, 10 mm nah2po4, ph 7.3) diluted 1:1 with ethanol. after a few sec, the earthworms extrude coelomocytes vigorous though their dorsal pores. then the coelomocytes are transferred to plastic tubes and washed by centrifugation in pbs-1 without alcohol at 500xg for 5 min (cooper et al., 1995). crustacea hemolymph sample is taken from the pericardic sinus of shrimps (p. elegans), hermit crabs (p. oculatus), lobster (h. americanus), norway lobster (n. norvegicus), squat lobster (m. rugosa) crayfish (a. leptodactylus) and mantis shrimps (s. mantis). blood samples are taken from the coxal sinus of crab. in the smallest species 50 µl of hemolymph is collected using a sterile 1 ml syringe fitted with a 25g needle, in the biggest animals up to 1 ml of hemolymph is obtained, depending on of the size of specimens (lorenzon at al., 1999, 2007, 2008; giulianini et al., 2007). insecta in the fly c. vomitoria the hemolymph is taken from a small incision in the ptilinum at the front of the head. by softly squeezing of the abdomen and thorax, a drop of hemolymph is forced out of the incision and collected with a pipette (franchini et al., 1996). the hemolymph in the g. mellonella larvae is collected by piercing with a small needle in one of the first prolegs (wittwer et al., 1999). 193 tunicata in solitary ascidians the tunic is cleaned from epiphytes and sterilized with ethyl alcohol. in s. plicata incurrent siphon is incised and the exuding hemolymph is collected, while in c. intestinalis a syringe is inserted directly into the hearth and the hemolymph is collected. in both cases, hemolymph is put in sterile tubes containing a 5-fold excess of calcium/magnesium-free artificial sea water (asw) (9 mm kcl; 0.15 m nacl; 29 mm na2so4, nahco3, ph 7.4) with 10 mm edta (asw-edta) as anticoagulant (1:9 medium/hemolymph ratio), in ice. after centrifuging at 400xg for 10 min at 4 °c, the hemocytes are washed three times in sterile aswedta. in the colonial ascidian b. schlosseri, blood is collected with a glass micropipette after puncturing, with a fine tungsten needle, the tunic marginal vessel of colonies previously rinsed in 0.38 % nacitrate or 10 mm l-cysteine in filtered seawater (fsw), ph 7.5, to prevent hemocyte clotting. it is then centrifuged at 780xg for 10 min and the pellet is re-suspended in fsw at the final concentration of 6-8x106 cells/ml. the cell concentration is evaluated with a burker’s hemocytometer. sixty μl of hemocyte suspension are placed in the centre of culture chambers, made by gluing teflon rings (15 mm internal diameter, 1 mm thick) on siliconized glass slides (fig. 1a). washed coverslips are laid over the teflon rings, smeared with vaseline and gently pressed down to touch the drop of the cell suspension (ballarin et al., 1994). the culture slides fig. 1 hemocyte culture. a) schematic drawing of a culture chamber with a drop of cell suspension in the center a: upper view; b: side view. b) living b. schlosseri hemocytes adherent to coverslips. bar = 15 µm. fig. 2 cryosection of matrigel supplemented with vegf. the mg sponge is infiltrated by ovoidal and agranular hematopoietic precursor cells stained with may-grünwald and giemsa solution. are kept upside-down for 30 min at rt to allow the cells to settle and adhere to the coverslips (fig. 1b). morphological methods optical and electron microscopy-based methods for characterization of circulating hemocytes (immunocytes) mollusca in freshwater snails and marine bivalves, the immunocytes are obtained in hemolymph drops on glass or by cytocentrifugation (cytospin ii, shandon instruments, uk) of hemolymph samples onto a slide at 500-1000 rpm for 5-10 min. they are observed by phase-contrast microscope and stained with or without fixation for morphological observations (ottaviani, 1983, 1989; ottaviani et al., 1998b). annelida may-grünwald and giemsa differential staining (bio-optica, milan, italy) and the vital dye acetylated low density lipoprotein labeled with 1,1’-dioctadecyl3,3,3’,3’-tetramethyl-indo-carbocyanine perchlorate (dil-ac-ldl) (biomedical technologies inc., ma, usa) are a quick method to characterize leech immunocytes (grimaldi et al., 2008). may-grünwald and giemsa procedure in in vivo experiments mg implant is removed from the animal, embedded in polyfreeze tissue freezing medium (polysciences, eppelheim, germany) and immediately frozen in liquid nitrogen. cryosections (7 μm), obtained with a cryotome, are immerse in 100 % may-grünwald for 4 min and then directly transferred to 4 % giemsa (diluted in tap water) for 4 min. slides are then washed in tap water, air dried, mounted with eukitt mounting medium (electron microscopy science, washington, pa, usa) and subsequently observed under a light microscope (fig. 2). in in vivo experiments immunocytes of leech, extracted from mg and plated in wells, are allowed to air dry onto slides and then they are stained as described above (fig. 3). 194 fig. 3 may-grünwald and giemsa staining micrographs showing cultured hematopoietic precursor cells. dil-ac-ldl procedure in in vivo experiments, injection of 10 µl of 10 µg/ml dil-ac-ldl in pbs-2 buffer (138 mm nacl, 2.7 mm kcl, 4.3 mm na2hpo4, 1.5 mm kh2po4, ph 7.4) is performed at the level of the 80th superficial metamere of the leech, where the mg is subsequently inoculated. after 1 week the mg implant is removed from the animal and quickly frozen. cryosections are mounted in citifluor (citifluor ltd, london, uk) and observed on fluorescence microscope through a rhodamine filter set (excitation/emission filters 550/580 nm) to visualize the dil (fig. 4). in in vitro experiments, leech immunocytes are cultured in dmem containing 10 µg/ml dil-ac-ldl according to the manufacturer’s suggestions and tamaki et al. (2002). after 4 h at rt, cells are washed several times with probe-free medium and directly observed using an inverted-fluorescence microscope (fig. 5). nuclei are colored with 4'-6-diamidino-2phenylindole (dapi) (sigma, st louis, mo, usa) (excitation/emission filters 410/460 nm). utrastructural procedures mg pellets (see above) are fixed for 2 h in 0.1 m cacodylate buffer ph 7.2, containing 2 % glutaraldehyde. specimens are then washed in the same buffer and postfixed for 2 h with 1 % osmic acid in cacodylate buffer, ph 7.2. after standard serial ethanol dehydration, specimens are embedded in an epon-araldite 812 mixture. semithin sections (750 nm) are obtained with an ultratome, stained by conventional methods (crystal violet and basic fuchsin) according to moore et al. (1960), and observed under a light microscope. thin sections (80 nm) are stained by uranyl acetate and lead citrate and observed with a transmission electron microscope (tem) (fig. 6). crustacea to characterize the different classes of blood cells in a. leptodactylus, by means of light and electron microscopy, 200 µl of hemolymph (about 2x105 hemocytes counted by using a bürker’s hemocytometer) are drawn into a sterile plastic 1 ml syringe filled with an equal volume of fixative (2.5 % glutaraldehyde, 1 % paraformaldehyde, 7.5 % saturated picric acid solution in 0.1 m cacodylate buffer, ph 7.4) and, after a fixation for a minimum of 10 min, the hemocytes are pelleted in 1 ml of fixative by 10,300xg centrifugation for 10 min at 20 °c. the pellets obtained are then washed in 0.1 m cacodylate buffer ph 7.4 and post-fixed in 1 % osmium tetroxide in the same buffer, serially dehydrated in ethanol and embedded, via propylene oxide, in embed812/araldite (electron microscopy sciences, fort washington, pa, usa) or, without post-fixation, they are serially dehydrated in ethanol and embedded in lr-white (sigma). for light microscopy, sections (2 µm thick) are collected on slides, baked for 5 min at 80 °c and stained with 0.5 % toluidine blue in 0.1 % carbonate solution at ph 11.1 at the same temperature. for tem, silver/goldcoloured sections are stained with uranyl acetate and lead citrate and observed with a tem philips em 208. for light microscopy, selected areas were observed with an olympus bx50 microscope, and images are acquired with an olympus dp11 photo camera at a resolution of 1712x1368 pixels. for tem, negative plates are digitized with an epson photo perfection scanner at 1200 dpi (optical resolution) and saved as a tagged image format file. all measurements, statistical analyses and photocomposition are performed with image-pro plus 4.5 software (media cybernetics, silver spring, md, usa) (giulianini et al., 2007). insecta for the morphological studies, hemolymph from several c. vomitoria flies are pooled to a volume of 50-100 μl and cytocentrifuged on a slide using a cytocentrifuge cytospin ii (shandon instruments) running at 500 rpm for 5 min. subsequently the immunocytes are stained with may-grünwald and giemsa (franchini et al., 1996). fig. 4 cryosection of matrigel supplemented with vegf. the implanted mg is invaded by precursor cells and identified with dil-ac-ldl. nuclei are stained with dapi (in blue). 195 fig. 5 characterization of cultured immunocytes of leech by dil-ac-ldl uptake. in macrophages the fluorochrome-conjugated probe showed a spotted localization a), while a diffused cytoplasmic localization was evident in the hematopoietic precursor cells b). nuclei are stained with dapi (in blue). tunicata in b. schlosseri, several morphological protocols have been set up for both fixed or living hemocytes. however, it is important to underline the possibility to obtain sub-populations from a pool of circulating hemocytes. blood harvested from large colonies (of about 1000 zooids) is centrifuged at 780xg for 10 min. pelleted hemocytes are re-suspended in 1 ml of fsw (final concentration: 50x106 cells/ml) and layered over a ficoll discontinuous gradient obtained by dissolving ficoll 400 (pharmacia, canada) in fsw to final concentrations of 10, 14.5, 20 and 34 % (modified after michibata et al., 1987) and sequentially overlayering 2 ml of each solution into a 10 ml centrifuge tube. tubes are centrifuged at 400xg for 10 min at 4 °c. blood cell fractions are collected with a glass micropipette from the top of the tube, diluted in an equal volume of fsw, and centrifuged at 780xg for 10 min. pelleted cells are re-suspended in 300 µl of fsw and the concentration of hemocytes is estimated with a bürker’s hemocytometer. the fixation procedures for cytochemical staining of hemocytes (or hemocyte sub-population) is performed as follows. after adhesion of the hemocytes to the coverslips, the debris-containing fsw is discarded and cell monolayers are washed by dipping the coverslips (see above) repeatedly in a large volume (100 ml) of fsw. the best and simplest fixative mixture for cell morphology preservation is represented by a solution of 1 % glutaradehyde (fluka, buchs sg, switzerland) and 1 % saccharose in fsw at 4 °c for 30 min. cells are then washed in pbs-3 (1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4, 0.065 m na2hpo4, ph 7.2) or in the buffers used for the cytochemical assays reported below. for b. schlosseri hemocyte identification and characterization, the following cytochemical staining methods can be used (cima et al., 2001; ballarin and cima, 2005): giemsa dye: fixed hemocytes adhering to coverslips are stained for 10 min with a 10 % giemsa (fluka) aqueous solution and then washed in distilled water and mounted in acquovitrex (carlo erba, milan, italy) on glass slides. with this dye, the nucleus appears blue and cytoplasm light blue or violet, due to metachromasia, under the light microscope. pappenheim’s panoptical stain: fixed hemocytes are stained for 3 min in may-grünwald’s dye (fluka). after washing in distilled water they are stained for 10 min with 5 % giemsa, washed again in distilled water and mounted in acquovitrex (c. erba) (mazzi, 1977; bancroft and gamble, 2002). with this technique, basophilic granules appear blue, neutrophils brown and acidophils dark pink (fig. 7a). ehrlich’s triacid mixture: this mixture is composed of 12 parts of saturated orange g aqueous solution, 8 parts of saturated acid fuchsin aqueous solution, 10 parts of saturated methyl green aqueous solution, 30 parts of distilled water, 18 parts of absolute ethanol and 5 parts of glycerin. fixed hemocytes are stained with this mixture for 15 min, washed in distilled water and mounted. basophilic granules appear light green, neutrophils violet and acidophils coppery red (mazzi, 1977; bancroft and gamble, 2002). toluidine blue staining: fixed hemocytes are stained for 10 s in a filtered aqueous solution of 0.5 % toluidine blue (fluka) and 0.5 % sodium tetraborate, to reveal the presence of mucosubstances and glucosaminoglycans which show pink-violet metachromasia. neutral red dye: after adhesion of hemocytes to coverslips, the fsw of the culture chambers is substituted with 60 µl of 8 mg/ml neutral red solution (merck, darmstadt, germany) in fsw. living hemocytes are directly observed. this dye specifically stains acid compartments (e.g., lysosomes or acid vacuolar contents) of living cells (mazzi, 1977; bancroft and gamble, 2002). sudan black for lipids: after adhesion of hemocytes, coverslips are dipped in 70 % ethanol for 30 s and stained with a saturated solution of sudan black (sigma) in 70 % ethanol for 15 min at 70 °c. cells are then rinsed in 70 % ethanol and washed in distilled water (mazzi, 1977; bancroft and gamble, 2002). black spots reveal the presence of lipids (fig. 7b). 196 periodic acid schiff (pas) reaction for polysaccharides: fixed hemocytes are incubated in 1 % periodic acid for 10 min, rinsed in tap water and stained with schiff’s reagent for 30 min at 37 °c. coverslips are then dipped in a solution of 0.6 % sodium metabisulfite in 0.02 m hcl for 6 min, washed in tap water for 10 min, and then rinsed in distilled water (mazzi, 1977; bancroft and gamble, 2002). positive sites appear primary red (fig. 7c). quinones: living hemocytes are incubated in a 2 mm solution of 3-methyl-2-benzothiazolinone hydrazone chloride (mbth) in fsw, containing 0.4 % dimethylformamide for 5 min, in the presence or in the absence (controls) of the po inhibitor 10 mm na-benzoate (winder and harris, 1991; ballarin et al., 1995). positivity is revealed by a marked red color (fig. 7d). dopa-containing proteins/quinoproteins: after the adhesion to coverslips, hemocytes are fixed as described above, washed in pbs-3 and incubated with a solution of 0.24 mm nitroblue tetrazolium (nb) (sigma) in 2 m potassium glycinate buffer (150 g/l glycine and koh 2 n to ph 10.0) and 20 mm sodium benzoate (flückiger et al., 1995). cells are then washed in pbs-3 and coverslips mounted with acquovitrex (c. erba). positive sites feature a dark blue color (fig. 7e). lectin cytochemistry fixed hemocytes are incubated for 30 min in pbs-3 containing 5 % powdered milk, washed three times for 10 min in pbs-3 and incubated for 60 min in a 50 µg/ml lectin solution in pbs-3 containing 0.1 m cacl2. both fitc-labeled and biotin-conjugated lectins can be used, e.g., ulex europaeus agglutinin-i (uea-i, specific for lfucose), datura stramonium lectin (dsl, specific for n-acetyl-β-d-glucosamine and n-acetyllactosamine), ricinus communis agglutinin-i (rca, recognizing β-d-galactosides), wheat germ agglutinin (wga, specific for n-acetyl-β-dglucosamine), helix pomatia agglutinin (hpa, recognizing n-acetyl-α-d-galactosamine), arachis hypogea agglutinin (pna, specific for galactosyl (β1,3) n-acetyl-galactosamine), concanavalin a (cona, recognising α-d-glucopyranosides and αd-mannopyranosides), vicia villosa agglutinin (vva, specific for n-acetyl-d-galactosamine), narcissus pseudonarcissus agglutinin (npa, specific for α-d-mannosyl carbohydrate residues). incubation with fitc-labeled lectins is followed by extensive washing in pbs-3, and coverslips are mounted with fluorsave (calbiochem, darmstadt, germany) on glass slides, and observed at a magnification of 1,250x with a fluorescence light microscope equipped with a filter block for fitc excitation (450-490 nm; emission at 525 nm). conversely, after incubation with biotin-conjugated lectins, hemocytes are washed in pbs-3, incubated for 30 min in avidin-biotin-peroxidase complex (abc) (vector lab., burlingame, ca, usa) in pbs-3, washed in pbs-3, incubated for 30 min in 0.5 mg/ml 3-3’ diaminobenzidine (dab) (sigma) in pbs-3 containing 0.04 % h2o2, mounted in acquovitrex (c. erba) and observed under microscope. fig. 6 tem micrographs. ultrastructural analysis of matrigel added with different cytokines. monocyte chemoactractant protein-1 (mcp-1/ccl2) a) selectively recruit cells characterized by cytoplasm filled with granules of diverse sizes while vascular endothelial growth factor (vegf) (b) recruits cells characterized by large nuclei with a scarce cytoplasm occupied by organelles and few small granules. 197 fig. 7 b. schlosseri hemocytes. a: fixed macrophage-lke cells (mlc) after pappenheim’s panoptical stain; b: living mlcs (arrowheads) stained with sudan black for lipids; c: fixed mlc positive for pas reaction; d: living morula cells treated with mbth to reveal quinones; e: fixed morula cell showing positivity at the reaction for dopacontaining proteins; f: actin cytoskeleton of fixed spreading phagocytes revealed with fitc-labeled phalloidin; g: tubulin cytoskeleton of fixed spreading phagocytes revealed, in immunofluorescence, with anti-tubulin antibodies; h: senescent living phagocyte with bleb after treatment with fluorescent annexin-v; i, j: fixed hyaline amoebocytes (spreading phagocyte; i) and mlc (j) showing tunel positivity for dna fragmentation; k: comet assay showing dna fragmentation in nuclei of senescent hemocytes; l: fixed hyaline amoebocyte showing positivity for βglucuronidase; m: fixed morula cell positive for phenoloxidase. bar = 10 µm. assay for cytosolic ca2+ sustained increases in cytosolic ca2+ are revealed as dark-blue precipitates by von kossa’s substitution method (callis and bone, 2002). glutaraldehyde-fixed monolayers are immersed in 5 % silver nitrate and exposed to ultraviolet light for 5 min. cells are then rinsed in distilled water, incubated for 2-4 min in a 5 % aqueous sodium thiosulfate solution, washed in distilled water, mounted in acquovitrex (c. erba), and observed under the light microscope. tem samples, represented by selected whole colonies or pellets of fixed hemocytes embedded in small pre-heated agar pieces, are fixed in a solution of 1.5 % glutaraldehyde in 0.2 m cacodylate buffer, ph 7.4, plus 1.6 % nacl for 2 h at 4 °c. specimens are then rinsed in cacodylate buffer containing 1.6 % nacl, post-fixed in 1.5 % oso4 in cacodylate buffer, dehydrated and embedded in epon. oneμm-thick sections are stained with toluidine blue and observed with light microscope. ultrathin sections, briefly stained with uranyl acetate and lead citrate, are examined under a tem at 75 kv. for enzymatic activity or antigen preservation, specimens are fixed by immersion in 4 % paraformaldehyde plus 0.1 % glutaraldehyde in saline buffer (sb) (0.2 m cacodylate buffer, ph 7.4, 1.7 % nacl and 1 % saccharose) for 2 h at 4 °c, and then washed three times in sb without postfixation in oso4. in the case of detection of enzymatic activity, a pre-embedding method is usually employed (cima et al., 2002a). specimens are pre-incubated in 0.1 % triton-x in sb for 10 min and then incubated overnight at rt in the reaction mixtures reported in the “cytoenzymatic assays” section. for each test, control specimens are prepared omitting the specific enzyme substrate. after incubation in the reaction and/or postincubation mixture, specimens are post-fixed in 1 % oso4 in 0.2 m cacodylate buffer, ph 7.4, dehydrated, and embedded in epon for sectioning. ultrathin sections are treated as reported above. in the case of immunocytochemistry, a postembedding method is applied. thin sections of fixed specimens, dehydrated and embedded in catalyzed lr white resin (polysciences, warrington, pa, usa) are collected on gold grids and processed for immunohistochemistry. after immersion in pbs-3 198 plus 10 % normal goat serum (vector lab., burlingame, ca, usa) for 10 min at rt, the grids are incubated in the primary antibody diluted in pbs-3 according to the manufacturer’s instructions overnight at 4 °c. they are then rinsed in pbs-3 and incubated in 10 nm gold-conjugated goat antirabbit igg (british biocell international, cardiff, uk), diluted 1:50, 1:100 in pbs-3 for 1 h at rt. in controls, the primary antibody is omitted. before observation with tem, the grids are also stained with uranyl acetate and lead citrate (fig. 8). scanning electron microscopy (sem) living hemocytes are left to adhere to poly-llysine-coated coverslips. they are then fixed for 30 min at 4 °c in the same fixative mixture for tem and post-fixed in 1 % oso4 in cacodylate buffer for 60 min. dehydration through a graded ethanol series is followed by critical-point drying in liquid co2 with absolute acetone as transitional fluid. cells are then sputtered with gold and observed with a sem. immunocytochemical procedures mollusca, annelida and insecta immunocytochemical assay is carried out on fixed or air-dried molluscan immunocytes in order to detect the presence of adrenocorticotropin hormone (acth) using a human acth polyclonal antibody (pab). after hemolymph cytocentrifugation, the immunocytes are air-dried, immersed in absolute ethanol followed to passages in 95 % ethanol (3 min each). endogenous peroxidase are inhibited by immersion in 0.3 % h202 in methanol for 15 min at rt, then the slides are immersed in running tap water for 10 min and rinsed in pbs-1 (3x5 min). incubate in normal goat serum (1:5) (vector lab.) for 30 min at rt; incubate in primary rabbit antihuman acth (1-24) pab (1:200) (dakopatts, denmark) overnight at 4 °c; wash in pbs-1 (3x10 min); incubate in secondary biotinylated anti-rabbit igg (1:200) (vector lab.) for 30 min at rt; wash in pbs-1 (3x10 min); incubate in abc (1:100) (vector lab.) in pbs-1 for 30 min at rt; wash in pbs-1 (3x10 min); incubate in a 0.025 % solution of dab tetrahydrochloride (sigma) in mcllvaine buffer at ph 5.5 containing 5 μl h202. stop the reaction by washing in tap water for 5 min and then rinse in distilled water; counterstain nuclei with hematoxylin; dehydrate through a graded ethanol series: 70, 80, 95 and 100 % (3 min each); clear in xylene and mount in eukitt (bio-optica, milan, italy). controls of immunocytochemical reaction are performed using the same procedure omitting the primary antibody or by pre-absorbing the primary antibody overnight at 4 °c with the homologous antigen (in excess) (ottaviani et al., 1990). the same immunocytochemical procedure is also applicable to the annelid e. foetida in studying the presence of cytokineand pomc-derived peptide-like molecules in the coelomocytes as well as for the immunocytes of the insect c. vomitoria (cooper et al., 1995; franchini et al., 1996). crustacea immunocytochemistry of neuroendocrine organs immunocytochemistry is performed on thick and semithin sections of the eyestalk of the test species to validate the specificity and cross-reactivity fig. 8 immunocytochemistry at tem: phagocyte with granular content labeled with colloidal gold. bar = 3 µm. of the purified antibody anti-nenchh (anti-n. norvegicus crustacean hyperglycaemic hormone) (giulianini et al., 2002). paraffin immunocytochemistry eyestalks dissected from n. norvegicus, a. leptodactylus, m. rugosa and s. mantis were fixed in bouin’s solution for 24 h, serially dehydrated in ethanol and embedded in paraffin. sections (7 μm thick) are collected on superfrost plus (bio-optica) slides and baked for 30 min at 50 °c. the slides are deparaffinized in xylene, hydrated, microwaved in 6 m urea at 800 w for 1 min, washed in ultra pure water and treated with tyramide amplification (nen, tsa-indirect kit, zavantem, belgium). endogenous peroxidases were blocked with 1 % h2o2 in pbs (sigma) for 15 min. slides are incubated in 10 % normal goat serum in 0.1 m tris-hcl, ph 7.5, 0.15 m nacl buffer containing 0.5 % tsa blocking reagent (tnb) for 1 h at rt. the sections are then incubated overnight at 4 °c with the primary antibody diluted 1:20,000 in tnb; concentrations of pre-immunization normal rabbit serum (nrs) as well as anti-gst (anti-glutathione-s-transferase) antibody are employed for parallel controls. after washing for 20 min in 0.1 m tris-hcl, ph 7.5, 0.15 m nacl buffer containing 0.05 % tween 20, they are incubate for 1 h in hrp-labelled goat anti-rabbit igg (nen) diluted 1:200 in tnb. afterwards, the slides are incubated in biotinyltyramide working solution for 10 min and in streptavidin-hrp diluted 1:100 in tnb for 30 min at rt. finally slides are developed in 50 mm tris-hcl buffer, ph 7.5, containing 0.05 % dab and 0.01 % h202 at dark for 20 min at 4 °c. sections are dehydrated and subsequently mounted in eukitt (bio-optica). resin immunocytochemistry eyestalks of n. norvegicus and p. elegans are fixed in 2 % glutaraldehyde in 0.1 m sodium cacodylate 199 and 0.4 m sucrose at ph 7.6 (and for n. norvegicus only post-fixed in 1 % oso4 in the same buffer), serially dehydrated in ethanol and embedded in lrwhite (sigma). one-μm-sections are collected on superfrost plus (bio-optica) slides and baked for 30 min at 80 °c. the slides are microwaved in 6 m urea at 800 w for 1 min, washed in ultra pure water, and only for n. norvegicus, deosmicated with 3 % h2o2 in pbs (sigma) for 15 min. slides are incubated in 10 % normal goat serum in 0.1 m trishcl, ph 8.3, 0.15 m nacl buffer (tris buffered saline, tbs-1) containing 1 % bovine serum albumin and 0.1 % tween 20 (tbb) for 30 min. sections are then incubated overnight at 4 °c with the primary antibody diluted 1:400 in tbb; concentrations of pre-immunization normal rabbit serum are employed for parallel controls. after washing for 20 min in tbs-1, the sections are treated with goat anti-rabbit igg labelled with 5 nm colloidal gold (british biocell international) diluted 1:200 in tbb. afterwards, the slides are washed in ultra pure water and developed with british biocell silver-enhancing kit (british biocell international) for 20 min at dark and mounted in eukitt (bio-optica). fluorescence-based morphological methods mollusca flurescence-based methods have been applied in molluscs especially to document cytoskeletal rearrangements following the exposure to chemotactic factors/cytokines (franchini and ottaviani, 1994; ottaviani et al., 2000). in the freshwater snail v. ater, acth is able to stimulate the motility of immunocytes. the withdrawn hemolymph plus acth are dropped onto a slide in which a small circle is delimited by a pvc adhesive strip. after 30 min of incubation in a humidified chamber, the supernatant is removed and the adhered immunocytes are left to air-dry for 30 min before the fixation in 1 % paraformaldehyde for 5 min at rt. after fixation, different procedures of permeabilization are followed on the basis of the target of the study. for the detection of actin and vinculin, the immunocyte membrane is permeabilized with an incubation in hepes-tritonx-100 for 5 min at rt, while for detection of microtubules, the permeabilization is achieved with a dehydration in cold absolute methanol (5 min at 20 °c), followed by cold acetone (5 min at -20 °c) and air drying. specific reactions are finally performed by incubating immunocytes for 30-60 min at 37 °c in a humidified chamber with either 2.5 μg/ml fluorescein isothiocyanate (fitc)-labeled phalloidin (a specific marker of f-actin) (sigma), or with 1-10 μg/ml mouse anti-vinculin mab (boeringher-mannheim, germany), or with rabbit anti-tubulin (chemicon). slides can be mounted with an ordinary aqueous mounting medium (e.g., 50 % pbs-1/glycerol) or with commercial anti-fading media. observations are performed under a fluorescence microscope (ex/em wavelengths 495/519 nm). in the bivalve m. galloprovincialis, the actin microfilament modifications in immunocytes exposed to 100 ng/ml human recombinant il-8 (pepro tech) have been analyzed by using a similar method. briefly, cell preparation is carried out as described above but fixation is performed with 4 % paraformaldehyde in pbs-1, ph 7.4, for 5 min at 4 °c. permeabilization is still achieved with a hepestriton x-100 solution (5 min at rt), then mussel immunocytes are incubated in a humidified chamber with 10 μg/ml fitc-labeled phalloidin at 37 °c for 30 min. finally, slides are again washed three times with pbs-1 and mounted in aqueous/anti-fading medium. insecta cytocentrifugation is the first-choice method for slide preparation when working with cell cultures growing in suspension. in the insect cell line iplbldfb, derived from the larval fat body of the lepidopteran l. dispar, it has been observed that cytocentrifugation has to be performed at a speed as low as possible, i.e., 200 rpm for 1 min. this is important since high speed cytocentrifugations can alter the morphology as well as the integrity of the cells and may alter results when studying cytoskeletal elements (malagoli et al., 2006). the cytoskeletal organization of iplb-ldfb cells has been evaluated by fitc-labelled phalloidin (sigma). after cytocentrifugation, the cells are fixed for 10 min at rt in 4 % paraformaldehyde, washed in pbs-1 for 5 min and incubated for 30 min at 37 °c in 30 nm fitc-labelled phalloidin in presence of 0.3 % triton x-100 in pbs-1. subsequently, nuclei are stained for 5 min at rt with 10 μm hoecsht 33342 and the cells are mounted in mowiol® (calbiochem). it should be remarked that fixation is a fundamental step not only for the preservation of cell morphology, but because it allows a tighter adhesion of cells to the slide. a similar cell preparation has been also applied for tunel assay (useful for detecting the presence of apoptotic nuclei) (malagoli et al., 2006). conversely, for the hoecsht 33342-propidium iodide (pi) staining, applied for the evaluation of cell membrane integrity, the cytocentrifugation is performed on unfixed cells after the incubation with hoecsht 33342 but before the incubation with pi. more in detail, iplb-ldfb cells are stained for 10 min at 26 °c with 10 μm hoecscht 33342 and subsequently cytocentrifuged. slides are then submerged at 26 °c in a 10 μm pi solution for 5 min and immediately observed (malagoli et al., 2005). the ex/em wavelengths for the cited fluorochromes are the following: 495/519 nm (fitc), 343/483 nm (hoecsht 33342), 536/617 nm (pi). tunicata some differences exist in fixation procedures for immunocytochemical protocols with respect to that described for characterization of circulating hemocytes of b. schlosseri. for antigen preservation in immunocytochemical assays, hemocytes are fixed in 4 % paraformaldehyde (serva, heidelberg, germany) plus 0.1 % glutaraldehyde in saline buffer (sb) for 30 min at 4 °c, and then washed in sb. fixed hemocytes are washed in pbs-3 plus 1 % sucrose and permeabilized with 0.1 % triton x100 (merck) in pbs-3 for 5 min. for the specific detection of f-actin, the monolayers are then incubated for 30 min at 25 °c in fitc-labeled phalloidin (sigma), 1 μg/ml in pbs-3. lastly, the coverslips are rinsed in 0.1 m carbonate buffer, ph 9.5, to amplify the fluorescent signal, mounted with fluorsave (calbiochem) on glass slides, and 200 observed at a magnification of 1,250x with a fluorescence light microscope equipped with a filter block for fitc excitation (450-490 nm; emission at 525 nm) (fig. 7f). to observe microtubules, hemocytes are preincubated in pbs-3 containing 3 % powdered milk and 0.5 % fetal calf serum for 30 min to block aspecific interactions of antibodies. coverslips are then incubated for 60 min in monoclonal anti-αtubulin antibody (sigma) (1:2,000 in pbs-3), washed in pbs-3 and incubated with 50 µg/ml fitc-labeled goat anti-mouse igg (sigma) in pbs3. slides are then mounted with fluorsave (calbiochem), as described above, and observed under the fluorescence microscope (fig. 7g). in addition to the analysis of the cytoskeleton, fluorescence-based methods have also been successfully applied to analyze apoptotic process of b. schlosseri hemocytes. we have analyzed both early-apoptosis and late-apoptosis markers, even if the boundary between these two phases is obviously not always clearly distinguishable. phosphatidilserine exposure (early apoptosis): living cells are exposed for 60 min to xenobiotics and then incubated with fitc-coupled annexin-v (annexin-v fluos roche diagnostics, indianapolis, in, usa), according to the manufacturer’s instructions, to detect the presence of phosphatidylserine in the outer leaflet of the plasma membrane of living cells, a marker of early apoptosis (martin et al., 1997). after 15 min, cells are observed under the fluorescence microscope, equipped with a filter block for fitc excitation, at the magnification of 1,250x. at least 300 cells/coverslip are counted to evaluate the apoptotic index, i.e., the percentage of fluorescent cells (fig. 7h). dna fragmentation (late apoptosis): to evaluate the occurrence of dna fragmentation, hemocytes ,exposed for 60 min to xenobiotics, are fixed for 30 min at 4 °c in 4 % paraformaldehyde plus 0.1 % glutaraldehyde in sb rinsed in pbs-3 and incubated for 30 min in 0.3 % h2o2 as blocking solution. then, cells are incubated in the permeabilization solution (0.1 % triton x-100 in 0.1 % na-citrate) for 2 min at 4 °c. samples are then rinsed twice with pbs-3 and incubated in the tunel reaction mixture (roche diagnostics) for 60 min at 37 °c according to the manufacturer’s instruction and immediately observed under the fluorescence microscope, equipped with a filter block for fitc excitation (abrams, 1997). the reaction product can also be longer preserved when, after this step, hemocytes are incubated with peroxidase-conjugated anti-fitc antibodies, stained with 0.05 % dab in pbs-3 containing 1.5 % h2o2, dehydrated, mounted with eukitt (bio-optica) and observed under the light microscope. the percentage of cells with fluorescent or stained (brown) nuclei (fig. 7i, j) is expressed as the dna fragmentation index. comet assay: this test for dna fragmentation is performed as described by singh et al. (1988) and modified by kamer and rinkevich (2002). ten µl hemocyte suspension, containing 104 cells are mixed with 90 µl of a 0.5 % solution of melted agarose (low-melting-point type), cast on a microscope slide pre-coated with 0.5 % regular agarose and followed, after solidification, by a layer of 0.5 % lowmelting-point agarose. slides are then immersed in lysis buffer (0.01 m tris-hcl, 2.5 m nacl, 0.1 m na2edta, 1 % triton x-100, 10 % dimethylsulfoxide, ph 10) for 2 h at 4 °c. the slides are placed on a horizontal gel electrophoresis unit filled with chilled electrophoretic buffer (0.3 m naoh, 1 mm na2edta) for 40 min, to denature dna. after electrophoresis (20 min at 20 v), the slides are washed in 0.4 m tris-hcl buffer (ph 7.5), blotted dry, stained with a 25 µg/ml solution of ethidium bromide, and observed under a fluorescence light microscope, with an excitation filter of 590 nm. the frequency of apoptosis is estimated by counting the percentage of cells showing nuclei transformed in typical apoptotic comets (fig. 7k) by counting at least 100 cells for each slide at 1,250x (bacsó et al., 2000). finally, a fluorescence-based immunocytochemical approach to identify stem hemocytes among a hemocyte population or subpopulation is here proposed. after adhesion to the coverslips, hemocytes are fixed for 30 min at 4 °c in 4 % paraformaldehyde plus 0.1 % glutaraldehyde in sb. they are washed in pbs-3, permeabilized with 0.1 % triton x-100 (merck) for 5 min, immersed for 30 min in a pbs-3 solution containing 10 % normal goat serum (vector lab.) to block aspecific reactions, and then washed again in pbs-3 for 5 min. cells are incubated in 50 µg/ml of anti-cd34 (calbiochem), anti-cd100 (alexis co, lausen, ch) or scf-r (sigma) mabs for 1 h at rt. cd34 is a highly glycosylated surface antigen of still unknown function, probably involved in cell adhesion (holyoake and alcorn, 1994); cd100 is also a transmembrane glycoprotein belonging to the igg superfamily, expressed by the majority of hemopoietic cells (hall et al., 1996); scf-r, receptor of stem cell growth factor (scf), also known as c-kit or cd117, seems to exert an essential role on vertebrate hemopoiesis (broudy, 1997). hemocytes are then washed in pbs-3 for 5 min and incubated for 30 min in 10 µg/ml fluoresceinated goat anti-mouse-ig antibody (sigma). lastly, they are washed for 5 min in pbs-3 and mounted in fluorsave (calbiochem). observations are carried out under a fluorescence light microscope equipped with a filter block for fitc excitation (ex/em wavelength 495/519 nm). in situ hybridization mollusca and insecta the following method has been successfully applied in molluscs (ottaviani et al., 1998c) as well as in the insect cell line crl-8003 (atcc 6538) from the cabbage moth m. brassicae (malagoli et al., 2002b). in the procedure reported for the detection of the expression of acth receptor-like mrna in molluscan immunocytes (fig. 9) plasmid pbluescript ii ks-phagemid containing about 3,000 bp bovine acth receptor cdna is used as probe (raikhinstein et al., 1994). the probe is labeled by incorporating digoxigenin (dig)-labeled deoxyuridine triphosphate (boehringer mannheim) by random primer dna-labeling, according to feinberg and vogelstein (1983). the reaction is stopped by adding 0.2 m edta, ph 8. the hybridization assay proceeds as follows: incubate unfixed and fixed immunocytes with pbs-1 plus glycine (0.7 %) and permeabilize with 0.3 % triton 201 fig. 9 in situ hybridization of the expression of acth receptor-like mrna in m. galloprovincialis immunocyte (a). negative control (b). bar = 10 μm. x-100 in pbs-1 for 15 min at rt; wash with pbs-1 and with a mixture containing 2x ssc (0.3 m nacl, 0.03 m sodium citrate, ph 7) for 10 min; incubate with pre-hybridization mixture (4x ssc), 40 % formamide and 1x denhardt’s solution for 1 h at rt; hybridization is performed by adding of a denaturated dig-labeled acth receptor cdna probe (50 μg/μl) to the pre-hybridization mixture overnight at 37 °c; rinse once in 2x ssc for 1 h at rt, once in 1x ssc for 30 min at rt, once 0.5x ssc for 30 min at 37 °c and once 0.5x ssc for 30 min at rt; incubate in a blocking solution [2 % normal sheep serum, 0.3 % triton x-100 in buffer 1 (100 mm tris-hcl, 150 mm nacl, ph 7.5)] for 30 min at rt; incubate with sheep anti-dig fab fragments conjugate to alkaline phosphatase diluted 1:500 in buffer 1 (boehringer mannheim) for 1 h at rt; wash twice in buffer 1 for 5 min and twice in buffer 3 (100 mm tris-hcl, 100 mm nacl, 50 mm mgcl2, ph 9.5) for 2 min at rt. the alkaline phosphatase activity is revealed by incubation in the following medium at rt: 45 μl nitro blue tetrazolium (75 mg/ml in dimethylformamide), 35 μl 5-bromo-4chloro-3-indolyl phosphate, toluidinium salt (50 mg/ml) (boehringer mannheim), 10 ml buffer 3, and 2.4 mg levamisole (sigma). the slides are mounted in glycerol diluted 50 % in te (10 mm tris-hcl, 1 mm edta, ph 8). controls are performed by mockhybridizing slides without probe. morpho-functional methods mollusca cell shape changes according to manske and bade (1994) cell motility is defined as cellular movements that do not result in translocation of the affected cell, while cell migration (chemotaxis) is a cell translocation over a clearly measurable distance as a consequence of the chemotactic activity exerted by a chemoattractant. cell shape changes can be easily evaluated by mean of a computer-assisted image analysis performed on digital pictures. when this procedure was proposed (schön et al., 1991) some specific tools were required. nowadays, the majority of laboratories has at least one digital camera installed on the microscope, while freeware softwares (e.g., imagej 1.32j, wayne rasband, national institute of health, usa, http://rsb.info.nih.gov/ij/) can be utilized to calculate the shape factor (sf), i.e., the circularity, of the cells. two are the true limiting factors in the application of this technique. the first is represented by the necessity to collect a reasonable amount of hemolymph (minimum 300 μl). the second is that the hemocytes have to remain functionally unaltered onto the slide for at least 30 min, after withdrawal. the hemolymph of m. galloprovincialis satisfies both of these requirements, thus it has been successfully utilized for cell shape analyses. the technique is based on the assumption that an inactive immunocyte has a round shape while, upon activation, it assumes a more elongated (ameboid) form. briefly, the sf is calculated as: ac/at = [lt/lc]2, where at is the area of an hypothetical circle with the same perimeter of the cell under measurement, lt is the perimeter of an hypothetical circle with the same area of the cell under measurement, and ac and lc are the actual area and perimeter, respectively, of the cell under measurement. the lower the sf value, the greater the perimeter of the cell with respect to its area, meaning that more the shape is ameboid. it descends that once a software able to calculate actual perimeter and area is available, it is possible to obtain the sf by relatively simple calculations. a b in cell shape change experiments, the hemolymph is collected form the mussel and 100 μl are spread onto the glass slide into a vaseline/rubber ring. the aim of the ring is to prevent the hemolymph from dropping out from the slide. a glass coverslip is then put over the ring, paying attention not to cover a small portion of the ring itself, so that it is possible to have access to the spread hemolymph in order to add molecules of interest, e.g., chemoattractants or signaling transduction pathway inhibitors (ottaviani et al., 1997b; malagoli et al., 2003). after 3-5 min for allowing immunocyte adherence, the operator has to select the cells (usually 5 for slide) that will be followed during the experiment. the photographs of these cells are taken at regular intervals (usually 5 min), and exposure to the light should be minimized. once the last photograph has been taken, it is strongly recommended to immediately perform the following controls. first, the cells that have been chosen must display a morphology similar to that of the majority of the other cells of the slide. if, for 202 example, the chosen cells are the only ones active in all the slide, the experiment should be discarded. second, in order to exclude any effect connected to the permanence of the slide under the microscope, it is important to immediately verify if a slide that was treated identically to that used for cell shape analysis but was not kept under the microscope, presents cells with a morphology almost overlapping to the first one. obviously, control slides in which the chemoattractant and/or the inhibitors are absent have also to be prepared and this explains why a relatively high amount of hemolymph is required for this method. the electronic images of the experiments can now be utilized for the actual evaluation of the sf, by following the edge of the cells with the pointer of the mouse and asking the software to directly evaluate the circularity or, as an alternative, the perimeter and the area of the chosen cells for each time point. chemotaxis the migration assay refers to the effect of human cytokines on molluscan immunocytes (ottaviani et al., 1995b). the collected hemolymph is centrifuged at 200xg for 15 min and the pellet resuspended in 1 ml of snail saline solution (sss) (na+ = 41.15 mm; k+ = 0.54 mm; ca2+ = 3.55 mm; mg2+ = 2.61 mm; ph 7.5; osmolarity = 109 ± 5 mos) (ottaviani, 1983) containing 0.1 % bovine serum albumin (bsa) (sigma). the test is carried out using 48-well microchemotaxis chambers (nucleopore, pleasanton, ca, usa), in which the upper and lower compartments are separated by a 5 μm pore, polycarbonate polyvinylpyrrolidone-free filter allowing the cell to migrate actively through the pores. fifty μl of immunocyte suspension is placed in the upper compartment, while in the lower one are placed different cytokines [concentrations: interleukin-1α (500, 50, 5 pg/ml), tumor necrosis factor (tnf)-α (100, 10, 1 u/ml)]. after 90 min incubation at 37 °c, the migrated immunocytes adhering to the distal part of the filter are fixed, stained, identified microscopically and counted using light microscopy (magnification 40x). data are expressed as migrate cells/field. controls are obtained measuring the immunocyte migration in the presence of sss. phagocytic tests mollusca a classical in vitro bacterial phagocytosis is reported in ottaviani et al. (1995b). one hundred μl of freshwater snail hemolymph, are placed on a slide in the form of a ring by applying an adhesive pvc strip, in which two holes (∅ 10 mm) are performed. the experiment is carried out by mixing the hemolymph with staphylococcus aureus (106108 bacteria/ml) in presence or in absence of different stimulators. the incubation is performed in humidified chamber and the phagocytic activity was tested after 40 min. the phagocytized bacteria is determined by immunocytochemical procedure (ottaviani, 1990) using a rabbit anti-s. aureus polyclonal antibody. the number of phagocytized bacteria is performed counting under the light microscopy (magnification 1,000x) 20 random immunocytes for each preparation. recently a different approach has been developed for the phagocytic test in the mussel m. galloprovincialis (malagoli et al., 2007). in this case, instead of bacteria, fluorescent micro-beads (diameter 1 μm) are used to assess phagocytic activity of immunocytes. after withdrawal, 100 μl of hemolymph (about 104 cells) are incubated at dark with 0.1 % v/v of 1 μm diameter green-fluorescent microspheres (fluospheres®, molecular probes, or, usa) in a 1.5 ml plastic tube and kept in gentle rotation for 30 min at rt. after incubation, immunocytes are placed on microscope slides and there left for a further 5 min to allow their adhesion. the number of phagocytic immunocytes out of 100 and the number of phagocytized microspheres from each phagocytic immunocyte are counted under a fluorescence microscope with the filter commonly used for fitc (ex/em wavelengths 495/519 nm). negative control, i.e., the discrimination between effectively phagocytized and non-specifically bound micro-beads, are performed by incubating the immunocytes with 1 % sodium azide alone for 5 min before the addition of the micro-beads. in this way it is possible to set a baseline percentage of phagocytic immunocytes that have to be taken into consideration when performing statistic calculations. insecta the in vitro bacterial phagocytosis is performed in 1.5 ml plastic tube, by adding s. aureus (108 bacteria/ml) to hemolymph collected from adult flies of c. vomitoria. the phagocytosis assay is determined after 30 min. the samples are cytocentrifuged, and the immunocytes are stained with 0.5 % toluidine blue. after staining, the cell phagocytic activity is recorded under the microscope (franchini et al., 1996). cytotoxicity the natural cytotoxicity is performed on freshwater molluscan immunocytes (franceschi et al., 1991) in a short term (4 h) 51cr release test following the classical protocol for human studies using the k562 cell line as a target (lozzio and lozzio, 1975; grimm and bonavida, 1979). target cells (t), are labeled overnight with na2 51cro4 solution (amersham int., amersham, uk) in complete medium. the effector (e)/t ratio varies from 100:1 to 6:1. the percentage of specific cytotoxicity is calculated according to the following formula: % s.c. = exp. rel.spontaneous rel. ________________________ max. rel. – spontaneous rel. x 100 s. c.= specific cytotoxicity exp. rel. = experimental release max. rel. = maximum release enzyme activity acid phosphatase activity has been examined in molluscan immunocytes (franchini and ottaviani, 1990). the activity is performed on fixed (1.5 % glutaraldehyde in 20 mm cacodylate buffer, ph 7.5) 203 and unfixed cytocentrifuged immunocytes. slides are incubated for 2 h at 37 °c in barka and anderson media (1962). after incubation, cells are treated with 1 % ammonium sulfate for 1 min and covered with acquovitrex (c. erba). flow cytometry the cytofluorimetric analysis has been applied to molluscan hemolymph in order to detect the different cell populations on the basis of physical characteristics, such as diameter, size, density, etc allowing to distinguish them on the basis of forward and side light scatters. a further phenotype characterization of immunocytes has been carried out using various antibodies, such as anti-humanacth pab and mab, anti-n-acetylmuramic acid pab and different mouse anti-human mabs recognizing different molecules (ottaviani and montagnani, 1989; franceschi et al., 1991; ottaviani et al., 1991). procedure using rabbit anti-humanacth pab and mouse anti-human acth mab: 400 μl of hemolymph are divided into two parts. one is incubated with 10 μl of anti-acth pab (dakopatts) or with mouse anti-acth mab (chemetron, milan, italy) for 20 min at 4 °c, while the second is taken as a negative control. the stained samples are then washed with cold pbs-1, incubated with 10 μl fitclabeled goat anti-rabbit igg (dakopatts) or with 10 μl phycoerythrin (pe)-labeled goat anti-mouse igg (becton-dickinson, san josè, ca, usa), washed with cold pbs-1 and analyzed with a flow cytometry (becton-dickinson). negative controls are prepared according to the above procedure, but omitting the anti-acth pab or anti-acth mab. samples are finally analyzed by using state of the art flow cytometry, taking into account the requirements for a precise setting of the fluorescence intensity and of the compensation, should one use two or more fluorochrome-conjugated antibodies to stain cells. tunicata trypan blue exclusion assay after cell adhesion, primary cultures of b. schlosseri hemocytes are exposed for 60 min to fsw containing xenobiotics at various concentrations. living hemocytes are then incubated for 5 min in a solution of 0.25 % trypan blue in fsw and observed under the light microscope at 1,250x. the percentage of stained hemocytes is estimated on a total count of at least 300 cells, and the concentration of xenobiotics able to cause mortality to 50 % of the hemocytes (lc50) is calculated according to the probit method (spss 11.0, spss corp., chicago, il, usa). adhesion assay since the capability of b. schlosseri hemocytes to adhere is fundamental for their role in immunity, adhesion assay can be usefully applied to acquire information on the effects of xenobiotics on this species. hemocytes are left to adhere for 60 min on clean coverslips in the presence of xenobiotics, at various concentrations. slides coated with 50 µg/ml poly-l-lysine are used as reference controls (100 % adhesion). after glutaraldehyde-fixation and staining for 5 min in 10 % giemsa solution, coverslips are mounted in acquovitrex (c. erba) and observed. in order to evaluate the ability of hemocytes to adhere to glass in the presence of xenobiotics, the total number of hemocytes in 10 optic fields at 1,250x is counted and expressed as the adhesion index, i.e., the ratio, expressed as percentage between the total hemocyte count in experimental slides and the cell count in similar conditions in poly-l-lysinecoated slides. cell spreading assay after 60 min-exposure to biocides at various concentrations, b. schlosseri hemocyte monolayers are fixed with glutaraldehyde, stained with giemsa solution and mounted in acquovitrex (c. erba). their morphology is then observed under the light microscope at 1,250x, and the cell-spreading index, i.e., the percentage of hemocytes with ameboid shape, is estimated after counting at least 300 cells per coverslip (cima and ballarin, 1999). in addition, computer-assisted image analysis (casting image nt, hp laboratories, palo alto, ca, usa) is performed to evaluate the sf, as defined above for cell shape change evaluation in molluscs. lower shape factors indicate larger perimeters with respect to the areas and, therefore, an increased ameboid shape. assay for reduced glutathione (gsh) content after treatment with xenobiotics, b. schlosseri hemocyte monolayers are washed in fsw, stained for 10 min at 37 °c in 40 mm chlorobimane (a fluorescent dye specific for gsh with λmax of 461 nm) (sigma) solution in fsw, obtained from a 20 mm stock solution in 95 % ethyl alcohol, and then rinsed in fsw (cookson et al., 1998). living cells are immediately observed under a fluorescence microscope equipped with an ultraviolet light filter block (ex/em 270-380/460 nm), at a magnification of 1,250x. positive sites appear fluorescent blue. the percentage of stained cells is then determined and expressed as the gsh index. morula cell (mc) degranulation assay fifty μl of b. schlosseri hemocyte suspension (15x106 cells/ml) are placed in the center of the culture chambers prepared as already described and left to adhere to washed coverslips for 30 min. after discarding the fsw, hemocytes are then incubated for 60 min at rt with 50 ml of autologous or heterologous blood plasma or with a suspension of escherichia coli (200x106 cells/ml), s. aureus (50x106 cells/ml), bacillus clausii spores (200x106 spores/ml) or ordinary baker’s yeast (saccharomyces cerevisiae) (50x106 cells/ml) in fsw, and washed in fsw (ballarin et al., 1998, 2005). the morphology of living mcs is then observed under a phase contrast microscope at a magnification of 1,200x (fig. 10). chemotactic assay migration of b. schlosseri hemocytes is analyzed using a 24-well chemotaxis chamber (transwell, costar, corning, ny, usa) with the lower and upper wells separated by 8-μm polycarbonate filters. the upper wells are filled with 100 μl of hemocyte suspension (5x105 cells) in fsw and 500 μl of fsw are added to the lower wells. solutions in upper and lower wells are allowed to equilibrate for 15 min, and then 20 μl of the lower solution are substituted with 20 μl of the putative chemoattractant at the concentration of 2 mg/ml 204 fig. 10 living morula cells incubated in the absence (a) or in the presence (b) of heterologous blood plasma. in this last case cells have degranulated and have released their vacuolar content. bar = 15 µm. (fsw in controls) (modified according to raftos et al., 1998). cells are allowed to migrate for 2 h at 18 °c. after incubation, the lower surface of the filters are flushed extensively with fsw, in order to detach adhering hemocytes and collect all the cells which have passed through the membrane. hemocytes in the lower wells are then collected in a 1.5 ml vial and centrifuged at 780xg for 10 min. the mean number of cells in 10 random optical fields, at a magnification of 1,250x, is used to define the migration stimulation index, i.e., the ratio between the mean number of migrated cells per field in experimental series and the mean number of migrated cells in controls. phagocytosis assay after adhesion, b. schlosseri hemocytes are incubated with 60 µl of a suspension in fsw of various test particles (ballarin et al., 1994) represented by living or autoclaved (15 min at 120 °c) cells of s. cerevisiae, sheep erythrocytes, zymosan (sigma), latex beads (1 and 3 µm diameter) (sigma), fluorescent e. coli cells (phagotest kit, orpegen, heidelberg, germany). the number of test particles is adjusted to a particle:hemocyte ratio of 10:1 after counting with a bürker’s hemocytometer. the best results are obtained with yeast suspensions in fsw since this type of target particles is both extensively engulfed by phagocytes and easily recognizable in phagosomes. cultures are kept upside-down for 5120 min at 25 °c and the uningested yeast is then removed by dipping repeatedly the coverslips in a large volume of fsw. viability, assessed by the trypan blue exclusion assay, exceeds 95 % after 2 h of incubation. when fluorescent test particles are used, uningested particles are quenched by quick immersion of coverslips in a solution of 2 mg/ml trypan blue and 2 mg/ml crystal violet in 0.02 m citrate buffer, ph 4.4 containing 33 mg/ml nacl. hemocyte monolayers are then fixed in 1 % glutaraldehyde and 1 % sucrose in fsw for 30 min at 4 °c and stained with 5 % giemsa for 5 min. the coverslips are mounted on glass slides with aquovitrex (c. erba) or fluorsave (calbiochem), the latter being used for assays with fluorescent test particles. at least 300 hemocytes are observed per slide under microscope, in ten optical fields, at the magnification of 1,250x, and, the phagocytic index, i.e., the percentage of hemocytes with ingested cells, is evaluated. the phagocytic index increases progressively with time reaching a plateau of 10-15 % after an incubation time of 60 min, which represents the optimum incubation time for this assay. in other experiments, at the end of the incubation time, the medium is collected and centrifuged to obtain a “conditioned” supernatant, which is used to detect enzymatic activity, reactive oxygen species, opsonins and cytokines released in the incubation medium (cima et al., 1996) and to study its effects on other phagocytosis assays (menin et al., 2005). finally, the phagocytosis assay can be also used for evaluating the immunosuppressant effects of xenobiotics added at various concentrations to the incubation medium containing the target particles. enzyme activity assays are carried out on glutaraldehyde-fixed hemocytes, previously exposed or not to xenobiotics. these methods can be applied either to investigations aimed at hemocyte characterization or to the study of xenobiotic effects on hemocyte functions. in controls, the specific enzyme substrate is omitted. after incubation in the reaction mixtures, monolayers are thoroughly washed in distilled water, coverslips mounted in acquovitrex (c. erba), and at least 300 cells per slide are counted to determine the fraction of positive hemocytes, expressed as the enzymatic index. acid phosphatase this is a lysosomal enzyme used as a marker of phagocytes. fixed hemocytes are washed in 0.1 m sodium acetate buffer, ph 5.2, for 10 min and incubated for 3 h at 37 °c in the reaction mixture made by 10 mg naphthol as-bi phosphate (sigma) in 400 μl dimethylformamide (dmf), 400 μl solution a (4 % new fuchsin (sigma), 2 % hcl in distilled water), 400 μl of a 4 % aqueous solution of nano2 solution b and 20 ml of 0.1 m sodium acetate buffer. positive sites appear red (lojda et al., 1979). alkaline phosphatase after fixation, hemocytes are washed in trishcl buffer 0.1 m, ph 9, for 10 min and incubated for 2 h at 37 °c in a reaction mixture similar to that used for acid phosphatase assay, but containing 20 ml of tris-hcl buffer instead of sodium acetate buffer (burstone, 1962). hemocytes are then washed in tris-hcl buffer for 10 min and mounted. positive sites stain red. β-glucuronidase it is another lysosomal hydrolytic enzyme, marker of phagocytes. fixed hemocytes, washed in 0.1 m sodium acetate buffer, ph 5.2, for 10 min, are incubated for 3 h at 37 °c in the reaction mixture made by 4 mg naphthol as-bi β-glucuronide (sigma) dissolved in 250 μl dmf, 400 μl solution a, 400 μl of aqueous solution of 4 % nano2 and 20 ml of sodium acetate buffer (lojda et al., 1979). positive sites stain red (fig. 7l). 205 5’-nucleotidase fixed hemocytes are washed for 10 min in tris–maleate buffer 0.2 m, ph 7.2 and incubated for 2 h at 37 °c in the following reaction mixture: 20 mg adenosine-5’-monophosphate (amp) (sigma), 22 ml distilled water, 20 ml tris-maleate buffer, 3 ml 2 % pbno3, 5 ml 2.5 % mgso4 (wachstein and meisel, 1957). hemocytes are then washed twice with distilled water and with ammonium sulfide solution (21 %) (fluka), diluted 1:100 in distilled water, for 2 min. they are then washed again with distilled water and mounted. positive sites stain black. acid esterase fixed hemocytes are washed for 10 min in phosphate-citric acid buffer 0.1 m, ph 5.5, and incubated for 16 h at 4 °c in the following reaction mixture: 3 mg naphthol acetate (sigma) dissolved in 500 µl acetone, 250 µl solution a, 250 µl solution b, 19 ml phosphate-citric acid buffer (lojda, 1977). hemocytes are then washed with distilled water and mounted. positive sites stain pinkish-brown. chloroacetylesterase hemocytes, washed in pbs-3 for 10 min, are incubated for 1 h at rt in the following reaction mixture: 6 mg naphthol chloroacetate (sigma) dissolved in 1 ml dmf and added to 19 ml pbs-3 containing 20 mg fast blue b (moloney et al., 1960). hemocytes are washed in pbs-3 for 10 min and mounted. positive sites stain blue. non-specific esterases non-specific esterases are reference hydrolytic enzymes, typical of phagocyte lysosomes. this assay was previously used to label the phagocytic line of b. schlosseri hemocytes (ballarin and cima, 2005) in acute toxicity assays with antifouling xenobiotics. hemocytes are washed in pbs-3 and incubated for 3 h at 4 °c in 100 ml of pbs-3 containing 2 ml of 1 % 1-napthyl acetate (sigma) as a substrate, previously dissolved in 1 ml acetone, 1 ml of hexazonium-p-rosaniline [500 µl of a solution of 0.4 g new fuchsin (c. erba) in 7.2 % hcl added to 500 µl of a 4 % solution of nano2 in distilled water] (davis et al., 1959). positive sites stain red. cytochrome-c-oxidase it is an enzyme of the mitochondrial respiratory chain, and its activity, related to mitochondrial functionality, is required for atp production. cells are washed in 0.1 m na-acetate buffer, ph 5.5, for 10 min, and incubated for 4 h at 37 °c in a solution of 0.2 % dab in 1 % mncl2 containing 0.01 % h2o2 (novikoff and goldfischer, 1969). positive sites stain brown. ca2+-atpase it is involved in the sequestration of ca2+ ions in intracellular stores. hemocytes are incubated for 20 min at 37 °c in 0.1 m na-barbital buffer, ph 9.0, containing 3 mm atp, 3.25 mm 2,4-dinitrophenol (dnp) and 0.1 m anhydrous cacl2. cells are then repeatedly washed in 1 % anhydrous cacl2, further rinsed in 1 % co(no3)2, immersed in 0.5 % ammonium sulfide for 1 min, and washed in distilled water (chayen et al., 1969). positive sites appear black. arylsulfatase hemocytes are washed in sodium acetate buffer 0.1 m, ph 5.2, for 10 min and incubated for 2 h at 37 °c in the following reaction mixture: 0.16 g p-nitrocatecholsulfate (sigma), 4 ml distilled water, 12 ml 0.1 m sodium acetate buffer, ph 5.5, 4 ml 8 % pbno3 (goldfischer, 1965). after incubation, hemocytes are washed with distilled water and then with 21 % ammonium sulfide solution (fluka) diluted 1:100 in distilled water, for 2 min. finally, they are washed with distilled water and mounted. positive sites stain brownish–black. peroxidase after fixation, hemocytes are washed in pbs-3 for 10 min and incubated for 2 h at 37 °c in the following reaction mixture (graham and karnovsky, 1966): 0.5 mg/ml dab in distilled water containing 0.02 % h2o2. hemocytes are then washed in distilled water and mounted. positive sites stain brown. phenoloxidase phenoloxidase is an oxidative enzyme with a cytotoxic activity involved in defense reactions in b. schlosseri, where it can be used as a marker for the cytotoxic line of blood cells. after fixation, hemocytes are incubated for 1 h in saturated dihydroxy-l-phenylalanine (l-dopa) solution in pbs3 (hose et al., 1987), washed in distilled water, and mounted in acquovitrex (c. erba). positive cells stain blackish-brown (fig. 7m). assay for lysozyme hemolymph is collected from the colonial marginal vessel with a fine micropipette and centrifuged at 780xg for 10 min. the supernatant, corresponding to the cell-free blood plasma, is immediately used for the lysozyme assay. pellets are resuspended in distilled water, sonicated for 5 min at 0 °c with a braün labsonic u sonifier at 50 % duty cycles, and centrifuged at 12,000xg for 15 min at 4 °c. supernatant, corresponding to the hemocyte lysate, is collected and immediately used for the lysozyme assay. one percent of methylene blue 0.13 % in distilled water is added to a 0.25 % suspension of micrococcus lysodeikticus (sigma) in 0.1 m phosphate/citrate buffer, ph 5.8, and incubated for 30 min at 25 °c to stain cells. seven ml of the above suspension are then added to 50 ml of 1.5 % melted agarose in 0.1 m phosphate/citrate buffer, ph 5.8, and 5 ml of the solution are poured into petri dishes. when the agarose solidifies, 5-mm wells are made and filled with 50 µl of hemocyte lysate. lysis diameter are observed after overnight incubation at 37 °c and compared with those obtained using known dilutions of lysozyme (5 and 10 mg/ml) from chicken egg white (sigma) in 0.1 m phosphate/citrate buffer, ph 5.8 (fig. 11a). protein concentration in hemocyte lysate and blood plasma is determined according to bradford (1976) using bsa as standard. hemagglutinating assay rabbit erythrocytes are washed three times by centrifugation at 500xg for 10 min in tris-buffered saline (tbs-2) (tris-hcl 50 mm, nacl 150 mm, ph 7.4) and incubated for 30 min at 37 °c in 0.1 mg/ml 206 trypsin in tbs-2 (ballarin et al., 1999). they are then washed again and resuspended in tbs-2 containing 0.2 % gelatin to get a 1 % solution (v:v). fifty µl of b. schlosseri blood plasma are serially diluted two-fold with tbs-2 in the wells of u-bottomed microtiter plates and an equal volume of 1 % rabbit erythrocyte suspension is added in each well. fsw is used in controls. tbs-2 containing 5 mm egta is also used to assess the ca2+-dependence of the reaction. in another experimental series blood plasma is incubated for 30 min with 1 % rabbit erythrocyte suspension in order to control the specificity of the interaction. the suspension is then centrifuged at 780xg for 10 min and the rabbit erythrocyte-absorbed supernatant is collected and used in the hemagglutination assay. plates are gently shaken, incubated for 1 h at 37 °c and then kept at 4 °c. the hemagglutination titre (ht), i.e., the reciprocal of the highest dilution giving positive hemagglutination is then evaluated (fig. 11b). fig. 11 a: lysis plaque of micrococcus lysodeikticus in agarose due to lysozyme activity; b: hemagglutination activity of blood plasma: absence and presence of agglutination in row a (control; fsw) and row b (hemagglutination titer = 128), respectively. agglutination activity is also evaluated in vitro by adding 50 µl of a 1 % trypsinized rabbit erythrocyte suspension in fsw to culture chambers. after 30 min of incubation at 25 °c, adhesion of erythrocytes is observable under the light microscope as rosette formation around some hemocyte types. in the solitary ascidian c. intestinalis erythrocytes from different species (i.e., rabbit, sheep, human abo group) were washed three times with tbs-2, 0.1 % (w:v) pig gelatin, suspended at 1 % in tbs-2 with or whitout 5 mm cacl2 ph 7.4, 0.1 % gelatin, and used in a microtitre plate hemagglutination assay (ha) in which 25 μl of serially diluted sample were mixed with an equal volume of erythrocyte suspension (fig. 12). serial dilutions (2-fold) of the tbs-2dialyzed and diluted serum (1:10) are performed with tbs-2 containing 0.1 % gelatin. the ht is evaluated after 1 h incubation (from 18 to 37 °c). also for c. intestinalis to increase the erythrocyte sensitivity to the hemagglutination assay, trypsintreated erythrocytes are prepared by resuspending an erythrocyte pellet (obtained from 1.0 ml blood) in 6 ml tbs-2 containing 300 μg trypsin (stock solution prepared in 10 mm hcl) (parrinello and canicattì, 1982). the reaction mixture is incubated at 37 °c for 15 min. the trypsin-treated erythrocytes are washed with tbs-2 and resuspended (1 %) in tbs-2. plaque-forming cell assay (pfc) a pfc assay using tunicate hemocyte effectors has been originally described by cunningham and szenberg (1968) for the human b cell/sheep red blood cells and subsequently modified for tunicate hemocytes (parrinello et al., 1996; cammarata et al., 1997). fifty µl of hemocytes suspension (1 x 106/ml) in marine solution (ms) (12 mm cacl2, 11 mm kcl, 26 mm mgcl2, 45 mm tris, 38 mm hcl, 0.45 m nacl, ph 7.4) are mixed with 50 µl of suspension of 5 % rabbit erythrocytes in ms. the reaction mixture is rapidly layered into the slide chamber by capillary action. the chamber has been constructed by placing three thin strips of doublestick tape placed between the borders and in the centre of a coverslip and another glass coverslip is then suspended onto the three pieces of tape forming a double chamber. after 15 min of incubation at 20 °c the cell mixture is examined under a phase contrast microscope. each slide chamber can hold just under 0.1 ml on either side of the tape (0.2 ml per slide) (fig. 13). phenoloxidase (po) activity determination of physical and chemical characteristics of lectins po activity is measured according to winder and harris (1991) using the reagent 3-methyl-2benzo-thiazolinone hydrazone (mbth) which reacts directly with dopaquinones. this method has the advantage of measuring the direct product of dihydroxyphenyl-l-alanine (l-dopa) oxidation since not all dopaquinones are converted to dopachromes which are measured when l-dopa only is used as substrate. briefly, 20 µl of hemolysate were incubated with 490 µl of pbs-3, 290 µl of 20.7 mm mbth in pbs-3 containing 4 % of dmf and 200 µl of l-dopa-saturated pbs-3. the reaction is read spectrophotometrically at 494 nm, each for 5 min. to examine divalent cation requirements for lectin binding, cacl2 or mgcl2 is added in the tbs-2 medium to obtain 5-10 mm final concentration. edta (1-10 mm) or egta (1-10 mm) are used to examine the effect of ca2+/mg2+ depletion on the hemoagglutinating activity. to examine the thermal stability of the protein, samples are incubated at different temperatures from 18 to 90 °c for 20 min and cooled down for 10 min on ice before the ha. susceptibility of the ha to freeze-thaw is examined by carrying out the ha on samples maintained at -20 °c for 2 months and thawed at rt. 207 serial dilution ct 2 4 8 16 32 64 128 256 512 1024 2048 1 2 cnt fig. 12 determination of hemoagglutinating titer. each well of the microtitre plate contained 25 μl of sample 1 and 2; 25 μl 1 % suspension of rabbit erythrocyte. the agglutinated erythrocytes form a carpet that cover completely the well, the last effective dilutions were indicated in blue (hemagglutination titer: sample 1=16; sample 2=512). in absence of agglutinin the cell form a button in the well. cnt= control in which sample has been substitute with tbs-2. carbohydrate specificity and chromatographic resin selection of lectins hemagglutinating activity is assayed with rabbit erythrocytes in the presence of saccharides as potential inhibitors of lectin binding. when the lectin activity has been detected in the hemolymph or extract of interest, the second step is to identify those carbohydrate ligands for which the lectin show the highest affinity for matrix selection in order to allow the lectin enrichments by affinity chromatography. inhibition experiments are carried out using decreasing concentrations (starting from 200 mm in tbs-2) of a large variety of monosaccharides that should include all four types of mäkelä’s classification (1957) plus sialic acid (i.e., l-fucose, d-arabinose d-galactose, d-glucose, dmannose, n-acetyl-galactosamine, and n-acetylglucosamine), oligosaccharides (i.e., maltose, lactose, d-raffinose, cellobiose), mannan and glycoprotein. the lectin solution is incubated with each one of the sugars at different concentrations for 60 min and the rabbit erythrocytes are then added. the agglutination titer in the presence of sugars are compared to the control where the sugars are substituted with tbs-2. the selected saccharide are coupled to the column support matrices by activation with cyanogen bromide or divinylsulfone. often other purification steps are required for an optimal lectin purity (i.e., size exclusion, ion exchange, hydrophobic chromatography). this additional protein purification step is an empirical process employing a large number of experiments, which often represent a limit especially for planning integrated project research. to overcome this limitation a plateor tube-based experiments could be used avoiding the preparation of numerous packed columns, the use of large volume of sample and test on chromatography systems. this parallel screening can facilitate the selection of media and development of optimal binding and elution conditions. for this test the media slurry can be incubated with the same volume of sample (i.e., hemolymph or extract of interest) and, after 1 h, centrifuged at 400xg for 5 min to remove not binding proteins. the binding activity can be detected analyzing the unbound proteins by electrophoresis or by centrifugation with the specific gel elution buffer for bound proteins recover. quantitative methods mollusca free amino acids (faa) the faa has been evaluated in molluscan hemolymph (ottaviani, 1984). the hemolymph is centrifuged at 3,000xg for 10 min and 100 μl of the supernatant are utilized to determine the faa using the amino acid analyzer liquimat iii (kontron, zurich, switzerland). radio-immuno assay (ria) test the following procedure has been utilized to quantify acth-like molecules in the molluscan serum and immunocytes (ottaviani et al., 1990). after hemolymph collection and the subsequently centrifugation at 600xg for 10 min, the serum is stored at -20 °c until use; the pellet must be resuspended in pbs-1, homogenized for 30 s and centrifuged at 1,500xg for 20 min at 4 °c before storing. the serum and pellet are freeze-dried, redissolved in pbs-1 and assayed via commercial ria kits for acth. high performance liquid chromatography (hplc) the hplc assay has been used in the determination of the biogenic amines (ba) in molluscan serum and immunocytes (ottaviani et al., 1992, 1993, 1994, 1995a, 1997a, 1998a). after centrifugation of the hemolymph, the ba are detected by hplc in the serum and pellet (immunocytes). the ba are extracted from the serum by using a clin-rep-catecholamine kit (recipe pharma vertriebs gmbh and co kg, münchen, germany) and the extracted sample is analyzed with a rapid and sample isocratic simultaneous determination of norepinephrine, epinephrine and dopamine. monoamine peaks are identified by comparing their retention times in the serum extracts with those of a standard solution. each ba is quantified using the internal standard 208 a b c figure 13 plaque forming cell assay. a: plaque of lysis observed by phase-contrast microscope. red arrowhead = plaque forming hemocytes capable of secreting lytic factors against rabbit erythrocytes; yellow arrows = erythrocyte ghosts; bar = 15 µm. b: low magnification observation, three plaques are marked in red; c: control of erythrocytes. (3,4-dihydroxybenzylamine, dhba) method with a correction factor. the pellet is washed with sss, homogenized with a fisher sonic dismembrator model 300 (globalspec inc., troy, ny, usa), centrifuged and the supernatant analyzed by direct injection into the chromatographic system. identification and calculation of the ba are performed by directly comparison areas of a standard calibration curve with those of the samples. the hplc system used consisted of a isocratic lc pump (mod. 250 perkin elmer, (waltham, ma, usa) equipped with a degasser (erc-3512-erma), an automatic injector (rheodyne 7125, 100 μl loop), an electrochemical detector (mod. 460 waters) and a data system pe nelson (mod. 1020 perkin elmer). spectrofluorimetry this method has been utilized to detect the neutral endopeptidase-24.11 (nep)-like activity in molluscan immunocytes (ottaviani and caselgrandi, 1997; caselgrandi et al., 2000). the collected hemolymph is centrifuged and the nep-like activity of the pellet is measured according to the deschodtlanckman et al. (1990) procedure. a maximum number of 700x103 immunocytes/ml have to be used for a correct evaluation since above this value enzymatic activity falls quickly in relation to a given number of cells. nep cleaves the substrate (sucala-ala-phe-amc) (sigma) to phe-amc. a fluorescent spectrometer (perkin elmer, mod. 204) at an excitation wavelength of 440 nm is utilized to detect the fluorescent product, free amc. the calibration curve is measured with pig nep enzyme. crustacea elisa to quantify the variation of chh (crustacean hyperglycaemic hormone) level in the eyestalks and hemolymph of p. elegans elisa test using an antibody against recombinant chh has been performed (lorenzon et al., 2004). in this method a group of 10 p. elegans is used for eyestalk ablation; animals are anaesthetized for 1 min on ice before ablation. the eyestalk was quickly frozen and the pigmented eyecup dissected. eyestalk homogenate is prepared from 20 eyestalks homogenized in 2 ml cold pbs (sigma) ph 8.0 and then centrifuged for 1 h at 930xg at 4 °c and the pellet is discarded. homogenates are quickly deep frozen at -20 °c and stored until required for study. hemolymph is withdrawn from groups of 10 animals, centrifuged for 1 min at 10,300xg at 4 °c and the supernatant is then stored at -20 °c. the standards are represented by a known concentration [from 1 to 0.001 µg in 100 µl of pbs (sigma)] of 6xhisnenchhwt (n. norvegicus chh wild type) recombinant protein (mettulio et al., 2004). one hundred µl of the samples are loaded onto a 96 microwells plate (costar) and incubated in duplicate overnight at 4 °c. the content of the wells is discarded and the wells are washed 4 times with 209 250 µl of pbs (sigma) + 0.1 % tween20, ph 7.4 (pbs-t); then filled with 100 µl of 3 % bsa (sigma) solution in pbs (sigma) ph 7.4 plus 5 % fetal calf serum (sigma) and left for 2 h at rt. the content is discarded and the plates are again washed 4 times as described above. one hundred µl of 1 µg/ml biotinylated anti-nenchh (anti n. norvegicus chh; giulianini et al., 2002) antibody is then added to each well and the plate is incubated for 3 h at 36 °c. after removal of the biotinylated antibody, plates are washed extensively with pbs-t, followed by the addition of 100 µl of streptavidin-peroxidase solution (sigma) diluted 1:5,000 and incubated for 1 h at rt. the plates are once again washed 4 times with pbs-t and developed with 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid solution (sigma) in darkness for 1 h at rt (100 µl per well). the absorbance is measured in a multiwell plate reader (anthos 2020 version 1.1, krefeld, germany) at 405 nm. tunicata spectrophotometric assay for detoxifying enzymes glutathione s-transferase (gst) this enzyme plays a multiple role in xenobiotic metabolism, catalyzing the conjugation reactions of reduced glutathione (gsh) and electrophilic xenobiotics. gst acts as a mediator of xenobiotic detoxification and it has recently been successfully used as a biomarker, since it appears to be important in oxidative stress protection (cima et al., 2002b). gst activity is determined according to the method of habig et al. (1974) using 1-chloro-2,4dinitrobenzene (cdnb) (sigma) as substrate (λ = 340 nm, ε = 9.6 mm-1 cm –1) on hemolysates obtained after sonication at 4 °c at 50 % duty cycles for 5 min of hemocytes previously exposed in vitro to xenobiotics, then centrifuged and resuspended in 20 mm tris-hcl buffer, ph 7.5. the reaction is performed in a final volume of 1 ml containing 160 µl hemolysate or fsw in controls (blank), 700 µl of 100 mm k3po4, ph 7.0, 70 µl of 20 mm cdnb dissolved in absolute ethanol, 70 µl of 20 mm gsh (sigma). absorbances are read at 10 s intervals for 2 min, and enzyme activities, measured as decrease of substrate, are expressed as nmol/min/mg protein (mean ± sd) with the following formula: enz. act. = enz. act. = enzyme activity abs = absorbance glutathione peroxidase (gpx) it is an antioxidant enzyme which protects from the effects of reactive oxygen species through reduction of hydrogen peroxide (or organic peroxides) to water (or alcohols), respectively, requiring gsh consumption. it also protects phagocytes from the deleterious effects of reactive oxygen species produced during the respiratory burst. its activity is measured by the consumption of nadph monitored at 340 nm (ε = 6.22 mm-1 cm-1) using h2o2 or cumene hydroperoxide as substrates, for the se-dependent or the sum of se-dependent and se-independent forms, respectively. the rate of blank reaction is substracted from the total rate (gunzler and flohe, 1985). the reaction is performed in a final volume of 1 ml containing 500 µl of 0.1 m phosphate buffer, ph 7.0, 1 mm edta, 0.1 % nahco3, 100 µl of 2.4 u/ml glutathione reductase (sigma), 100 µl of 10 mm gsh, 100 µl hemolysate. the reaction is initiated by the addition of 100 µl of 0.03 % h2o2 in distilled water or 100 µl of 25 mm cumene hydroperoxide (sigma) in ethyl alcohol, respectively. absorbances are read at 10 s intervals for 2 min, and enzyme activities is expressed as nmol/min/mg protein (mean ± sd) and it is calculated with the formula reported above. protein concentration is measured according to bradford (1976) using bsa as standard. spectrophotometric assay for caspases whole blood from large colonies of b. schlosseri (≥ 10 systems) that have been previously blotted dry is collected and centrifuged at 780xg for 10 min. supernatants are discarded and the pellets resuspended in 100 μl of the lysis buffer from colorimetric activity assay kits for caspase-3 and -8 (chemicon). after a 10 min incubation, samples are centrifuged at 10,000xg for 10 min. supernatants, referred to as hemocyte lysates, are collected and their protein content evaluated according to bradford (1976). in the wells of a 96-well microtiter plate, 50 μl hemocyte lysate are incubated for 1 h at 37 °c with 10 μl of the specific colorimetric substrates n-acetyl-asp-glu-val-asp-p-nitroaniline or n-acetyl-ile-glu-thr-asp-p-nitroaniline, for caspase-3 and -8, respectively, according to the manufacturer’s instructions. the release of pnitroaniline is measured at 405 nm in a microplate reader. a p-nitroaniline reference curve is obtained by serial dilution of a 10 mm standard solution in dimethylsulfoxide. one unit of caspase activity is defined as the amount of enzyme able to cleave 1 nmole substrate per hour at 37 °c. the results are expressed as specific activities (u/mg protein). spectrophotometric assay for lysozyme activity lysozyme activity is quantified in cell lysate. in this case, hemolymph is collected from blood vessels without previous dipping in na-citrate, placed in 1.5 ml plastic tubes, and centrifuged at 780xg for 10 min. hemocytes are then incubated at 20 °c for 60 min in fsw and then centrifuged at 780xg for 10 min, re-suspended in 1 ml of distilled water, sonicated at 0 °c for 2 min at 50 % duty cycles, and centrifuged at 12,000xg for 15 min at 4 °c. supernatant, corresponding to cell lysate, is collected for the lysozyme assay. fifty μl of cell lysate, from both controls and treated hemocytes, are added to 950 μl of a 0.15 % suspension of m. lysodeikticus (sigma) in 66 mm phosphate buffer, ph 6.2, and the decrease in absorbance (da)/min is continuously recorded at 450 nm for 5 min at 20 °c, according to santarém et al. (1994). standard solutions containing 1, 2.5, 5 and 10 μg/ml lysozyme in phosphate buffer are prepared from crystalline hen egg white lysozyme (sigma). the average decrease in absorbance/min is determined for each enzyme solution and a standard curve of enzyme concentration versus da/min is drawn. one unit of lysozyme is defined as the amount of cell lysate equivalent to 1 μg of lysozyme, in the conditions (abssample – absblank) x final volume ______________________________ 1000 x ε x mg protein x ml substrate 210 described above. results are expressed as μg lysozyme/mg protein. protein concentration in cell lysate is quantified according to bradford (1976). assays for detection of reactive oxygen species superoxide anion in cytochemical assay, modified after song and hsieh (1994), yeast cells prepared for phagocytosis assay are resuspended in 0.3 % nitroblue tetrazolium (sigma) in fsw. sixty µl of this suspension are added to each culture chamber. after incubation for 60 min, hemocyte monolayers are fixed, washed and mounted as previously described for the phagocytosis assay. positive sites stain blue. yeast is omitted in negative controls, while 150 u superoxide dismutase (sigma) are added to the incubation medium as a control of specificity. the superoxide anion production is evaluated as percentage of hemocytes containing dark blue spots of precipitated formazan. in spectrophotometric assay, after incubation with 0.3 % nitroblue tetrazolium the hemocyte monolayers are washed and dipped in absolute methanol for 2 min before being air-dried. eighty µl of a solution of 2 m koh and dimethylsulfoxide (ratio 6:7) are added to the monolayers to dissolve formazan precipitates and recovered after 5 min. the absorbance at 620 nm is then read with a microplate reader. hydrogen peroxide collected hemocytes are centrifuged at 780xg for 15 min and resuspended in a freshly prepared hydrogen peroxide reaction mixture [200 µl of 1 % phenol red and 200 µl of 200 u/mg peroxidase (grade ii, roche) in 9.6 ml of fsw] (pick, 1986) containing yeast. at the end of the incubation period, suspensions are centrifuged at 780xg for 15 min, 100 µl of 1 n naoh are added to the supernatants and their absorbance at 620 nm is read with a microplate reader. hypochlorite ions washed hemocytes are resuspended in 1.87 % taurine (sigma) and yeast in fsw. after 60 min, 0.5 µl of a 50 mg/ml solution of catalase (sigma) are added to stop the reaction and suspensions are centrifuged at 780xg for 15 min. twenty mm ki is added to the supernatants and absorbance at 350 nm is read with a spectrophotometer (gressier et al., 1994). the thiol-containing antioxidant sodium 2mercaptoethane sulfonate (mesna; sigma) is added to the incubation medium at a concentration of 10 mm as a control for specificity. nitrite ions washed hemocytes are resuspended in yeastcontaining fsw and, at the end of the incubation period, they are centrifuged at 780xg for 15 min and 100 µl of supernatants are incubated for 10 min with 100 µl of griess reagent [equal volumes of 0.1 % naphtylethylenediamine (sigma) in distilled water and 1 % sulfanilamide (sigma) in 5 % h3po4]. the absorbance at 550 nm is then read with a microplate reader (shen et al., 1994). a precalibrated standard curve, with nano2 as standard, is used to calculate nitrite concentrations. yeast is omitted in controls. protein purification and analysis protein analysis by mean of electrophoresis and immunoblotting is usually applied in invertebrate models following protocols that are very similar to those realized in mammals. for instance, the common sds-page method (laemmli, 1970) followed by the blotting procedure (towbin, et al., 1979) has been successfully applied in mollusca (malagoli et al., 2003), insecta (malagoli et al., 2002a) and tunicata (gasparini et al., 2008). therefore, we report here only a method that has been applied to purify specific lectins from b. schlosseri hemolymph, since from protein purification onwards, the methods used do not significantly differ from invertebrate and mammalian models. affinity chromatography for β-galactosiderecognizing lectins various pools of 10-20 large (500-800) zooid colonies of b. schlosseri are homogenized in a homogenizer in 10 ml of a solution of 10 mm nabenzoate (sigma) in fsw, containing 0.1 mg/ml pepstatin and 1 mg/ml leupeptin (sigma). the homogenate is centrifuged at 2,000xg for 10 min at 4 °c and the supernatant is collected (average protein concentration: 2.5 mg/ml). affinity chromatography is carried out on acid-treated sepharose cl-6b (ge healthcare, chalfont st. giles, uk), previously equilibrated with pbs-3 according to parrinello and canicattì (1982). the column is washed with 1 m nacl, and absorbed proteins are eluted with a solution of 0.2 m dgalactose (sigma) in 1 m nacl. the flow rate is kept constant at 20 ml/h, and 2 ml fractions are collected, the absorbance of which is read at 280 nm. fractions corresponding to single absorbance peaks are pooled, dialyzed overnight against distilled water, vacuum-dried in a vacuum concentrator, and stored at -20 °c. protein content is evaluated according to bradford (1976). concluding remarks the majority of procedures developed for invertebrates and here reported find their counterpart in vertebrates, even if modifications to the canonic vertebrate protocols are needed. however, on the whole it emerges an important observation, i.e., the conservation in invertebrate and vertebrate species of the molecules involved in the immuneneuroendocrine interactions. this allows, for instance, that antibodies raised against components of vertebrate molecules can in most cases be used also for evidencing invertebrate molecules. however, nowadays, the immunoreactivity alone cannot be considered a sufficient indication to prove the existence of homologies between vertebrate and invertebrate molecules. the fundamental results collected by mean of careful morphological observations, as well as functional assays, should be integrated with molecular-biology and/or bioinformatics approaches whenever possible. 211 references abrams jm. the drosophila embryo as a model for the study of programmed cell death. in: cotter tg, martin sj (eds), techniques in apoptosis. a user’s guide. portland, london, pp 211-224, 1997. alper s, laws r, lackford b, boyd wa, dunlap p, freedman jh, et al. identification of innate immunity genes and pathways using a comparative genomics approach. proc. natl. acad. sci. usa 105: 7016-7021, 2008. bachère e, gueguen y, gonzalez m, de lorgeril j, garnier j, romestand b. insights into the antimicrobial defense of marine invertebrates: the penaeid shrimps and the oyster crassostrea gigas. immunol rev. 198: 149-168, 2004. bacsó z, everson rb, eliason jf. the dna of annexin v-binding apoptotic cells is highly fragmented. cancer res. 60: 4623-4628, 2000. ballarin l, cima f. cytochemical properties of botryllus schlosseri haemocytes: indications for morpho-functional characterisation. eur. j. histochem. 49: 255-264, 2005. ballarin l, cima f, sabbadin a. phagocytosis in the colonial ascidian botryllus schlosseri. dev. comp. immunol. 18: 467-481, 1994. ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri. dev. comp. immunol. 22: 479-492, 1998. ballarin l, menin a, franchi n, bertoloni g, cima f. morula cells and non-self recognition in the compound ascidian botryllus schlosseri. inv. surv. j. 2: 1-5, 2005. ballarin l, tonello c, guidolin l, sabbadin a. purification and characterization of a humoral opsonin, with specificity for d-galactose, in the colonial ascidian botryllus schlosseri. comp. biochem. physiol. 123b: 115-123, 1999. bancroft jd, gamble m. theory and practice of histological techniques. churchill livingstone, london, 2002. barka t, anderson pj. histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. j. histochem. cytochem. 10: 741-753, 1962. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. anal. biochem. 72: 248-254, 1976. bréhelin m, roch p. specificity, learning and memory in the innate immune response. inv. surv. j. 5: 103-109, 2008. broudy vc. stem cell factor and hematopoiesis. blood, 90: 1345-1364, 1997. burstone ms. enzyme histochemistry and its application in the study of neoplasms. new york, academic press, 1962. callis g, bone m. in: bancroft jd, gamble m (eds), theory and practice of histological techniques. churchill livingstone, london, pp 269–301, 2002. cammarata m, arizza v, parrinello n, candore g, caruso c. phenoloxidase-dependent cytotoxic mechanism in ascidian (styela plicata) hemocytes active against erythrocytes and k562 tumor cells. eur. j. cell. biol. 74: 302307, 1997. caselgrandi e, kletsas d, ottaviani e. neutral endopeptidase-24.11 (nep) deactivates pdgf and tgfβ-induced cell shape changes in invertebrate immunocytes. cell biol. int. 24: 8590, 2000. chayen j, bitensky l, butcher rg, poulter lw. a guide to practical histochemistry. oliver and boyd, edinburgh, 1969. cima f, ballarin l. tbt-induced apoptosis in tunicate haemocytes. appl. organometal. chem. 13: 97-703, 1999. cima f, ballarin l, sabbadin a. new data on phagocytes and phagocytosis in the compound ascidian botryllus schlosseri. ital. j. zool. 63: 357-364, 1996. cima f, brena c, burighel p. multifarious activities of gut epithelium in an appendicularian (oikopleura dioica: tunicata). mar. biol. 141: 479-490, 2002a. cima f, dominici d, ballarin l, burighel p. influence of tributyltin on activity of detoxifying enzymes from haemocytes of a colonial ascidian. fresenius environ. bull. 11: 573-577, 2002b. cima f, perin a, burighel p, ballarin l. morphofunctional characterization of haemocytes of the compound ascidian botrylloides leachi (tunicata, ascidiacea). acta zool. (stockh.) 82: 261-274, 2001. cookson mr, slamon nd, pentreath vw. glutathione modifies the toxicity of triethyltin and trimethyltin in c6 glioma cells. arch. toxicol. 72: 197-202, 1998. cooper el, franchini a, ottaviani e. earthworm coelomocytes possess immunoreactive cytokines and pomc-derived peptides. anim. biol. 4: 25-29, 1995. cunningham aj, szenberg a. further improvements in the plaque technique for detecting single antibody-forming cells. immunology 14: 599-600, 1968. davis bj, ornstein l. high resolution enzyme localisation with a new diazo reagent, “hexazonium pararosaniline”. j. histochem. cytochem. 7: 297-298, 1959. deschodt-lanckman m, vanneste y, loir b, michel a, libert a, ghanem g, et al. degradation of alpha-melanocyte stimulating hormone (alphamsh) by calla/endopeptidase 24.11 expressed by human melanoma cells in culture. int. j. cancer 46: 1124-1130, 1990. feinberg ap, vogelstein b. a technique for radiolabeling dna restriction endonuclease fragments to high specific activity. anal. biochem. 132: 6-13, 1983. flückiger r, paz ma, gallop pm. redox-cycling detection of dyalizable pyrroloquinoline quinone and quinoproteins. meth. enzymol. 258: 140149, 1995. 212 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22cammarata%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22arizza%20v%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22parrinello%20n%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22candore%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22caruso%20c%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'eur%20j%20cell%20biol.'); franceschi c, cossarizza a, monti d, ottaviani e. cytotoxicity and immunocyte markers in cells from the freshwater snail planorbarius corneus (l) (gastropoda, pulmonata): implications for the evolution of natural killer cells. eur. j. immunol. 21: 489-493, 1991. franchini a, miyan ja, ottaviani e. induction of acthand tnf-α-like molecules in the hemocytes of calliphora vomitoria (insecta, diptera). tissue cell 28: 587-592, 1996. franchini a, ottaviani e. fine structure and acid phosphatase localization of hemocytes in the freshwater snail viviparus ater (gastropoda, prosobranchia). j. invertebr. pathol. 55: 28-34, 1990. franchini a, ottaviani, e. modification induced by acth in hemocyte cytoskeleton of the freshwater snail viviparus ater (gastropoda, prosobranchia). eur. j. histochem. 38: 145150, 1994. gasparini f, franchi n, spolaore b, ballarin l. novel rhamnose-binding lectins from the colonial ascidian botryllus schlosseri. dev. comp. immunol. 32: 1177-1191, 2008 gerber s, cadet p, sheehan m, stefano gb, mantione kj. vertebrate interleukins originated in invertebrates? inv. surv. j. 4: 95-100, 2007. giulianini pg, bierti m, lorenzon s, battistella s, ferrero ea. ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge. micron 38: 49-57, 2007. giulianini pg, pandolfelli n, lorenzon s, ferrero ea, edomi p. an antibody to recombinant crustacean hyperglycaemic hormone of nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan crustacea. cell tissue res. 307: 243-254, 2002. goldfischer s. the cytochemical demonstration of lysosomal aryl sulfatase activity by light and electron microscopy. j. histochem. cytochem. 13: 520–523, 1965. graham rc, karnovsky m j. the early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. j. histochem. cytochem. 14: 291302, 1966. gressier b, cabanis a, lebegue s, brunet c, dine t, luyckx m, cazin m, cazin jc. decrease of hypochlorous acid and hydroxyl radical generated by stimulated human neutrophils: a comparison in vitro of some thiol-containing drugs. meth. find. exp. clin. pharmacol. 16: 913, 1994. grimaldi a, bianchi c, greco g, tettamanti g, douglas mn, valvassori r, de eguileor m. in vivo isolation and characterization of stem cells with diverse phenotypes using growth factor impregnated biomatrices. plos one 3: e1910, 2008. grimm e, bonavida b. mechanism of cell-mediated cytotoxicity at the single cell level. i. estimation of cytotoxic t lymphocyte frequency and relative lytic efficiency. j. immunol. 123: 28612869, 1979. gunzler wa, flohe l. glutathione per-oxidase. in: greenwald er (ed) crc handbook of methods for oxygen radical research. crc press, boston, pp 285-290, 1985. habig wh, pabst, mj, jacoby wb. glutathione stransferases the first enzymatic step in mercap-turic acid formation. j. biol. chem. 249: 7130-7139, 1974. hall kt, boumsell l, schultze jl, boussiotis va, dorfman dm, cardoso aa, et al.. human cd100, a novel leukocyte semaphorin that promotes b-cell aggregation and differentiation. proc. natl. acad. sci. usa 93: 11780-11785, 1996. holyoake tl, alcorn mj. cd34+ positive haemopoietic cells: biology and clinical applications. blood rev. 8: 113-124, 1994. hooper c, day r, slocombe r, handlinger j, benkendorff k. stress and immune responses in abalone: limitations in current knowledge and investigative methods based on other models. fish shellfish immunol. 22: 363-379, 2007. hose je, martin gg, nguyen va, lucas j, rosenstein at. cytochemical features of shrimp hemocytes. biol. bull. 173: 178-187, 1987. kamer i, rinkevich b. in vitro application of the comet assay for aquatic genotoxicity: considering a primary culture versus a cell line. toxicol. in vitro 16: 177-184, 2002 kleinman hk, mcgarvey ml, hassell jr, star vl, cannon fb, et al. basement membrane complexes with biological activity. biochemistry 25: 312-318, 1986. laemmli uk. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227: 680-685, 1970. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. lojda z. studies on glycyl-proline naphthylamidase. i. lymphocytes. histochemistry 54: 299-309, 1977. lojda z, gossrau r, schieblet th. enzyme histochemistry, a laboratory manual. springerverlag, berlin, heidelberg, new york, 1979. lorenzon s, de guarrini s, smith vj, ferrero ea. effects of lps injection on circulating haemocytes in crustaceans in vivo. fish shellfish immunol. 9: 31-50, 1999. lorenzon s, edomi p, giulianini pg, mettulio r, ferrero ea. variation of crustacean hyperglycemic hormone (chh) level in the eyestalk and hemolymph of the shrimp palaemon elegans following stress. j. exp. biol. 207: 4205-4213, 2004. lorenzon s, giulianini pg, libralato s, martinis m, ferrero ea. stress effect of two different transport systems on the physiological profiles of the crab cancer pagurus. aquaculture 278: 156-163, 2008. lorenzon s, giulianini pg, martinis m, ferrero ea. stress effect of different temperatures and air exposure during transport on physiological profiles in the american lobster homarus americanus. comp. biochem. physiol. 147a: 94-102, 2007. 213 lozzio cb, lozzio bb. human chronic myelogenous leukemia cell-line with positive philadelphia chromosome. blood 45: 321-334, 1975. mäkelä o, hemagglutinins of leguminous seeds. ann. med. exp. biol. fenn. 35 (suppl. 11): 1156, 1957. malagoli d, casarini l, sacchi s, ottaviani e. stress and immune response in the mussel mytilus galloprovincialis. fish shellfish immunol. 23: 171-177, 2007. malagoli d, conte a, ottaviani e. protein kinases mediate nitric oxide-induced apoptosis in the insect cell line iplb-ldfb. cell. mol. life sci. 59: 894-901, 2002a. malagoli d, gobba f, ottaviani e. effects of 50-hz magnetic fields on the signalling pathways of fmlp-induced shape changes in invertebrate immunocytes: the activation of an alternative "stress pathway". biochim. biophys. acta 1620: 185-90, 2003. malagoli d, iacconi i, marchesini e, ottaviani e. cell death mechanisms in the iplb-ldfb insect cell line: a nuclear located bcl-2-like molecule as a possible controller of 2-deoxy-d-ribosemediated dna fragmentation. cell tissue res. 320: 337-343, 2005. malagoli d, mandrioli m, ottaviani e. cloning and characterisation of a procorticotrophin-releasing hormone in the izd-mb-0503 immunocyte line from the insect mamestra brassicae. peptides 23: 1829-1836, 2002b. malagoli d, marchesini e, ottaviani e. lysosomes as the target of yessotoxin in invertebrate and vertebrate cell lines. toxicol. lett. 167: 75-83, 2006. malagoli d, ottaviani e. helical cytokines and invertebrate immunity: a new field of research. scand. j. immunol. 66:484-485, 2007. manske m, bade eg. growth factor-induced cell migration: biology and methods of analysis. int. rev. cytol. 155: 49-96, 1994. martin sj, reutelingsperger cpm, green dr. annexin v: a specific probe for apoptotic cells. in: cotter tg, martin sj (eds), techniques in apoptosis. a user’s guide. portland, london, pp 107-119, 1997. mazzi v. manuale di tecniche istologiche e istochimiche. piccin ed., padova, 1977. menin a, del favero m, cima f, ballarin l. release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian. mar. biol. 148: 225230, 2005. mettulio r, giulianini pg, ferrero ea, lorenzon s, edomi p. functional analysis of crustacean hyperglycemic hormone by in vivo assay with wild-type and mutant recombinant proteins. regul. pept. 119: 189-197, 2004. michibata h, hirata j, uesaka m, namakunai t. separation of vanadocytes: determination and characterization of vanadium ions in the separated blood cells of the ascidian, ascidia ahodori. j. exp. zool. 24: 33-38, 1987 moloney wc, mcpherson k, fliegelman l. esterase activity in leukocytes demonstrated by the use of naphthol as-d chloroacetate substrate. j. histochem. cytochem. 8: 200-207, 1960. moore rd, mumaw v, shomberg md. optical microscopy of ultrathin tissue sections. j. ultrastruct. res. 4: 113-116, 1960. novikoff ab, goldfischer s. visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. j. histochem. cytochem. 17: 675-680, 1969. ottaviani e. composition of the serum haemolymph of planorbis corneus (gastropoda, pulmonata). comp. biochem. physiol. 78b: 585-589, 1984. ottaviani e. the blood cells of the freshwater snail planorbis corneus (gastropoda, pulmonata). dev. comp. immunol. 7: 209-216, 1983. ottaviani, e. haemocytes of the freshwater snail viviparus ater (gastropoda, prosobranchia). j. moll. stud. 55: 379-382, 1989. ottaviani, e. immunocytochemical study on bacterial elimination from the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). zool. jb. anat. 120: 57-62, 1990. ottaviani e, caselgrandi e. neutral endopeptidase24.11 (nep)-like activity in molluscan hemocytes. peptides 18: 1107-1110, 1997. ottaviani e, caselgrandi e, franceschi c. cytokines and evolution: in vitro effects of il-1α, il-1β, tnf-α and tnf-β on an ancestral type of stress response. biochem. biophys. res. commun. 207: 288-292, 1995a. ottaviani e, caselgrandi e, franchini a, franceschi c. crf provokes the release of norepinephrine by hemocytes of viviparus ater (gastropoda, prosobranchia): further evidence in favour of the evolutionary hypothesis of the "mobile immune-brain". biochem. biophys. res. commun. 193: 446-452, 1993. ottaviani e, caselgrandi e, kletsas d. effect of pdgf and tgfβ on the release of biogenic amines from invertebrate immunocytes and their possible role in the stress response. febs lett. 403: 236-238, 1997a. ottaviani e, caselgrandi e, kletsas d. the crhacth-biogenic amine axis in invertebrate immunocytes activated by pdgf and tgf-β. febs lett. 427: 255-258, 1998a. ottaviani e, caselgrandi e, petraglia f, franceschi, c. stress response in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata): interaction between crf, acth and biogenic amines. gen. comp. endocrinol. 87: 354-360, 1992. ottaviani e, cossarizza a, ortolani c, monti d, franceschi c. acth-like molecules in gastropod molluscs: a possible role in ancestral immune response and stress. proc. r. soc. lond. b 245: 215-218, 1991. ottaviani e, franchini a, barbieri d, kletsas d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two aging-related hemocyte stages. ital. j. zool. 65: 349-354, 1998b. ottaviani e, franchini a, caselgrandi e, cossarizza a, franceschi c. relationship between corticotropin-releasing factor and interleukin-2: evolutionary evidence. febs lett. 351: 19-21, 1994. ottaviani e, franchini a, cassanelli s, genedani s. cytokines and molluscan immune responses. biol. cell 85: 87-91,1995. ottaviani e, franchini a, hanukoglu i. in situ localization of acth receptor-like mrna in molluscan and human immunocytes. cell. mol. life sci. 54: 139-142, 1998c 214 ottaviani e, franchini a, malagoli d, genedani s. immunomodulation by recombinant human interleukin-8 and its signal transduction pathways in invertebrate hemocytes. cell. mol. life sci. 57: 506-513, 2000b. ribeiro c, brehélin m. insect haemocytes: what type of cell is that? j. insect physiol. 52: 417429, 2006. santarém mm, robledo jaf, figueras a. seasonal changes in hemocytes and serum defense factors in the blue mussel mytilus galloprovincialis. dis. aquat. org. 18: 217-222, 1994. ottaviani e, montagnani g. immunodetection of haemocytes subpopulations by nacetylmuramic acid antibody in planorbarius corneus (l.) (gastropoda, pulmonata). histochem. j. 21: 675-678, 1989. schön jc, torre-bueno j, stefano gb. microscopic computer-assisted analysis of conformational state: reference to neuroimmunology. adv. neuroimmunol. 1: 252-259, 1991. ottaviani e, petraglia f, montagnani g, cossarizza a, monti d, franceschi c. presence of acth and β-endorphin immunoreactive molecoles in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata) and their possible role in phagocytosis. regul. pept. 27: 1-9, 1990. shen hm, sha lx, kennedy jl, ou dw. adrenergic receptors regulate macrophage secretion. int. j. immunopharmacol. 16: 905-910, 1994. singh np, mccoy mt, tice rr, scneider el. a simple technique for quantitation of low levels of dna damage in individual cells. exp. cell. res. 175: 184-191, 1988. ottaviani e, franchini a, caselgrandi e, cossarizza a, franceschi c. relationship between corticotropin-releasing factor and interleukin-2: evolutionary evidence. febs lett. 351: 19-21, 1994. söderhäll i, kim ya, jiravanichpaisal p, lee sy, söderhäll k. an ancient role for a prokineticin domain in invertebrate hematopoiesis. j. immunol. 174: 6153-6160, 2005. ottaviani e, franchini a, cassanelli s, genedani s. cytokines and molluscan immune responses. biol. cell 85: 87-91,1995. song yl, hsieh yt. immunostimulation of tiger shrimp (penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species. dev. comp. immunol. 18: 201-209, 1994. ottaviani e, sassi d, kletsas d. pdgfand tgf-βinduced changes in cell shape of invertebrate immunocytes: effect of calcium entry blockers. eur. j. cell biol. 74: 336-341, 1997b. tamaki t, akatsuka a, ando k, nakamura y, matsuzawa h, et al. identification of myogenicendothelial progenitor cells in the interstitial spaces of skeletal muscle. j. cell biol. 157: 571-577, 2002. parrinello n, cammarata m, arizza v. univacuolar refractile hemocytes from the tunicate ciona intestinalis are cytotoxic for mammalian erythrocytes in vitro. biol. bull. 190: 418-425, 1996. towbin h, staehelin t, gordon j. electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. proc. natn. acad. sci. usa 76: 4350-4354, 1979. parrinello n, canicattì c carbohydrate binding specificity and purification by biospecific affinity chromatography of ascidia malaca traust. hemagglutinins. dev. comp. immunol. 6: 53-64, 1982. wachstein m, meisel e. histochemistry of hepatic phosphatases at a physiological ph with special reference to the demonstration of bile canaliculi. am. j. clin. pathol. 27: 13-23, 1957. pick e. microassay for superoxide and hydrogen peroxide productionandnitroblue tetrazolium reduction using an enzyme immunoassay microplate reader. meth. enzymol. 132: 407421, 1986. winder aj, harris h. new assays for tyrosine hydroxylase and dopa oxidase activities of tyrosinase. eur. j. biochem. 198: 317-326, 1991. raftos da, stillman dl, cooper el. chemotactic responses of tunicate (urochordata, ascidiacea) hemocytes in vitro. j. invertebr. pathol. 72: 44-49, 1998. wittwer d, franchini a, ottaviani e, wiesner a. presence of il-1and tnf-like molecules in galleria mellonella (lepidoptera) haemocytes and in an insect cell line from estigmene acraea (lepidoptera). cytokine 11: 637-642, 1999. raikhinstein m, zohar m, hanukoglu i. cdna cloning and sequence analysis of the bovine adrenocorticotropic hormone (acth) receptor. biochim. biophys. acta 1220: 329-332, 1994. 215 isj 5: 43-xx, 2008 isj 5: 43-49, 2008 issn 1824-307x review effects of interleukin-2 on nitric oxide production in molluscan innate immunity r barcia, ji ramos-martinez departamento de bioquímica y biología molecular, facultad de veterinaria, universidad de santiago de compostela, lugo, spain accepted april 29, 2008 summary the hemocytes are the cells responsible for the immunity in molluscs. cytokines, growth factors, etc, present in the soluble fraction of the hemolymph modulate the immune response. the inflammatory cytokine interleukin-2 (il-2) induces the synthesis of nitric oxide (no), responsible for oxidizing processes. in hemocytes of mollusks, the presence of il-2 induces the synthesis of the three subunits of the receptor of high affinity, and the subsequent activation of the signaling pathway where the camp-dependent protein kinase (pka) plays the main role, with a secondary role of pkc. in the presence of il-2, pka activates no synthesis through the constitutive enzyme mc-nos. in winter, the action of pkc on a novel inducible form mw-nos leads to an increase of the il-2-induced no synthesis. according to our studies, this set of metabolic reactions explains the seasonal variations in no production by hemocytes of mytilus galloprovincialis. however, after two years of studies, and coinciding with the prestige oil spill in november 2002, this effect was cancelled for at least the two years following the catastrophe. key words: hemocyte; immune response; interleukin-2; nitric oxide; mollusc; mytilus galloprovincialis introduction molluscs constitute the widest phylum existing on earth after arthropods, with over 100,000 species present in all the ecosystems. many mollusc species, mainly marine, are very important from the economic point of view because of their use as human food resource. molluscs are also interesting because of their susceptibility to infection by parasites, bacteria, and viruses, which makes them transmitters of many diseases affecting different vertebrate species. finally, their ability to accumulate diverse toxins allows considering several mollusc species as excellent biosensors of the biologic quality of the ecosystems. unlike vertebrates, molluscs only have an ancestral defensive line, comparable to that known as immune innate system in vertebrates (medzhitov and janeway, 2000; plows et al., 2005), and constituted by a cellular component and the products that it generates (i.e. humoral component). ___________________________________________________________________________ corresponding author: juan ignacio ramos-martinez departamento de bioquímica y biología molecular, facultad de veterinaria, universidad de santiago de compostela, e-27002 lugo, spain e-mail: bniramos@lugo.usc.es the circulating cells, called hemocytes, are responsible for the phagocytosis, cytotoxic reactions, and the synthesis of the humoral factors, comprising antimicrobial peptides, agglutinins, lectins, cytokines, nitric oxide, etc. (ottaviani, 2006). due to their immune activity, hemocytes can be also referred to as immunocytes. although molluscan immunocytes show a wide heterogeneity, the cells involved in the defensive processes are similar to the macrophages from vertebrates. their morphological features include an irregular shape, a small nucleus in relation to cell size and presence of numerous granules (krupa et al., 1977; joky et al., 1983; ottaviani and franchini, 1988; ottaviani et al., 1998a; cao et al., 2003). moreover, molluscan hemolymph contains other hemocytes, roundish and with a bigger nucleus. in the particular case of mytilus galloprovincialis, both cell types seem to correspond to two maturation stages of the same cell (ottaviani et al., 1998a). as far as the humoral component is concerned, it promotes several responses involving cell shape changes, chemotaxis, phagocytosis, cytotoxicity, encapsulation, and neuroendocrine responses, thus suggesting the involvement of stress mediators in the immune response (for review see, ottaviani, 2006; ottaviani et al., 2007). 43 mailto:bniramos@lugo.usc.es in invertebrates, physiologic responses suggesting the presence of molecules with cytokinelike functions have been observed in molluscs, insects, annelids, echinoderms, and tunicates (ottaviani et al., 1995a, 2004; ottaviani, 2006). as for molluscs, cytokine-like molecules with immune and neuroendocrine functions have been detected in both marine and freshwater species. in this sense, heterologous cytokines provoking cell shape changes, chemotaxis or phagocytosis are also capable to induce the synthesis of biogenic amines (ba), nitric oxide (no) or oxygen radicals. these results suggest the existence of an ancestral immune-mobile brain that imitates the hypothalamicpituitary-adrenal axis (hpa) from vertebrates (ottaviani et al., 1993a; ottaviani, 2004, 2006). heterologous cytokines, growth factors, and cell differentiating factors increase epinephrine, norepinephrine and dopamine synthesis in molluscan hemocytes, both in those freshly extracted and those cultured for several days (franchini et al., 2000; cao et al., 2003, 2004b, 2007b). interleukin-2 (il-2) receptor in molluscan hemocytes early studies on the involvement of diverse cytokines in the activation of the immune response in molluscan hemocytes suggest the existence of a receptor with low specificity (ottaviani et al., 1994, 1995a, b). il-2 was one of the cytokines assayed, as it induces phagocytosis and provokes the strongest response in the synthesis of ba, no, etc (ottaviani et al., 1995a, b). studies on the interference of the inflammatory cytokine and corticotrophin-releasing hormone (crh) on ba production provided the initial clues about the possible presence of an il-2 receptor (ottaviani et al., 1994). subsequent studies on hemocytes of m. galloprovincialis allowed the detection of the three subunits constituting the il-2 receptor (barcia et al., 1999). flow cytometry confirmed the presence of a subunit of high affinity, termed il-2rα, and two of low affinity, termed il-2rβ and il-2rγ, which only one group of hemocytes expresses (barcia et al., 1999). also lipopolysaccharide (lps) induces the expression of the il-2 receptor, which can be explained as a collateral effect, similar to that detected in vertebrates, as lps might stimulate the hemocytes to synthesize molecules comparable to tnf-α, and the necrosis factor might in its turn induce il-2 the synthesis of molecules structurally/functionally related to il-2 (barcia et al., 1999). nitric oxide production in molluscs nitric oxide (no) is a gas that performs multiple biologic functions. among them outstands its character of intra and extracellular signal (moncada et al., 1991). in recent years, several studies have proved the presence of nitric oxide synthase (nos) and the involvement of no in cellular signaling in organisms dispersed through the whole animal kingdom (palumbo, 2006). the involvement of no in fig. 1 effect of different representative protein kinase inhibitors on the stimulation of no production by il-2. control: non-activated hemocytes. *compared with control p< 0.001. ** compared with il-2 p< 0.001. *** compared with il-2. (modified from novas et al., 2004). such processes as memory (korneev et al., 2005), bioluminescence (trimmer et al., 2001), sight (elphick et al., 1996), or cell proliferation (kuzin et al., 1996) was studied in invertebrates. particularly, in molluscs, no and the different forms of nos seem to be involved in the modulation of neuron activity (gelperin, 1994; moroz et al., 1996; hurst et al., 1999; stefano and ottaviani, 2002), metamorphosis (leise et al., 2004) and preferably in processes related to the immune defense (ottaviani, 2006). no may act directly, but it also reacts with free oxygen radicals, thus generating peroxinitrite (onoo-), a potent oxidizing agent whose action affects the peroxidation of the membrane lipids, the inhibition of tricarboxylic acid (tca) cycle, and the mitochondrial respiration (nappi and ottaviani, 2000). molluscan hemocytes remove bacteria by means of phagocytosis and no production. both processes are interdependent, although phagocytosis seems to happen before no synthesis. this means that the bacterial clumping takes place prior to no elimination. hemocyte incubation with sodium nitroprusside provokes bacterial clumping, and lps also display a similar action (ottaviani et al., 1993b; franchini et al., 1995; tafalla et al., 2002). in addition, incubation of hemocytes from the mussel m. galloprovincialis or the oyster crassostrea gigas with phorbol myristate acetate (pma) or laminarin leads to the synthesis of no and superoxide ions. these induce the synthesis of peroxinitrite, which has the cytotoxic properties commented above (arumugan et al., 2000, 44 fig. 2 no synthesis by haemocytes of mytilus galloprovincialis collected in winter and summer. a) the cells were cultured for 3 days in l-15 medium and then incubated for 24 h with the referred cytokines as expressed in novas et al., 2007c. the bars represent the standard deviation of six individual assays. results of a representative assay of each season. tumor necrosis factor-α (tnf-α); platelet-derived growth factor (pdgf); transforming growth factor-β1 (tgf-β1). 2001; gourdon et al., 2001; torreilles and romestand, 2001). these authors, however, discard lps as inducer of no synthesis. the existence of nos activity in molluscan hemocytes was proved in viviparus ater. the activity was stimulated by lps and was located in the soluble fraction of hemolymph (conte and ottaviani, 1995). the use of nos inhibitors promoted the canceling of the bacterial clumping induced by lps in the molluscan hemocytes of m. edulis and v. ater (ottaviani et al., 1993b). studies performed in hemocytes from freshwater molluscs show that the incubation with different cytokines induces a significant increase of no production (ottaviani et al., 1995a). similar works performed with hemocytes from marine molluscs offer similar results, with il-2 as the cytokine inducing the maximal response (ottaviani et al., 1995a). the results are similar, either if the hemocytes were freshly extracted or if they were subjected to culture for several days, and this was related to the capability of molluscan hemocytes to express the complete structure of the il-2 receptor (barcia et al., 1999; novas et al., 2004). during culturing, the hemocytes from m. galloprovincialis keep their ability to synthesize no induced by il-2. the results about the effect of lps on no production are different, maybe because of the use of lps of diverse origins lps does not induce the no synthesis in hemocytes of mytilus (arumugan et al., 2000, 2001; gourdon et al., 2001; torreilles and romestand, 2001), a different result to the obtained with cells of other models (conte and ottaviani, 1995). mytilus hemocytes respond to the presence of mammalian il-2 synthesizing ba and no, although the difference between the kinetics of no and ba synthesis (cao et al., 2004a, b; novas et al., 2004, 2007b) suggest different pathways of signal internalization. apparently, ba synthesis involves preferably the enzyme protein kinase c (p105) (cao et al., 2004a, 2007a), whereas the camp-dependent protein kinase (pka) plays a secondary role (cao et al., 2004a). both enzymes were purified from different tissues of m. galloprovincialis (mercado et al., 2002a, b, 2003; diaz-enrich et al., 2003; bardales et al., 2004), which led to the obtaining of specific antibodies that allowed learning more about the function of these enzymes in the action of il-2 on no production. as commented above, phorbol 12-myristate 13acetate (tpa) induces no synthesis, thus involving pkc in the process (arumugan et al., 2000, 2001; gourdon et al., 2001; torreilles and romestand, 2001). since il-2 has the same effect of tpa, all suggests that pkc is involved in the action induced by il-2. similar works on freshwater molluscs such as lymnaea stagnalis link pkc and erk (extracellular signal-regulated kinase) to the signaling mechanisms of the regulation of nos activity (wright et al., 2006). despite the involvement of pkc in mediating the signal borne by il-2, further experiments with specific pk inhibitors indicate that the signaling pathway by which il-2 induces no synthesis preferably involves pka. the action of inhibitors specific for these protein kinases suggests that the signal of the il-2-induced no synthesis preferably involves pka (fig. 1). the kinase activates a form of nos specific of mytilus hemocytes (novas et al., 2004), initially identified as a form of 130 kda and immunologically similar to inos from vertebrates (novas et al., 2004). the different kinetics of ba and no production that the cell shows in the presence of il-2 seems to have its base on the different protein kinase involved in each process. as it happens in many aspects of the physiology of marine molluscs (robledo et al., 1995; ramos-martinez et al., 1993), hemocyte immune response shows a seasonal variation in basal and il-2-induced no synthesis (novas et al., 2007c). fig. 3 il-2-induced production of no. control: basal non-stimulated hemocytes. il-2: hemocytes incubated with il-2. the pkc inhibitor bisindolylmaleimide (bsm) was added to the cells simultaneously with il-2. error bars are the sd of six assays as described in novas et al., 2007b. 45 fig. 4 schematic diagram illustrating the hypothetical implication of pka and pkc in the two seasonal pathways of hemocyte il-2-induced no synthesis. dotted lines represent the inhibiting effect of down-regulation (novas et al., 2007b). no basal production is higher during the summer months, whereas il-2-induced no synthesis by cells obtained in winter is 5-fold that of the cells obtained in summer (fig. 2). when studying the effect of pkc inhibitors on the seasonal variation of il-2-induced no production, pka appeared as the major activating agent of the mytilus constitutivenos (mc-nos) (novas et al., 2004). also, the results about no seasonal production in the presence of bisindolylmaleimide (bsm) suggest a participation of pkc that would be compatible with the hypothesis expressed in fig. 4 (novas et al., 2007b). pkc appears as a potent mc-nos inhibitor. in summer, and as a consequence of the presence of il-2, takes place pkc inhibition by downregulation, which implicates pka in keeping an activating action of no synthesis. the action described persists in winter, but in this season, a new nos-inducible enzyme was detected. this was termed mytilus winter-nos (mw-nos) and has similarities with the constitutive form of vertebrates susceptible of activation by pkc (fig. 4). pkc activating action on the new mw-nos inducible enzyme explains the result presented in fig. 3. concluding remarks in marine molluscs, there is a remarkable biologic paradox such as the fact that the widest mortalities occur in the seasons with most abundant food and when the immunologic potential is seems at its maximum (summer). apparently, the reason for the high mortality could be the increase of not only food but also parasites and pathogens occurring in phytoplankton blooms (robledo et al., 1995; lacoste et al., 2001; hernroth, 2003). when considering the basal no synthesis, the low production detected in winter is still compatible with a frame of reduced immune response in winter, hich may seem rather odd, considering the high degree of mortality registered in summer. indeed, this supposed immunodepression during wintertime is false, because an inflammatory phenomenon, and especially the increased production of molecules related to il-2 and to the activation of mw-nos trigger the mechanism that ensures the response, despite the low basal levels on no. fig. 5 seasonal variation of nitric oxide production by hemocytes of mytilus galloprovincialis lmk. in 2001 and 2002 the assays were performed during winter (w) and summer (s) (novas et al., 2007c). empty bars represent the basal production and solid bars represent no production of hemocytes incubated with 10 μg/ml il-2 for 24 h. error bars are the sd of 6 assays. 46 the results of the assay on no basal production and, mainly, hemocyte response against il-2 accounts for the utility of mytilus as biosensor, to judge by the consequences of the prestige oil spill in the immune response of the mollusc (ruizvillareal et al., 2006; soriano et al., 2006). as shown in fig. 5, no synthesis ability was stable in the years before the catastrophe; then it drops and does not recover until two years later. the process is especially dramatic if assayed no production by hemocytes treated with il-2. pollution inactivates no basal production, but it also cancels the no production mechanism induced by il-2 during the assay. hemocyte lack of response during assay time is parallel to other assays, such as the study of cell necrosis, apoptosis and others (novas et al., 2007a), and is compatible with a situation of immunodepression (laffon et al., 2006). finally, the test on il-2-induced no production by molluscan hemocytes may join other parameters assayed for the study of the effect of diverse stressing factors on the viability of edible molluscs, considered as biosensors (malagoli et al., 2006). references arumugan m, romestand b, torreilles j, roch p. in vitro production of superoxide and nitric oxide (as nitrite and nitrate) by mytilus galloprovincialis haemocytes upon incubation with pma or laminarin or during yeast phagocytosis. eur. j. cell biol. 79: 513-519, 2001. arumugan m, romestand b, torreilles j. nitrite relase in haemocytes from mytilus galloprovincialis, crassotrea gigas and ruditapes decussatus upon stimulation with phorbol myristate acetate. aquat. living resour. 13: 173-177, 2000. barcia r, cao a, arbeteta j, ramos-martínez, ji. the il-2 receptor in hemocytes of the sea mussel mytilus galloprovincialis lmk. iubmb life 48: 419-423, 1999 bardales jr, díaz-enrich mj, ibarguren i, villamarín ja. isoforms of camp-dependent protein kinase in the bivalve mollusc mytilus galloprovincialis: activation by cyclic nucleotides and effect of temperature. arch. biochem. biophys. 432: 71-78, 2004. cao a, mercado l, ramos-martínez ji, barcia r. primary cultures of hemocytes from mytilus galloprovincialis lmk: expresión of il-2rα subunit. aquaculture 216: 1-8, 2003. cao a, novas a, ramos-martínez ji, barcia r. seasonal variations in haemocyte response in the mussel mytilus galloprovincialis lmk. aquaculture 263: 310-319, 2007a. cao a, ramos-martínez ji, barcia r. implication of pka and pkc in the activation of the haemocytes of mytilus galloprovincialis lmk by lps and il-2. mol immunol. 41: 45-52, 2004a. cao a, ramos-martínez ji, barcia r. in hemocytes from mytilus galloprovincialis lmk., treatment with corticotropin or grow factors conditions catecholamine release int. imunopharmacol. 7: 1395-1402, 2007b. cao a, ramos-martínez ji, barcia r. in vitro effects of lps, il-2, pdgf and crf on haemocytes of mytilus galloprovincialis lmk. fish shelfish immunol. 16: 215-225, 2004b. conte a, ottaviani e. nitric oxide synthase activity in molluscan hemocytes febs lett. 365: 120124, 1995. díaz-enrich mj, ibarguren i, hellman u, villamarín ja. characterization of a type i regulatory subunit of camp-dependent protein kinase from the bivalve mollusc mytilus galloprovincialis. arch. biochem. biophys. 416: 119-127, 2003. elphick m, kemenes g, staras k, o’shea m. new features of the locust optic lobe: evidence of a role for nitric oxide in insect vision. j. exp. biol. 199: 2395-2407, 1996. franchini a, fontanilli p, ottaviani e. invertebrate immunocytes: relationship between phagocytosis and nitric production. comp. biochem. physiol. 110b: 403-407, 1995. franchini a, ottaviani e. repair of molluscan tissue injury: role of pdgf and tgf-β. tissue cell 32: 312-321, 2000. gelperin a nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc. nature 369: 61-63, 1994. gourdon i, guerin mc, torreilles j, roch p. nitric oxide generation by hemocytes of the mussel mytilus galloprovincialis. nitric oxide 1: 1-6, 2001. hernroth b. the influence of temperature and dose on antimicrobial peptide response against lipopolysaccharide in the blue mussel mytilus edulis l. fish shellfish immunol. 14: 25-37, 2003. hurst wj, moroz ll, gillette mu, gillette r. nitric oxide synthase imunolabeling in the molluscan cns and peripheral tissues. biochem. biophys. res. commun. 262: 545-548, 1999. joky a, matricon-gondran m, benex j. fine structural differences in the amoebocytes of biomphalaria glabrata. dev. comp. immunol. 7: 217-228, 1983. korneev sa, straub v, kemenes i, korneeva ei, ott sr, benjamin pr, et al. timed and targeted differential regulation of nitric oxide synthase (nos) and anti-nos genes by reward conditioning leading to long-term memory formation. j. neurosci. 25: 1188-1192, 2002. krupa pl, lewis lm, del vecchio p. schistosoma haermatobium in bulinus guerneri: electrón microscopy of hemocyte-sporocyst interactions. j. invertebr. pathol. 30: 35-45, 1977. kuzin b, roberts i, peunova n, enikolopov g. nitric oxide regulates cell proliferation during drosophila development. cell 87: 639-649, 1996. lacoste a, jalabert f, malham s, cueff a, gelebart f, cordevant c, et al. a vibrio spledidus strain is associated with summer mortality of juvenile oysters crassotrea gigas in the bay of morlaix. dis. aquat organ. 46: 139-145, 2001. laffon b, rábade t, pasaro e, mendez j. monitoring of the impact of prestige oil spill on mytilus galloprovincialis from galicia coast. env. int. 32: 342-348, 2006. 47 http://www.ncbi.nlm.nih.gov/pubmed/15519298?ordinalpos=8&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/15519298?ordinalpos=8&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/12859988?ordinalpos=9&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/12859988?ordinalpos=9&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum leise em, kempf sc, durham nr, gifondorwa dj. induction of metamorphosis in the marine gasteropod ilyanassa obsoleta: 5ht, no and programmed cell death. acta biol. hung. 55: 293-300, 2004 malagoli d, casarini l, sacchi s, ottaviani e. stress and immune response in the mussel mytilus galloprovincialis. fish shellfish immunol. 23: 171-177, 2007. medzhitov r, janeway cajr. innate immune recognition: mechanisms and pathways. immunol. rev. 173: 89-97, 2000. mercado l, cao a, barcia r, ramos-martínez ji. phorbol esters induce translocation of the npkc p105 to membrane in mussel hemocytes. mol. cell biochem. 250: 41-45, 2003. mercado l, cao a, barcia r, ramos-martínez ji. purification of a lipid-activated and ca2+independent protein kinase from the mantle tissue of mytilus galloprovincialis lmk. mol. cell biochem. 233: 99-105, 2002a. mercado l, cao a, barcia r, ramos-martínez ji. regulatory properties of p105: a novel pkc isoenzyme in mantle tissue from marine mussels. biochem. cell biol. 80: 1-5, 2002b. moncada s, palmer rm, higgs ea. nitric oxide: physiology, pathophysiology, and pharmacology. pharmacol. rev. 43: 109-142, 1991. moroz ll, chen d, gillette mu, gillette r. nitric oxide synthase activity in the molluscan cns. j.neurochem. 66: 873-876, 1996. nappi aj, ottaviani e. cytotoxicity and cytotoxic molecules in invertebrates. bioessays 22: 469480, 2000. novas a, barcia r, ramos-martínez ji. after the prestige oil spill modifications in no production and other parameters related to the immune response were detected in hemocytes of mytilus galloprovincialis. aquat. toxicol. 85: 285-290, 2007a. novas a, barcia r, ramos-martínez ji. implication of pkc in the seasonal variation of the immune response of the hemocytes of mytilus galloprovincialis lmk. and its role in interleukin2-induced nitric oxide synthesis. iubmb life 59: 659-663, 2007b. novas a, barcia r, ramos-martínez ji. nitric oxide production by haemocytes from mytilus galloprovincialis shows seasonal variations. fish shellfish immunol. 23: 886-891, 2007c. novas a, cao a, barcia r, ramos-martínez ji. nitric oxide release by hemocytes of the mussel mytilus galoprovincialis lmk was provoked by interleukin-2 but not by lipopolysaccharide. int. j. biochem. cell biol. 36: 390-394, 2004. ottaviani e, caselgrandi e, franceschi c. cytokines and evolution: in vitro effects of il-1α, il-β, tnf-α and tnf-β on an ancestral type of stress response. biochem. biophys. res. commun. 207: 288-292, 1995b. ottaviani e, caselgrandi e, franchini a, franceschi c. crf provokes the release of norepinephrine by hemocytes of viviparus ater (gastropoda, prosobranchia): further evidence in favour of the evolutionary hypothesis of the “mobile immune-brain”. biochem. biophys. acta 193: 446-452, 1993a. ottaviani e, franchini a, ultrastructural study of haemocytes of the freshwater snail planorbarius corneus (l.) (gasteropoda, pulmonata). acta zool. (stock.) 69: 157-162, 1988. ottaviani e, franchini a, barbieri d, kletsas, d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two aging-related hemocytes stages. ital. j. zool. 65: 349-354, 1998a. ottaviani e, franchini a, caselgrandi e, cossarizza, a, franceschi c. relationship between corticotrophin-releasing factor and interleukin-2: evolutionary evidence. febs lett. 351: 19-21, 1994. ottaviani e, franchini a, cassanelli s, genedani s. cytokines and invertebrate immune response. biol. cell 85: 87-91, 1995a. ottaviani e, malagoli d, franceschi c. common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidence to in silico approaches. trends immunol. 28: 497-502, 2007. ottaviani e, paemen lr, cadet p, stefano gb. evidence for nitric oxide production and utilization as a bacteriocidal agent by invertebrate immunocytes. eur. j. pharmacol. 248: 319-324, 1993b. ottaviani e. the mollusc as a suitable model for mammalian immune-neuroendorine investigations. inv. surv. j. 1: 2-4, 2004. ottaviani e. molluscan immunorecognition. inv. surv. j. 3: 50-63, 2006. palumbo a. nitric oxide in marine invertebrates: a comparative perspective. comp. biochem. physiol. 142a: 241-248, 2006. plows ld, cook rt, davies aj, walker aj. carbohydrates that mimic schistosome surface coat components affect erk and pkc signalling in lymnaea stagnalis haemocytes. int. j. parasitol. 35: 293-302, 2005. ramos-martínez ji, villamarín ja, barcia r, vazquez-illanes md, ibarguren i. the role of fructosa 2,6-bisphosphate in the regulation of marine mussels (mytilus galloprovincialis and mytilus edulis) glycolisis/gluconeogenesis. comp. biochem. physiol. 104b: 641-647, 1993. robledo jaf, santarem mm, gonzalez p, figueras a. seasoanl variations inn the biochemical composition of the serum of mytilus galoprovincialis lmk. and its relation to the reproductive cycle and parasitic load. aquaculture 263: 310-319, 1995. ruiz-villarreal m, gonzález-pola c, diaz del rio g, lavin a, otero p, piedracoba s, et al. oceanographic conditions in north and northwest iberia and their influence on the prestige oil spill. mar. pollut. bull. 53: 220-38, 2006. soriano ja, viñas l, franco ma, gonzález jj, ortiz l, bayona jm, et al. spatial and temporal trends of petroleum hydrocarbons in wild mussels from the galician coast (nw spain) affected by the prestige oil spill sci. total environ. 370: 80-90, 2006. 48 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22ruiz-villarreal%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplusdrugs1 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22gonz%c3%a1lez-pola%20c%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplusdrugs1 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http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22bayona%20jm%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplusdrugs1 stefano gb, ottaviani e. the biochemical substrate of nitric oxide signaling is present in primitive non-cognitive organisms. brain res. 924: 8289, 2002. tafalla c, novoa b, figueras a. production of nitric oxide by mussel (mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions. comp. biochem. physiol. 132b: 423-431, 2002. torreilles j, romestand b. in vitro production of peroxynitrite by haemocytes from marine bivalves: c-elisa determination of 3nitrotyrosine level in plasma proteins from mytilus galloprovincialis and crassotrea gigas. bmc immunol. 2: 1-5, 2001. trimmer ba, aprille jr, dudzinski dm, lagace cj, lewis sm, michel t, et al. nitric oxide and the control of firefly flashing. science 292: 24862488, 2001. wright b, lacchini ah, davies aj, walker aj. regulation of nitric oxide production in snail (lymnaea stagnalis) defence cells: a role for pkc and erk signalling pathways. biol. cell 98: 265-278, 2006. 49 interleukin-2 (il-2) receptor in molluscan hemocytes nitric oxide production in molluscs concluding remarks isj 4: xx-yy, 2007 isj 4: 92-94, 2007 issn 1824-307x technical report a full-length protocol to test hemolytic activity of palytoxin on human erythrocytes d malagoli department of animal biology, university of modena and reggio emilia, modena, italy accepted september 18, 2007 abstract the hemolytic assay protocols currently utilized to test the presence of the marine biotoxin palytoxin (ptx) are deeply analyzed. in some points, slight modifications and rearrangements have been realized, to obtain an exhaustive protocol suitable to test ptx activity on human erythrocytes. key words: palytoxin; hemolysis; method introduction palytoxin (ptx) is a non-protein toxin firstly isolated from the soft coral of the genus palythoa more than 30 years ago (moore and scheuer, 1971). ptx is considered one of the most toxic molecules occurring in nature (lenoir et al., 2004), and it has also recently been studied also as a skin tumor promoter (wattenberg, 2007). since marine animals display a certain degree of resistance to its action (gleibs and mebs, 1999), ptx can accumulates in the food chain therefore provoking severe, or even lethal, intoxications to humans (taniyama et al., 2003). even though ptx is not an hemolysin (habermann, 1989), its interaction with na+/k+ atpase on erythrocyte membranes (artigas and gadsby, 2003; hilgemann, 2003) provokes a delayed release of hemoglobin. this activity is the base of the hemolytic assay, proposed by bignami (1993) as a more sensitive alternative to enzymelinked immunoassay. bignami (1993) realized his protocol taking advantage from the already known hemolytic activity registered for ptx in mammalian erythrocytes (habermann et al., 1981). the text based its specificity on the capability of the glycoside ouabain to inhibit ptx activity (habermann and chhatwal, 1982; habermann, 1989). since then, several studies indirectly identified ptx in invertebrate (gleibs and mebs, 1999) and vertebrate (taniyama et al., 2001, 2003) extracts by mean of the hemolytic assay. however, some considerations have to introduced before analyzing the numerous report concerning the use of ptx in hemolytic assays. ___________________________________________________________________________ corresponding author: davide malagoli department of animal biology via campi, 213/d, 41100 modena italy e-mail: davide.malagoli@unimore.it different mammalian species exhibit a specific degree of sensitivity to ptx (habermann, 1989) and different modalities are utilized by the researcher for quantifying hemolytic activity of the samples, thus introducing slight modifications in the method described in each report (bignami, 1993; gleibs et al. 1995; gleibs and nebs, 1999; taniyama et al., 2001, 2003; lenoir et al., 2004; riobó et al., 2006). on these basis, it is difficult to obtain a complete and detailed protocol for evaluating the hemolytic activity of ptx, especially for human erythrocytes, from a single report. the aim of this technical report, is to present an exhaustive and detailed protocol to evaluate the hemolytic activity of ptx on human a+ erythrocytes and to confront the collected results with those already present in literature. materials and methods hemolysis assay human a+ whole blood was used within 24 h after bleeding and washed three times (gleibs et al., 1995) in 9 volumes of sterile 0.9 % nacl saline solution. after each washing, cells were pelleted by centrifugation at 150xg for 5 min and the supernatant was discarded. the final pellet was diluted 1:9 (v/v) in sterile 0.9 % nacl saline solution than 1:24 (v/v) in sterile dulbecco’s phosphate buffer saline (d-pbs), ph 7.0 (bignami, 1993) containing 0,5 mm boric acid and 1 mm calcium chloride (taniyama et al., 2001). red cell suspensions (1 ml of final volume) were incubated with an aqueous solution (taniyama et al., 2003) of ptx standard, from 10-3 to 103 ng/ml. the following times of incubations were tested: 4, 6, 8 and 24 h. however, since no differences were observed from samples incubated for 6 h or more, all the repetitions have been performed with incubation 92 fig. 1 dose-dependent hemolytic effects of ptx standard (blue) on human erythrocytes and their reversal in presence of 100 μm ouabain (purple). dose-ranging experiments were performed from 10-3 to 103 ng/ml of ptx. however, since for concentrations ≤ 100 ng/ml, no effects were observed, the 100 ng/ml concentration is represented as the first value considered. error bars represent sd. time of 6 h. incubation temperature was set at 37 °c (taniyama et al., 2001), and during the incubation the samples were occasionally (maximum once per h) resuspended by inversion. for negative controls, red blood cell suspension was added with 100 μm ouabain and incubated for 30 min at 37 °c, prior to the addition of ptx (gleibs et al., 1995). in order to avoid any possible differences due to a diverse manipulation, also samples that did not contain the glycoside ouabain were incubated for 30 min at 37 °c before the addition of ptx. total lysis of erythrocyte suspension was obtained by incubating the cells with 0,1 % v/v tween 20. in order to evaluate the degree of spontaneous lysis, also tubes containing exclusively the red blood cell suspension in d-pbs were set. for each concentration and control, the experiments were set in triplicate. a total of eight independent experiments have been performed. hemolysis evaluation after the incubation, the cell suspensions were centrifuged at 900xg for 10 min (taniyama et al., 2003) and the supernatant was carefully collected, by paying attention not to disturb the pellet. the absorbance at 405/540 nm of supernatant was measured with a “helios β" spectrophotometer (spectronic, unicam, cambridge, uk). the value of absorbance of erythrocytes maintained exclusively in d-pbs has been utilized to set the 0 value before reading the samples that contained ptx. hemolytic levels were expressed by percentage of hemolysis, calculated with the ratio between the value measured for each sample and that registered for the total hemolysis (taniyama et al., 2003). chemical reagents human erythrocytes were obtained from laboratorio medicina trasfusionale (policlinico, azienda ospedaliero universitaria, modena, italy), ptx standard was from wako chemicals (neuss, germany). a second ptx standard solution was generously gifted by centro di ricerche marine (cesenatico, fc, italy). all the other chemicals, including d-pbs and ouabain, came from sigmaaldrich (st louis, ca, usa). all the plastics used were sterile and cell-culture tested (sarstedt, nümbrecht, germany). results and discussion the applied protocol confirms that ptx is able to induce hemolysis in human erythrocytes (fig. 1), and the levels of the lysis are comparable with those observed by other groups using the same source for red blood cells (taniyama et al., 2001, 2003). it is worth noting that the value here presented indicated a discrepancy with those already published, since the effects of ptx were detectable only from 101 ng/ml, while at this value taniyama and collaborators found a clear activity of ptx (taniyama et al., 2001). in order to verify if present data could be due to an erroneous preparation of the ptx standard, experiments were repeated with a second ptx solution coming from centro di ricerche marine. however, data collected with this second standard substantially confirmed those collected with the first one. differently from this first result, there is a substantial agreement between data presented here and those collected by taniyama et al (2001) for what concerns the 93 maximum levels of hemolysis detectable in human erythrocytes. as it has been observed from a long time (habermann and chhatwal, 1982), ptx effects on human erythrocytes are completely reverted by the glycoside ouabain (taniyama et al., 2001). the results obtained by using 100 μm ouabain confirm this indication even if a small percentage of erythrolysis have been observed in the presence of the glycoside (fig. 1). also other concentrations have been suggested for ouabain (habermann and chhatwal 1982, taniyama et al., 2001), but that adopted here was the most effective and did not significantly influence the basal levels of erythrolysis. as far as data analysis is concerned, fig. 1 presents data collected by using the absorbance wavelength of 405 nm, following gleibs and collaborators (1995). different values of wavelength, such as 540 nm, have been utilized to evaluate the hemolysis of mouse erythrocytes (lenoir et al., 2004). however, even if also the latter wavelength has been utilized in the present research, the most repeatable data were obtained at 405 nm, after imposing to the spectrophotometer the value 0 for the samples maintained only in d-pbs. if some differences in the protocols used to test the hemolytic activity of ptx can be retrieved, probably the most notable ones concern the unity chosen to measure the levels of hemolysis. gleibs et al (1995), working on palythoa and other marine specimen, describe the hemolytic unit (hu) as the amount of material necessary to produce 50 % hemolysis in human erythrocytes within 4 h incubation ptx. a similar definition of the hu, without references to the time of incubation, is given also by lenoir et al (2004), that worked with mouse red cells. working with human erythrocytes, gleibs and mebs (1999) quantified one hemolytic unit (hu) in 0,4 μg of ptx, but due to the different sensitivity of mammalian erythocytes to ptx (habermann, 1989), this value should probably be referred only to human red cells. taniyama et al. (2001) quantify ptx effects with the percentage of hemolysis, calculated with the ratio between the value measured for a sample and that registered for the total hemolysis. the percentage is a pure number that can be applied independently from the source of the erythrocytes, therefore it has been chosen here to evaluate ptx activity from commercially available standards. however, since the percentage of hemolysis should be properly calculated as follows: [absorbance in test absorbance in dpbs]/[absorbance in 100 % hemolysis absorbance in d-pbs] (hubert et al., 1997), the value of absorbance in d-pbs has been utilized to set the 0 value on the spectrophotometer. only after this procedure, the ratio proposed by taniyama et al. (2001) can be properly applied. acknowledgements the author wish to thank laboratorio medicina trasfusionale (policlinico, modena, italy) that kindly supplied the human blood and centro di ricerche marine (cesenatico, fc, italy) for the ptx solution. the author gratefully acknowledges also prof. enzo ottaviani (university of modena and reggio emilia, italy) for the critical reading of the manuscript. references artigas p, gadsby dc. na+/k+-pump ligands modulate gating of palytoxin-induced ion channels. proc. natl. acad. sci. usa 100: 5015055, 2003. bignami gs. a rapid and sensitive hemolysis neutralization assay for palytoxin. toxicon 31: 817-820, 1993. gleibs s, mebs d, werding b. studies on the origin and distribution of palytoxin in a caribbean coral reef. toxicon 33: 1531-1537, 1995. gleibs s, mebs d. distribution and sequestration of palytoxin in coral reef animals. toxicon 37: 1521-1527, 1999. habermann e. palytoxin acts through na+,k+atpase. toxicon 27: 1171-1187, 1989. habermann e, ahnert-hilgert g, chhatwal gs, béress l. delayed hemolytic action of palytoxin. general characteristics. biochim. biophys. acta 649: 481-486, 1981. habermann e, chhatwal gs. ouabain inhibits the increase due to palytoxin of cation permeability of erythrocytes. naunyn schmiedebergs arch. pharmacol. 319: 101-107, 1982. hilgemann dw. from a pump to a pore: how palytoxin opens the gates. proc. natl. acad. sci. usa 100: 386-388, 2003. hubert f, cooper el, roch p. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloproíincialis. biochim. biophys. acta 1361: 29–41, 1997. lenoir s, ten-hage l, turquet j, quod jp, bernard c, hennion mc. firstevidence of palytoxin analogues from an ostreopsis mascarenensis (dinophyceae) benthic bloom in southwestern indian ocean. j. phycol. 40: 1042-1051, 2004. moore re, scheuer pj. palytoxin: a new marine toxin from a coelenterate. science 1971 172: 495-498, 1971. riobó p, paz b, franco jm. analysis of palytoxinlike in ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection. anal. chim. acta 566: 217-223, 2006. taniyama s, mahmud y, tanu mb, takatani t, arakawa o, noguchi t. delayed hemolytic activity by the freshwater puffer tetraodon sp. toxin. toxicon 39: 725-727, 2001. taniyama s, arakawa o, terada m, nishio s, takatani t, mahmud y et al.. ostreopsis sp., a possible origin of palytoxin (ptx) in parrotfish scarus ovifrons. toxicon 42: 29-33, 2003. wattenberg ev. palytoxin: exploiting a novel skin tumor promoter to explore signal transduction and carcinogenesis. am. j. physiol. cell physiol. 292: c24-c32, 2007. 94 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj 6: s46-s57, 2009 issn 1824-307x review focusing on ciona intestinalis (tunicata) innate immune system. evolutionary implications n parrinello laboratory of marine immunobiology, department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 abstract phylogenetic analyses based on molecular data provide compelling evidence that ascidians are of critical importance for studying chordate immune system evolution. the ciona intestinalis draft genome sequence allows searches for phylogenetic relationships, gene cloning and expression of immunorelevant molecules. acidians lack of the pivotal components of the vertebrate recombinatory adaptive immunity, i.e., mhc, tcrs and dimeric immunoglobulins. however, bioinformatic sequence analyses recognized genic elements indicating the essential features of the ig superfamily and ancestor proto-mhc genes, suggesting a primitive pre-duplication and pre-recombination status. c. intestinalis genes for individuality in the absence of mhc could encode diverse molecular markers, including a wide panel of complement factors that could be responsible for self-nonself discrimination. genome analysis reveals a number of innate immunity vertebrate-like genes which encode toll-like and virus receptors, complement pathways components and receptors, cd94/nk-receptor-like, lectins, tnf, il1-r, collagens. however, pure homology seeking for vertebrate-specific immunorelevant molecules is of limited value, and functional screening methods may be a more promising approach for tracing the immune system evolution. c. intestinalis, which displays acute and chronic inflammatory reactions, is a model organism for studying innate immunity genes expression and functions. key words: immunoevolution; genome; ciona intestinalis; ascidians; innate immunity; inflammatory response; gene expression evolutionary relevance of ascidian immunity studies ascidians (urochordata), including cosmopolitan compound and non-colonial species, occupy a key position in the phylogenetic line leading to the vertebrates (swalla et al., 2000; zeng and swalla, 2005; delsuc et al., 2006), therefore they have attained importance for immunity evolution studies recently promoted by available genome sequences (dehal et al., 2002b; satou et al. 2002; satoh, 2003; yokobori et al., 2003; kasahara et al., 2004; litman and cooper, 2007; ben-shlomo, 2008). bioinformatic approach and extensive in silico search of immunorelevant genes have been in part validated by expression patterns and biological properties of their products (davidson and swalla, 2002; nonaka and miyazawa, 2002; fujita, 2002; azumi et al., 2003; ___________________________________________________________________________ corresponding author: nicolò parrinello laboratory of marine immunobiology department of animal biology university of palermo via archirafi 18, 90123 palermo, italy e-mail: nicpar@unipa.it shida et al., 2003; terajima et al., 2003; fujita et al., 2004; du pasquier, 2004; kasahara et al., 2004; litman and cooper, 2007). botryllids provided of allorecognition reaction (de tomaso and weissman, 2004; de tomaso et al. 2005; ballarin , 2008; gasparini et al., 2008) and ciona intestinalis which displays acute inflammatory responses (parrinello, 1981; parrinello and patricolo, 1984; parrinello et al., 1984) are model organisms for studying chordate evolution. the whole genome of c. intestinalis has been sequenced and analyzed, genes have been annotated and some expression studies carried out, whereas adequate botryllid genome sequencing is lacking. adaptive immunity pivotal genes in vertebrates, the emergence of the adaptive immune system is linked to the acquisition of the enzyme machinery encoded by the recombination activating genes (rag) that provide to the rearrangement of immunoglobulin (ig) and t cell receptor (tcr) genes. both b and t cells carry s46 mailto:nicpar@unipa.it receptor molecules to recognize and respond to the antigens. b cell receptor is a prototype of the antibody, and tcr on the surface of t lymphocytes is responsible for recognizing antigens bound to major histocompatibility complex (mhc) molecules. these receptors are composed of two polypeptides of the ig superfamily (ig heavy h and light l chains; tcr α and β or γ and δ chains) with recognizable constant (c) and variable (v) domains. the c domain of c1-set characterizes only ig, tcr, and mhc class ii, class i and class i-related molecules, whereas the c2 set domain occurs in both vertebrates and invertebrates (see kasahara et al., 2004). the domain provided with variability is generated by somatic recombination of multiple elements scattered in the genic locus. in lymphocytes, rags cut dna at the recombinant signal sequence (rss) for mediating recombination of v(d)j regions through the joining gene segments. somatic hypermutation, rags and activation-induced cytidine deaminase (aid), regulators of secondary ig diversification, are crucial components of vertebrate adaptive immunity (manis et al., 2002; bransteitter et al., 2003) producing an unlimited repertoire of diversity that enables the cells to recognize and respond to unpredictable pathogens. analysis of c. intestinalis genome sequences did not reveal the pivotal genes and molecules for adaptive immunity, such as mhc genes, tcrs, or dimeric igs (dehal et al. 2002b; azumi et al., 2003; shida et al., 2003). nevertheless, bioinformatic sequence analyses have recognized two ig domaincontaining regions, key v regions, the essential feature of an ig superfamily vc1-like core trait, presumptive proto-mhc regions scattered throughout the genome, and three types of genes with receptor-like v-c architecture (du pasquier, 2004). in addition, there are indicators of v domains that could be targets for rag-mediated transposition. these genes belong to two families with recognizable homologs in vertebrates: the cortical thymocyte marker of xenopus/junctional adhesion molecules family (ci-ctx/jam) and the nectin ci-nec2 family. signal of ancestor proto-mhc genes can be drawn from the human mhc genome paralogy (kasahara, 2000). more than 100 genes are located in the human leukocyte complex (hla) and about 40 of them have paralogous copies on 1, 9 and 19 chromosomes. c. intestinalis genome contains a single copy gene with features of a precursor of multiple human mhc paralogous suggesting the existence of a pre-duplication region (bodmer, 1972; kasahara et al., 2004). chromosome duplications could be took place before the emergence of a common ancestor of vertebrates as indicated by mhc paralogous emergence. mhcbased allorecognition seems to be a unique feature of jawed vertebrates, and finding of variable lymphocyte receptors in agnates (pancer et al., 2004) indicate that different groups of molecules could be responsible for similar functions. accordingly, allorecognition machinery in urochordates has nothing in common with the mhcbased histocompatibility reaction of vertebrates (de tamaso et al., 2005; klein, 2006), and observations indicate that different ascidian species could have evolved their own independent allorecognition strategies. in example, the hystocompatibility fu/hc receptor of b. schlosseri has no direct homologs neither in vertebrates nor in c. intestinalis, indicating that self-nonself discrimination systems have branched off into a variety of unique and specialized systems during evolution. in the absence of vertebrate-like adaptive immunity, ciona‘s genes reflect a primitive preduplication and/or pre-recombination status leading to the genesis of mhc, tcr and ig (yu et al., 2005). moreover, it is generally accepted that c. intestinalis activating and inhibitory receptors of immunocytes have mhc-independent functions. presumptive molecular codes for sef-nonself discrimination in the absence of mhc although most animals are able to distinguish self from non-self (buss, 1987; chadwick-furman and rinkevich, 1994; cadavid et al., 2004), allorecognition molecular mechanisms do not seem of monophyletic origin and remain to establish if they evolved independently. different groups of invertebrates could have developed their own histocompatibility system and could express taxonspecific self-nonself determinants. ciona is a valuable model for deepening the question. it is hermaphrodite producing eggs and sperm simultaneously, but self-fertilization normally does not occur because the follicle cells accept only allogeneic sperm (rosati and de santis, 1978; pinto et al., 1995; murabe and hoshi, 2002). it can be assumed that a receptor, potentially involved in selfincompatibility, should have sequence and expression variable in oocytes from different individuals. based on this assumption, novel models have been proposed upon a c. intestinalis molecular code for individuality in the absence of mhc. to search for molecular markers of individuality, a suppression subtractive hybridisation approach has been used for comparing the somatic transcriptomes of two ciona individuals and for identifying individually variable cdnas (khalturin et al., 2005). the results show that two genes, cimeta2 and cis7, encode two classes of soluble proteins which exhibit high degree of interand intra-individual variability and contain multiple domains suitable for protein-protein interaction. both classes of individually variable proteins are coded in the genome by several gene copies organized in clusters. one class consists of secreted protein thrombospondin type 1-like domains (thrombospondin type 1 repeat, tsr superfamily), the second one consists of secreted proteins with multiple epidermal growth factor (egf)-like domains. although the functional significance is unknown, the authors suggest that these gene loci may participate in controlling non-self recognition. cis7 and cimeta2 isoforms with a high degree of aminoacid sequence variations are expressed in hemocytes and gametes which are mediators of recognition events. individuals have several genes and transcribe several mrnas coding for similar but s47 not identical proteins of each gene family. apparently each individual carries an unique repertoire (haplotype) in each locus, probably established by crossing over events during gamete maturation and/or fertilization. individuality may be encoded by the haplotype and the individual-specific combination of genes in the genome locus. this phenomenon resembles the variability of vertebrate killer cell ig-like receptors (kir), and fit with the predictions of self-nonself recognition molecules provided with individual variability, allorecognition and block of self-fertilization. to shed light on c. intestinalis specific receptors for allorecognition and self fertilization avoidance, kurn and colleagues (2007) subctracted gonadal cdna from three genetically unrelated c. intestinalis individuals by suppression subtractive hybridisation. individualspecific genes encoding variable transmembrane complement receptor-like protein (vcrl) have been identified. interestingly, they contain several complement controlling protein domains (scr/ccp). one of these genes reveals a high degree of inter-individual vcrl amino acid variation, and it is expressed in follicle cells and hemocytes. diverse vcrl1s, highly variable between individuals, show sequence similarity varying from 70 % to 93 %. each animal has its own version of vcrl1, therefore cells in different individuals are marked by non-overlapping receptors and corresponding ligands. intraindividual variants most likely are due to the two alleles of a single locus, and several alternative intracellular vcrl1 domains may be produced by alternative splicing. the cells within one individual are appropriately marked and will be referred to as “self” whereas any cell of genetically different individual will be distinguished as “non self”. in mammals, the complement components are not variable between individuals. in ciona, the number of genes encoding complement system components is greatly expanded compared to mammals and, together the vcrl high variability, allow to suppose that the ciona allorecognition reactions involve complementrelated receptors in a “missing self” mechanism. intriguingly, complement receptors are also expressed in human gametes and are involved in sperm/oocyte interaction (seya et al., 1999). the ciflrt transmembrane receptor gene expressed in hemocytes (kurn et al., 2007) was also analyzed. the corresponding protein consists of a signal peptide, nine leucine-rich repeats (lrrs, protein interaction modules), fibronectin type iii domain (mammalian fniii repeats on cell surface and extracellular) and a transmembrane domain (khalturin et al. 2005). the ciflrts from two individuals were amplified, sequenced and compared to sequences reported by khalturin and colleagues (2005). the distance between the ciflrt proteins of the four individuals is much smaller than that between vcrl1. the high degree of ccrl1 variation clearly exceeds the naturally occurring variations normally found in ciona genes. toll-like receptors the toll-like receptor (tlr) multigene family encodes recognition receptors of innate immune system conserved in invertebrates and vertebrates (kaisho and akira, 2000; medzhitov and janeway, 2000; imler and hoffmann, 2001; vasselson and detmers, 2002). tlrs recognize a variety of endogeneous and exogeneous ligands, they are pattern recognition receptors that bind molecules broadly shared by pathogens collectively referred to as pamps, many of which are conserved molecules essential for pathogen survival (zhong and kyriakis, 2007). c. intestinalis genome sequence analysis disclosed three distinct tlrs expressed genes and the corresponding signal transduction cascades (azumi et al., 2003; terajima et al., 2003; roach et al., 2005; shida et al., 2005). in invertebrates, tolllike receptors mediated antibacterial, antifungal and antiviral systems. recently, c. intestinalis inflammatory responses challenged by lps have been reported. the triggering mechanism presumably includes toll-like receptors (tlrs) which bind lps (a component of the pamps) initiating the inflammatory reactions. responses stimulated by lps including complement activation and products, galectincytokine-like and cd94-nk-c-type lectin-like receptor genes expression, and collagen synthesis have been reported (see below). genes for intracellular immunoreceptors, tyrosine-based inhibition motifs (ciitims) and tyrosine-based activation motifs (ciitams) may be responsible of the cell signaling pathways (azumi et al. 2003; shida et al., 2003). vertebrate itims and itams are short conserved sequences in the cytoplasmic tails of many immune cells inhibitory and activating receptors (including tcrs), respectively. virus receptors in c. intestinalis genome, homologs to vertebrate adhesion molecules members of membrane ig superfamily show ancestral features of antigen receptors (du pasquier, 2004). they include the junction adhesion molecule (reovirus receptor) and the cortical thymocyte marker of xenopus (adenovirus receptor) which are members of cictx/jam family, and the poliovirus receptor (pvr) members of the cinec family. pvr genes contain one distal v domain followed by two c domains, transmembrane and cytoplasmic segments. vertebrate pvrs are known to allow endocytosis of different viruses through the interaction with v domain. since in humans 4 paralogous groups exist, the ciona set of genes could correspond to a preduplication status. it has been suggested that the virus binding property of the members of this family were recruited in the vertebrate immune system following the introduction of the somatic rearrangement machinery. complement system the innate complement system evolved long s48 before the origin of vertebrate somatically rearranging antibodies. in vertebrates, there are about 30 complement protein components and three distinct pathways by which the complement system can be activated (nonaka and yashizaki, 2004a, b): 1. classical, antibody-mediated pathway; 2. lectin mediated pathway; 3. alternative pathway, triggered the pathogen surface. all the activation mechanisms converge to the c3 component proteolysis, and generate a same set of activation products. c3 is the central component, equipped with an unique intramolecular thioester bond which, upon activation, is exposed to the molecular surface and forms a covalent bond with invading microorganisms (lambris, 1990). c3 proteolysis enhances the phagocytosis (opsonin) of pathogens through some c3b fragment and the chemotaxis by the c3a-fragment which binds to macrophage and leucocyte receptors, furthermore c3b contribute in forming c5 convertase that activates c5-c9 late complement components leading to the formation of a cytolytic complex (membrane attack complex or mac). a large number of complement components are conserved between higher vertebrates and urochordates. in c. intestinalis, a wide and detailed architecture of the primitive complement system has been described (nonaka and yoshizaki, 2004a, b; fujita et al., 2004), and complement-like genes, most of which are transcriptionally active, indicate potential activity of lectin and alternative pathways. the ciona lectin mediated complement pathway has been revealed by genome sequence analysis, est, cloning and expression studies that identified nine mannose-binding lectins (cimbl), nine ficolins and four cimbl-associated serine proteases (cimasps c1r/c1s-like). the carbohydrate-binding collectin pathway (cimbls/ficolins as pattern recognition proteins), initiated by recognition of pamps and activated by cimasps, leads to cic4, cic2 and cic3 cleavage. several soluble components and receptors (four cimasps, cic3 and corresponding cicr3/cr4 receptors) have been recognized in the genome, and ests disclosed transcripts in hemocytes (fujita, 2002; azumi et al., 2003; fujia et al., 2004; wakoh et al., 2004). cimasps could exert trypsinlike activity, and the identified cimbl collectin genes encode proteins composed of the collagen and lectin-like domains. recently a cimbl has been cloned and sequenced, and its expression was promptly enhanced by lps (bonura et al., submitted). the deduced amino acid sequence (221 aa) showed a n-cysteine-rich terminal domain, a type 2 collagen domain, and a c-type mannose/glucose-specific crd. comparative analysis reveals 56.5 % similarity and 37.8 % identity with human mbl. marino and colleagues (2002) cloned two cic3-like genes (cic3-1 and cic3-2 cdna) from ciona hemocyte mrna. the deduced aminoacid sequences of both cic3 proteins show an overall similarity to the c3 molecules from vertebrate species, and exhibit a canonical processing site for αand β-chains, including a typical thioester site with the his residue required for nucleophilic activation. lps activates the complement, presumably via cimbl and cic3-1 proteolysis leading to the cic3a fragment which is a pro-inflammatory peptide akin to the vertebrate anaphylatoxin (pinto et al., 2003). the inflammatory challenge upregulates the cic3-1 expression in hemocytes and cic3-1a production. the recombinant cic3-1a exerts in vitro chemotactic effect on hemocytes interacting with a receptor molecule cic3ar coupled with gi protein and homologous to the mammalian receptor (melillo et al., 2006). an ancestor of the two cic3 seems to have diverged from a common ancestor of vertebrate c3/c4/c5 and has duplicated in two genes. accordingly, sequence phylogenetic analysis indicates that cimasps have diverged from the common ancestor of vertebrate masp/c1r/c1s. in addition, the genome presents two α2macroglobulin-like (ciα2m) genic elements (azumi et al., 2003). mammalian α2m is able to inactivate an enormous variety of proteinases, inhibits complement activation by masp and it is conceivable that it plays a regulatory role in mbpderived complement activation (terai et al., 1995). c3/c4/c5 and α2m form a molecular family different from other complement components and they do not show clear domain structure. in mammals, the factor b (bf) is the central serine esterase of the alternative pathway of complement activation, and in the active form (bb) is component of c3 convertase. in ciona three bflike genes, supported by cdna evidence, have also been identified and they, presumably responsible for the alternative pathway, encode predicted proteins with domain structures similar to the vertebrate bf/c2 gene family basic domain structure (fujita, 2002; azumi et al. 2003; fujita et al. 2004). bf and c2 are catalytic subunits of the c3 convertases of the alternative and vertebrate classic pathways respectively. the cibf genes are longer than those of jawed vertebrates, the deduced amino-acid sequences of cibf1-3 contain the usual domains of bf and, in addition, three extra domains at the n-terminus. similarly to vertebrate masp-2, masp-3 and c1r/c1s, cibf presents one additional short consensus repeat domain, and two low-density lipoprotein receptor (ldlr) domains. overall deduced amino-acid identity between cibf-1 and cibf -2 was 88 %, whereas cibf-3 showed 49 % identity to both cibf-1 and cibf-2. the cibf serine protease domain shared characteristic features with the complement components c1r/c1s, masp-2 and masp-3, and the active site appears to be of the agy codon type for the catalytic serine residue, and lacks of a histidine loop disulfide bridge (fumiko et al., 2005; yoshizaki et al., 2005). phylogenetic analysis indicates that cibf genes could be the result of duplication and gene conversion occurred after the divergence of the urochordate lineage from the vertebrate subphylum as also shown by genomic organization and intron/exon composition. presumably they have diverged from the common ancestor of vertebrate bf s49 and c2 before the divergence of bf and c2. these results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. exons 3 and 5 of the three cibf genes show an extremely high degree of nucleotide identity, indicating duplication and gene conversion, since its divergence from the vertebrate bf/c2 gene. the ciona genome analysis also allowed the identification of eleven presumptive genes with mac/perforin domain, nine of them exhibit domain structures similar to those of late complement components. although, activation mechanism and a functional linkage between cic3 and the possible lytic components have not been demonstrated, the lytic function is strongly suggested by the presence of the mac/perforin-like domain. combination of several domains assign the lytic components to the c6-c9 family. however, cic6 and cic7 lack the several c-terminal domains of the corresponding human components, and their functional link to cic3 remain unclear. finally, a group of 132 presumptive genes with complement control module (src domain) have been identified (azumi et al., 2003). in mammals, regulators for inhibiting undesirable complement activation against self cells are composed of repeat of short consensus repeats src domain. in brief, three functional units may be distinguished in activating cic3 and active factors production: 1. collectins, composed of collagen and lectin-like domains which recognize miscroorganisms, associated with serine proteases that presumably activate cic3 leading to a chemotactic product; 2. cibf and cic3, components of the vertebrate alternative pathway, and the activation product may have an opsonic activity; 3. presumptive cic6-c9 molecules forming the mac/perforin complex with cytolytic activity. cytokine-like molecules and receptors pleiotropic and multifunctional proinflammatory cytokines (tumour necrosis factor tnf, interleukins il1 and il6) play a pivotal role in innate immune responses, in cell proliferation, differentiation, apoptosis, and stimulation of the collagen synthesis in wound healing and tissue repair. in invertebrates, cytophilic humoral molecules with functional similarities to vertebrate cytokines have been reported (beck, 1998; beshin et al., 2001; ottaviani et al., 2008). in ascidians, cytokine-like molecules active in stimulating cell proliferation, phagocytosis and opsonisation, have been revealed by immunological and biochemical methods (see beshin et al., 2004). genome sequencing, cdna/est derived from c. intestinalis hemocytes, and identification of the corresponding genes in genome sequences (shida et al., 2003; terajima et al., 2003) revealed the existence of ciil1 receptor, ciil17 receptor genes and an ectodysplasin/tnf-like multigene family. the presumptive il6 gene was also identified. three interleukine-1-recepor-like (ciil-1r) genes present an extracellular ig and an intracellular tir domain which is the conserved toll/il-1 receptor (tir) domain of the two families of receptors (tong, 2005). this domain was first characterized due to the homology between the intracellular region of the mammalian il-1rs and the drosophila tlrs. like tlrs, il-1r signaling pathways are key mediators of the innate immune response to bacteria (lps), fungi, cytokines and growth factors. in mammals, the signalling pathways mediated by tlr, il-1r and tnfr share common components. the possibility exists that these signalling cascades are indeed functioning in c. intestinalis hemocytes. hemocyte citnfα gene expression is challenged by lps vertebrate tnfα is a component of a wide tnf family, it is a type ii transmembrane protein with an extracellular homotrimeric c-terminal domain. a membrane-bound form may be cleaved, and the mature cytokine may be released as soluble form by a variety of cell types including macrophages, monocytes, granulocytes, nk-cells. tnfs are promptly expressed, and regulate inflammatory reactions by interacting with other pro-inflammatory cytokines recruiting and activating inflammatory cells (arika et al., 1990). in the ciona genome, one citnf-like and three citnf-receptors–like genes have been identified (terajima et al., 2003). the c. intestinalis tnfα-like cdna (citnfα) has been cloned from the pharynx excised at 4 h after lps inoculation (parrinello et al., 2008). comparative analysis of the deduced aminoacid sequence discloses that the cloned citnfα-like clusters at a phylogenetic position close to vertebrate tnfα, whereas a considerable distance separates citnfα from drosophila melanogaster tnf-related eiger isoforms and earthworm ccf. like the vertebrate tnfα, citnfα is constitutively expressed in the hemocytes and it is promptly (4 h) increased by lps both in the pharynx after in vivo inoculation and in hemocytes challenged in vitro. western blot analysis with monoclonal antibodies specific for human recombinant tnfα, showed a cell bound form (43 kda) in hemocytes and a 15 kda soluble form in the serum suggesting the role of this cytokine in both local and systemic responses to inflammation. densitometry analysis of these bands confirms the gene upregulation. in particular the cell bound form is enhanced at 2 h post lps injection, whereas later (4 h pi.) the soluble form appears to be enhanced in the serum in accordance with the gene expression disclosed by real-time pcr analysis of the pharynx. the anticipated expression of the cellbound form in hemocytes challenged in vitro appears to be congruent with the presumed maturation process of the soluble one. similarly to vertebrates, different cell types can secrete the same cytokine. at 4 h after lps inoculation, amebocytes with large granules, contained in the pharynx vessels and in the connective tissue lining the tunic, as well as circulating hyaline amebocytes and granulocytes express the citnfα mrna as revealed by in situ s50 hybridization and immunohystochemistry with anti-human rtnfα monoclonal antibody. hemolymph galectins with il1α epitopes are modulated by lps the direct homologue of mammalian il-1 has not been found in the ciona genome. in mammals, il-1α and -β interleukines as well as galectins are pro-inflammatory molecules, furthermore many cytokines are bifunctional molecules containing a receptor-binding domain and an evolutionary conserved carbohydrate recognition domain (crd) that is typical of lectins. the carbohydrate binding is requested for the cytokine biological activity (beschin et al., 2004). in this regard, il1α and β can be considered as lectins (cebo et al., 2001, 2002) directly interacting and contributing to pathogen elimination via opsonization and/or leukocyte activation. recently, inducible galectin-like molecules with human recombinant il-1 epitopes and opsonic properties have been found. parrinello and colleagues (2007) have shown that ca2+independent cigalectin-like molecules, specific for d-galactose and d-galactosides, present human ril1α epitopes. the lps inoculation challenges a promptly (4 h) enhanced serum concentration of this lectin that has been related to the augmented serum opsonizing and hemagglutinating activities assayed with yeast and rabbit erythrocytes respectively. furthermore, human il-1 epitopes are involved in the opsonizing and hemagglutinating processes which are blocked by anti-human recombinant ilα monoclonal antibodies. the western blot pattern showed that, within the initial phase of the inflammatory response (4 h), several serum proteins (59, 37, 30, 23, 15 kda) cross-reacted with the antibody suggesting an oligomerization process of the opsonin/lectin. ciil-17 receptor hemocytes express an interleukine ciil-17r gene that encode a predicted polypeptide of 769 aminoacid residues (dehal et al., 2002b; shida et al., 2003). the c-terminal half of this protein shows homology to the cytoplasmic region of mammalian il-17r (27 % identity/40 % similarity). the central portion is rich in hydrophobic aminoacid residues presumably correspondent to a trans-membranous region, and 22 residues at the extreme n-terminal portion could be a signal peptide sequence. the nterminal portion, probably an extracellular region, shows no homology to the corresponding region of mammalian il-17r. il-17 is produced by mammalian t-lymphocytes whereas its receptor is expressed in a variety of cell types such as fibroblasts and stromal cells disclosing a wide spectrum of activity. a possible ligand of ciil17r has not been predicted. emergence of nk cells receptors natural killer (nk) cells are critical in the evolution of the innate immune system being active in discriminating and killing “normal” and virusinfected, tumor or allogeneic cells. in mammals, nk cells monitor mhc class i expression on target cells by means of inhibitory nk cell receptors (nkrs) (lanier, 2000; vivier et al., 2002). the nkrs transmit an inhibitory signal that cancels a program for cytotoxic action previously triggered by the target cell contact. nkrs belong to two distinct groups of molecules: ig-like receptors or c-type lectin receptors including human cd94 (boyington, 1999). c-type lectin receptors are type ii transmembrane glycoproteins with a c-type lectin domain (ctld) in the extracellular region that bind proteins in a ca2+independent manner rather than sugars. they are known to be a hallmark of surface markers for nk cells (biassoni et al., 2007). c-type lectin superfamily includes carbohydrate-binding proteins (lectins) involved in pathogen recognition and neutralization, leukocyte trafficking, phagocytosis, antigen uptake and processing, and apoptosis. the ca2+-dependent binding of their carbohydrate recognition domain (crd) characterized the ctdl. however, many c-type lectins included in the superfamily lack critical amino acid residues required for crd to bind carbohydrates (rogers and wong, 2003). in this respect it has been hypothesized that divergent evolution, acting on the ctld fold, has generated the lectin-like natural killer (nk) receptors that bind proteins in a ca2+independent manner, rather than sugars. hemocyte cicd94 gene expression modulated by lps is involved in phagocytosis c. intestinalis cd94 (cicd94-1) protein containing cictld is a homolog of the botryllus schlosseri (50/66 % identity/similarity) bscd94/nkr-p-1 molecule (khalturin et al., 2003), and human (30/46 % i/s) cd94. cicd94-1 has been cloned and sequenced, and hemocytes have been stimulated in vitro with lps (zucchetti et al., 2008). even though sequence homology situates the cicd94-1 molecule close to cd94, several features do not suggest that they are complete orthologs. cicd94-1 could be considered a c-type lectin which lacks ca2+-binding property and its carbohydraterecognition (mannose/galactose and related sugars) capacity, and it could be located along the evolutionary line leading to the nk receptors functionally related to the human cd94 which recognizes peptides in the groove of mhc class i molecules. on the other hand, the lack of mhc genes in c. intestinalis genome should indicate that cicd94 is functionally similar to the mice nk cells mhc-independent cd94 (iizuka et al., 2003; mcnerney et al., 2005). however the involvement of cicd94 in cytotoxic mechanism has not been shown, whereas it appears to be involved in phagocytosis of polystyrene latex beads by granular amebocytes inhibited in the presence of anticicd94-1 specific antibodies (zucchetti et al., 2008). no assays have been reported to disclose a cicd94-1 dependent cytotoxic activity by unilocular refractile granulocytes which are known to be cytotoxic (parrinello, 1996; parrinello et al., 1996). the cicd94-1 as receptor on phagocytes can be up-regulated by lps, presumably part of a s51 mechanism of self-nonself recognition (zucchetti et al., 2008). interestingly, granular amebocytes, together with compartment cells, are engaged in the production of cic3-1 and express cic3a-r following inoculation of lps. the presence in the genome of a cd94 homolog and molecules with inhibition (ciitim) and activation (ciitam) motifs reasonably sustain the activity of precursors of nk cells in ciona. the cicd94-1 protein has been found in about 20 % of the granular amebocytes of naïve ascidians suggesting the existence of a cell population that constitutively express cicd94-1 presumably acting as a self-nonself sentinel. altogether these results indicate that hemocytes are provided with a complex array of surface receptors and effector molecules, enabling them to be active in several immune responses. alternatively, distinct hemocyte populations originated from a same hemocyte type (lymphocytelike cells) could express distinct receptors and exert different activities following an inflammatory challenge. ca2+-dependent hemocyte cytotoxicity seems to be cicd94-independent apparently, a cicd94-independent a ca2+dependent cytotoxic activity of c. intestinalis hemocytes has been shown. the hemocyte type named unilocular refractile granulocytes (urg, the unique large granule occupies the cytoplasm), constitutively display cytotoxic activity and lyse rabbit erythrocytes and k562 tumour cell line. zucchetti and colleagues (2008) did not report any urg that express cicd94-1, furthermore immunocytochemical staining of percoll gradient separated hemocytes shows that, after a short incubation with lps, about 80 % granular amebocytes express the cicd94-1 protein. no signs of the receptor in hyaline amebocytes, that have been reported to be phagocytes (rowely, 1981), have been found. the receptor involved in hemocyte cytotoxic activity remain unknown. a plaque forming cell assay with rabbit erythrocytes showed that the cellkilling mechanism requires effector-target cells contacts for challenging the release in vitro of soluble cytolysins. the cytolysin is ca2+-dependent and is inhibited by sphingomyelin and carbohydrates (unpublished). the unique granule displays phenoloxidase activity, and urgs are components of the inflammatory reaction and densely populate the tunic matrix after lps inoculation (parrinello, 1996; parrinello et al., 1996; cammarata et al., 2008). although, any evidence exists on the involvement of cicd94-1-nkr in urg mediated cytotoxicity, in line with the observation that some c-type lectins bind carbohydrates in a ca2+independent manner (brown and gordon, 2001), carbohydrates could be still be potential ligands for cicd94-1, and the possibility exists that a coreceptor may be involved. since lectins have also been claimed as recognition molecules (quesenberry et al., 2003), presumably different self-nonself recognition molecules characterize ascidian hemocytes and tissues. cifacit-collagen expression as a component of the inflammatory reaction inflammation plays an important role in many processes, protecting organisms against pathogens, and also potentially promoting damage progression (henson, 2005). collagens are major structural components of extracellular matrix in tissues of vertebrates and invertebrates, involved in defence and reparative processes (singer and clark, 1999). in the mammalian acute inflammatory reaction, collagen fibres bundles are organized for tissue repair during the remodelling phase (nwomeh et al., 1998), moreover, the total collagens present in normal tissue, is increased from 2to 9-fold in the chronically inflamed tissue (narayanan et al., 1983). in this respect, activation of the innate immune response leads to the production of proinflammatory cytokines that can promote collagenolysis. collagen degradation, and collagen fragments modulate inflammation either augmenting or suppressing interleukine production from peripheral-blood cells (thomas et al., 2007). a family of non-fibrillar collagens, including type ix (facit) collagen, contain short triple helical domains, composed of three genetically distinct polypeptide-chains, interrupted by short non-helical domains (ricard-blum et al., 2005). this collagen type does not form fibrils, and interacts with fibrillar collagen (fibril-associated collagen of cartilage extracellular matrix) and with other extracellular matrix partners (eyre and wu, 2005). since type-ix collagen interacts with the cellular receptor integrins it may have an important function as mediator of cell adhesion to collagen fibrils (käpylä et al., 2004). a c. intestinalis type ix-like collagen cdna (citypeix-col 1α chain), with features of fibril associated collagens formed of interrupted triple helices (facit) has been cloned and sequenced (vizzini et al., 2002). the involvement of this collagen in the inflammatory response has been shown by real-time pcr analysis, ish assay and immunohistochemical methods. in addition flow cytometry with anti-ci-typeix-col 1α chain specific antibodies, showed a prompt (1-4h) and enhanced collagen expression in the circulating hemocytes treated in vitro with lps, and in epidermis cells after in vivo lps inoculation (vizzini et al., 2008). morula cells with large granules (morular feature) express this collagen revealing a fibroblast-like role. conclusions the barrel-shaped sea squirt c. intestinalis (non-colonial ascidian, tunicata) has its life cycle in shallow sea and ocean waters around the world. one day after an egg is fertilized, it develops into a swimming small tadpole that settles down and metamorphoses into an immobile adult. the settled adult feeds by siphoning seawater using a basketlike filter to capture particulate food and oxygen. despite the adult humble appearance, the tadpole larva, comprised of about 2,500 cells, is provided of notochord and dorsal neural tube revealing kinship s52 to vertebrates. the importance of tunicates as models for the vertebrate ancestor was recognized by kowalevsky, who first identified them as chordates, and they have played an important role in various evolution scenarios (see gee, 1996). due to the very simple ascidian body plan and an apparently increased body complexity of cephalochordates, ascidians were previously thought to be living form of the earliest chordate lineage. in the december 13, 2002, an issue of the journal science, an international consortium of researchers reported on the draft sequencing, assembly, and analysis of the c. intestinalis genome (dehal et al., 2002a). from then on, sequence comparison, cdnas, est and gene modulation and function studies provided new insights about the evolution of key vertebrate systems including immune system and development. recently, taking advantage of the genomes sequencing, delsuc and colleagues (2006) proposed that tunicates and not cephalochordates are the closest living relatives of vertebrates stimulating further research on chordates evolution. simplified form of vertebrate gene families were typically found in ciona, whereas the lancelet lineage diverged before the tunicates and vertebrates. genome sequences show that these relationships, reflected at the molecular level, indicates how similar systems and gene sets evolved in different ways from a common ancestor. the close relationship to vertebrates along with its compact genome (about 160 million base pairs, 1/20 the size of the human), makes this sea squirt an ideal model organism for studying chordate evolution. sequence analysis revealed that c. intestinalis genome contains about 16,000 genes, about 80 % of which are also present in humans and other vertebrates. however, the total number of ciona genes is only about half the number in vertebrates, presumably due to the fact that it has single copies of a large number of genes whereas they are present in multiple copies in vertebrates (francino, 2005). anyway, it can be retained that comparative analysis of genes and knowledge of the immunity genes evolution models are consistent with darwin’s 1871 suggestion that ascidians and vertebrates diverged from a common ancestor. increase in the extent of genome resources as well as understanding of transcription at both transcriptosome and spliceosome levels may unveil conserved features on the coding and non coding dna that sustain genetic stability or promote changes (litman and cooper, 2007) either they mutate away and disappear, or they evolve to perform other functions and advance in complexity. altogether adaptive and innate immune system, form a tremendous complex system to recognize non-self and provide protection from a wide variety of pathogens. the innate immune system is the most ancient of the two systems, and the adaptive immune system appeared more recently developing a high degree of complexity and interconnectivity. many components of the innate immune system in vertebrates can be reliably traced to urochordates. for example, genome analysis reveals a number of innate immunity vertebrate-like genes, including toll-like and virus receptor genes, complement pathways components and receptors, cd94/nkreceptor-like, lectins, tnf, il1-r, collagens. however, pure homology seeking for vertebratespecific immunorelevant molecules in invertebrates is of limited value, and functional screening methods may be a more promising approach. accordingly, the expression analysis of humoral and receptorial molecules in ciona’s tissues and hemocytes following a challenge indicate their involvement in ciona’s inflammatory response. there is no evidence of mhc orthologs, tcr, igs in urochordates and agnathans, and no evidence have been reported on the hundreds of key genes involved in vertebrate adaptive immunity. there is a lack of evidence for a gradual transition from the invertebrate innate immune system to the recombinatorial immune system of higher vertebrates (khalturin et al., 2004) and how the adaptive immune system emerged is still obscure. in the genome of c. intestinalis, genes that encode molecules with membrane receptor features have been found among many members of the ig superfamily. they contain the v, and c1-like domains typical of vertebrate antigen receptors and mhc class i and ii. the human homologs of these genes segregate in a single unit of four paralogous segments on chromosomes 1q, 3q, 11p, and 21q. in these regions there are several genes involved in the adaptive immune system, and mhc paralogs with some related members. presumably, an ancestral receptor emerged before the ragmediated rearrangement originated the ancestor of ig and tcr provided with a v domain, in which v and j regions were encoded by a single exon. it has been hypothesized that the simplest ancestral receptor could be a single chain made up of v and c domain, every one linked to a transmembrane segment and a short cytoplasmic tail for signaling (azumi et al., 2003; du pasquier, 2004; kasahara et al., 2004). the status of the urochordate genes reflects perhaps a primitive pre-duplication/prerecombination-activating gene (rag) stage that foreshadow the pathway leading to the genesis of the t-cell receptor (tcr) and antibodies (du pasquier et al., 2004). the absence of antigen-presenting molecules of the mhc-linked class i and ii types, raise questions on the ancestral chordates self-nonself self and allorecognition constituents. recently novel perspectives have been proposed upon a c. intestinalis molecular code for individuality in the absence of mhc (khalturin et al., 2005) as well as on the involvement of complement components. kurn and colleagues (2007) proposed that early during chordate phylogenesis the components of the complement system in addition to their role in pathogen elimination may be involved in allorecognition. so far the phylogenetic analysis of complement components indicates that gene expansion was generated by duplication events that occurred independently in the ascidian and vertebrate lineages. furthermore, since diverse lectin-crd repertoires in tunicates mediate broad recognition (quesenberry et al., 2003), molecular diversity in s53 non self recognition could be due to the glycome code. in addition, mounting evidence indicates that invertebrate immune-type receptors may undergo somatic diversification through elaborate rna processing mechanism (zhang et al., 2004; kalturin et al., 2005; watson et al., 2005; sadd and schimidhemplel, 2006). nowadays, much is known about the evolution of the immune system, but the details of its origin and ancestral genes functions remain to be elucidated (loker et al., 2004). obviously, we are expecting insight that will come by recognizing gene product functions and identifying the function of noncoding sequences of dna lying between the genes, which could regulate gene expression. finally, understanding the complex orchestration of ciona gene networks is crucial to much of biomedical research, while the comparative genomic studies are helping to determine the function and modulation of the genes and other dna regions in the human genome. acknowledgements this work was supported by the miur princofin 2006-2008 and the university of palermo research grant to np. references arika s, hirano t, taga t, kishimoto t. biology of multifunctional cytokines: il6 and related molecules (il1 and tnf) faseb j. 4: 28602867, 1990. azumi k, de santis r, de tomaso a, rigoutsos i, yoshizaki f, pinto mr, et al. genomic analysis of immunity in a urochordate and the emergence of the vertebrate immune system: "waiting for godot”. immunogenetics 55: 570581, 2003. ballarin l. immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art. inv. surv. j. 5: 54-74, 2008. beck g. macrokines: invertebrate cytokine-like molecules. front. biosci. 3: 559-569, 1998. ben-shlomo r. the molecular basis of allorecognition in ascidians. bioessay 30: 1112, 2008. beschin a, bilej m, torreele e, de baetselier p. on the existence of cytokines in invertebrates. cell. mol. life sci. 58: 801-814, 2001. beschin a, bilej m, lucas r, de baetselier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on similar lectin-like activity. in: beshin a (ed), progress in molecular and subcellular biology. invertebrate cytokines, springer, berlin, heidelberg, new york, pp 145-163, 2004. biassoni r, cantoni c, marras d, giron-michel j, falco m, moretta l, et al. human natural killer cell receptors: insights into their molecular function and structure j. cell. mol. med. 7: 376387, 2007. bodmer wf. evolutionary significance of the hla system. nature 237: 139-145 1972 bonura a, vizzini a, salerno g, parrinello n, longo v, colombo p. the expression of a cloned mbl-like collectin is enhanced in the inflamed body wall of the ascidian ciona intestinalis as an effect of lps injection. mol. immunol. [submitted]. boyington jc, riaz an, patamawenu a, coligan je, brooks ag, sun pd. structure of cd94 reveals a novel c-type lectin fold: implications for nk cell-associated cd94/nkg2 receptors. immunity 10: 75-82, 1999. bransteitter r, pham p, scharff md, goodman mf. activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded dna but requires the action of rnase. proc. nat. acad. sci. usa 100: 4102-4107, 2003. brown gd, gordon s. immune recognition. a new receptor for beta-glucans. nature 413: 36-37, 2001. buss l. the evolution of individuality. princeton university press, princeton, nj, 1987. cadavid lf, powel ae, nicotra ml, moreno m, buss lw. an invertebrate histocompatibility complex. genetics 167: 357-365, 2004. cammarata m, arizza v, cianciolo c, parrinello d, vazzana m, vizzini a, et al. the prophenoloxidase system is activated during the tunic inflammatory reaction of ciona intestinalis. cell tissue res. 333: 481-492, 2008. chadwick-furman n, rinkevich b. a complex allorecognition system in a reef-building coral: delayed responses, reversal and nontransitive hierarchies. coral reef 13: 57-63, 1994. cebo c, dambrouck t, maes e, laden c, strecker g, michalski jc, et al. recombinant human interleukins il1 alfa, il1 beta, il4, il6, and il7 show different and specific calciumindependent carbohydrate-binding properties. j. biol. chem. 276: 5685-5691, 2001. cebo c, vergoten g, zanetta jp. review. lectin activities of cytokines: functions and putative carbohydrate-recognition domains. bioch. biophys. acta 1572: 422-434, 2002. darwin c. the descent of man, and selection in relation to sex. hurst, new york, 1874. davidson b, swalla bj. a molecular analysis of ascidian metamorphosis reveals activation of an innate immune response. development 129: 4739-4751, 2002. dehal p, satou y, campbell rk, chapman j, degnan ba, de tomaso b, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157–2167, 2002a. dehal p, satou y, azumi k, branno m, campbell b, degnan b, et. al. the complete genome sequence of the ascidian, ciona intestinalis: insights into the evolutionary origins of chordates and vertebrates. science 298: 2157-2167, 2002b. delsuc f, brinkmann h, chourrot d, hervé p. tunicate and not cephalochordates are the clotest living relatives of vertebrates. nat. lett. 439: 965-968, 2006. de tomaso aw, weissman il. evolution of protochordate allorecognition locus. science 303: 977, 2004. de tomaso aw, nyholm sv, palmeri kj, ishizuka kj, ludington wb, mitchel k, et al. isolation and characterization of a protochordate s54 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&term=de%20tomaso+a http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&term=nyholm+s http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&term=palmeri+k http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&term=ishizuka+k http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&term=ludington+w http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&term=mitchel+k histocompatibility locus. nature 438: 454-459, 2005. du pasquier l. innate immunity in early chordates and the appearance of adaptive immunity. c.r. biol. 327: 591-601, 2004. du pasquier l, zucchetti i, de santis r. immunoglobulin superfamily receptors in protochordates: before rag time. immunol. rev. 198: 233-248, 2004. eyre dr, wu j-j. collagen cross-links. top. curr. chem. 247: 207-229, 2005. francino mp. an adaptive radiation model for the origin of new gene functions. nat. genet. 37: 573-578, 2005. fumiko y, yoshizaki fy, ikawa s, satake m, satoh n, nonaka m. structure and the evolutionary implication of the triplicated complement factor b genes of a urochordate ascidian, ciona intestinalis. immunogenetics 56: 930-942, 2005. fujita t. evolution of the lectin-complement pathway and its role in innate immunity. nature rev. immunol. 2: 346-353, 2002. fujita t, endo y, nonaka m. primitive complement system-recognition and activation mol. immunol. 41: 103-111, 2004. gasparini f, franchi n, spolaore b, ballarin l. novel rhamnose-binding lectins from the colonial ascidian botryllus schlosseri. dev. comp. immunol. 32: 1177-1191, 2008. gee h. before the backbone: views on the origin of vertebrates. chapman & hall (eds), london, 1996. henson pm. dampening inflammation. nat. immunol. 6: 1179-1181, 2005. imler jl. hoffmann ja. toll receptors in innate immunity. trends cell biol. 11: 304-311, 2001. iizuka k, naidenko ov, plougastel bf, fremont dh, yokoyama wm. genetially linked c-type lectinrelated ligands for nkrp1 family of natural killer receptor. nat. immunol. 4: 801-807, 2003. käpylä j, jäälinoja j, tulla m, ylöstalo j, nissinen l, viitasalo t, et al. the fibril-associated collagen ix provides a novel mechanism for cell adhesion to cartilaginous matrix. j. biol. chem. 279: 51677-51687, 2004. kaisho t, akira s. critical roles of toll-like receptors in host defense. crit. rev. immunol. 20: 393405, 2000. kasahara m. genome paralogy: a new perspective on the organization and origin of the major histocompatibility complex. curr. top. microbiol. immunol. 248: 53-66, 2000. kasahara m, suzuki t, du pasquier l. on the origin of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates. trends immunol. 25: 105-111, 2004. khalturin k, becker m, rinkevich b, bosch tcg. urochordates and the origin of natural killer cells: identification of a cd94/nkr-p1-related receptor in blood cells of botryllus. proc. natl. acad. sci. usa 100: 622–627, 2003. khalturin k, kurn u, pinnow n, bosch tcg. towards a molecular code for individuality in the absence of mhc: screening for individuality variable genes in the urochordate ciona intestinalis. dev. comp. immunol. 29: 759-773, 2005. khalturin k, pancer z, cooper md, bosch tcg. recognition strategies in the innate immune system of ancestral chordates. mol. immunol. 41: 1077-1087, 2004. klein j. the grapes of incompatibility. dev. cell 10: 2-4, 2006 kürn u, sommer f, bosch tc, khalturin k. in the urochordate ciona intestinalis zona pellucida domain proteins vary among individuals. dev. comp. immunol. 31: 1242-1254, 2007. lambris jd. the third component of complement chemistry and biology. springer, berlin, heidelberg, new york, 1990. lanier ll. turning on natural killer cells. j. exp. med. 191: 1259-1262, 2000. litman g, cooper md. why study the evolution of immunity? nat. immunobiol. 8: 329-331, 2007. loker es, adema cm, zhang s-m, kepler tb. invertebrate immune systems not homogeneous, not simple, not well understud. immunol. rev. 198: 10-24, 2004. manis jp, tian m, frederick w. mechanism and control of class-switch recombination trends immunol. 23: 31-39, 2002. mcnerney me, guzior d, kumar v2b4 (cd244)cd48 interactions provide a novel mhc class iindependent system for nk-cell self-tolerance in mice. blood 106: 1337-1340, 2005. medzhitov r, janeway ca. innate immune recognition: mechanisms and pathways immunol. rev. 173: 89-97, 2000. melillo d, sfyroera g, de santis r, graziano r, marino r, lambris jd, et al. first identification of a chemotactic receptor in an invertebrate species: structural and functional characterization of ciona intestinalis c3a receptor. j. immunol. 177: 4132-4140, 2006. marino r, kimura y, de santis r, lambris jd, pinto mr. complement in urochordates: cloning and characterization of two c3-like genes in the ascidian ciona intestinalis. immunogenetics 53: 1055-1064, 2002. murabe n, hoshi m. re-examination of sibling cross-sterility in the ascidian, ciona intestinalis: genetic background of the self-sterility. zool. sci. 19: 527-538, 2002. narayanan as, engel ld, page rc. the effect of chronic inflammation on the composition of collagen types in human connective tissue. coll. relat. res. 3: 323-334, 1983. nonaka m, yoshizaki f. evolution of the complement system mol. immunol. 40: 897902, 2004a. nonaka m, yoshizaki f. primitive complement system of invertebrates. immunol. rev. 198: 203-215, 2004b. nonaka m, miyazawa s. evolution of the iniziating enzymes of the complement system. genome biol. 3, reviews1001, 2002. nwomeh bc, yager dr, cohen ik. physiology of the chronic wound. clin. plast. surg. 25: 341356, 1998. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. in: beschin a, muller weg. (eds), s55 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22narayanan%20as%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22engel%20ld%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22page%20rc%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract javascript:al_get(this,%20'jour',%20'coll%20relat%20res.'); invertebrate cytokines and the phylogeny of immunity: facts and paradoxes. progr. mol. subcell. biol., springer-verlag, berlin, heidelberg, vol. 34, pp 1-25, 2004. pancer z, amemya ct, ehrhardt gr, ceitlin j, gartland gl, cooper md. somatic diversification of variable lymphocyte receptors in agnathan sea lamprey. nature 430: 174-180, 2004. parrinello n. the reaction of ciona intestinalis l. (tunicata) to subcuticular erythrocyte and protein injection. dev. comp. immunol. 5 (sup.1): 105-110, 1981. parrinello n. cytotoxic activity of tunicate hemocytes. in rinkevich b, müller weg (eds), invertebrate immunology, springer-verlag, berlin. pp 190-217, 1996. parrinello n, patricolo e. inflammatory-like reaction in the tunic of ciona intestinalis (tunicata). ii. capsule components. biol. bull. 167: 238-250, 1984. parrinello n, patricolo e, canicatti c. inflammatorylike reaction in the tunic of ciona intestinalis (tunicata). i. encapsulation and tissue injury. biol. bull. 167: 229-237, 1984. parrinello n, cammarata m, arizza v. univacuolar refractile hemocytes from the tunicate ciona intestinalis are cytotoxic for mammalian erythrocytes in vitro. biol. bull. 190: 418-425, 1996. parrinello n, arizza v, cammarata m, giaramita ft, pergolizzi m, vazzana m, et al. inducible lectins with galectin properties and human il1α epitopes opsonize yeast during the inflammatory response of the ascidian ciona intestinalis. cell tissue res. 329: 379-390, 2007. parrinello n, vizzini a, arizza v, salerno g, parrinello d, cammarata m, et al. enhanced expression of a cloned and sequenced ciona intestinalis tnfα-like (citnfα) gene during the lps-induced inflammatory response cell and tissue res. 334: 305-317, 2008. pinto mr, de santis r, marino r, usui n. specific induction of self discrimination by follicle cells in ciona intestinalis oocytes. dev. growth differ. 37: 287-291, 1995. pinto mr, chinnici c, kimura y, melillo d, marino r, spruce la, et al. cic3-1a-mediated chemotaxis in the deuterostome invertebrate ciona intestinalis (urochordata). j. immunol. 171: 5521-5528, 2003. quesenberry ms, ahmed h, elola mt, o’leary n, vasta gr. diverse lectin repertoires in tunicates mediate broad recognition and effector innate immune responses. integr. comp. biol. 43: 323-330, 2003. ricard-blum s, ruggiero f. the collagen superfamily: from the extracellular matrix to the cell membrane. pathol. biol. 53: 430-442, 2005. roach jc, glusman g, rowen l, kaur a, purcell mk, smith kd, et al. the evolution of vertebrate toll-like receptors proc. natl. acad. sci. usa 102: 9577-9582, 2005. rogers s, wong syc. c-type lectin and lectin-like receptors in the immune system. in wong syc, arsequell g. (eds), immunobiology of carbohydrates, landes bioscience/ eurekah.com, georgetown, texas, usa and kluwer academic/plenum publishers, new york, usa, pp 101-118, 2003. rosati f, de santis r. studies on fertilization in the ascidians. i. self-sterility and specific recognition between gametes of ciona intestinalis. exp. cell res. 112: 111-119, 1978. rowley, af. the blood cells of the sea squirt ciona rntestinalis. morphology, differential counts, and in vitro phagocytic activity. j. invertehr. pathol. 37: 91-100, 1981. sadd bm, schmid-hempel p. insect immunity shows specificity in protection upon secondary pathogen exposure. curr. biol. 16: 1206-1210, 2006. satoh n. the ascidian tadpole larva: comparative molecular development and genomics. nat. rev. genet. 4: 285-295, 2003. satou y, yamada l, mochizuki y, takatori n, kawashima t, sasaki a, et al. a cdna resource from the basal chordate ciona intestinalis. genesis 33: 153-154, 2002. seya t, hirano a, matsumoto m, nomura m, ueda s. human membrane cofactor protein (mcp, cd46): multiple isoforms and functions. int j. biochem. cell biol. 31: 1255-1260, 1999. shida k, terajima d, uchino r, ikawa s, ikeda m, asano k, et al. hemocytes of ciona intestinalis express multiple genes involved in innate immune host defense. biochem. biophys. comm. 302: 207-218, 2003. singer aj, clark raf. mechanisms of desease: cutaneous wound healing. n. engl. j. med. 341: 738-743, 1999. swalla bj, cameron cb, corley ls, garey jr. urochordates are monophyletic within the deuterostomes. syst. biol. 49: 52-64, 2000. terai i, kobayashi k, matsushita m, fujita t, matsuno k. α2-macroglobulin binds to and inhibits mannose-binding protein-associated serine protease. int. immunol. 7: 1579-1584, 1995. terajima d, yamada s, uchino r, ikawa s, ikeda m, shida k., arai y., wang h.g. et al. identification and sequence of seventy-nine new transcripts expressed in hemocytes of ciona intestinalis, three of which may be involved in characteristic cell-cell communication. dna res. 10: 203-212, 2003. thomas ah, edelman er, stultz cm. collagen fragments modulate innate immunity exp. biol. med. 232: 406-411, 2007. tong l. toll/interleukin-1 receptors and innate immunity. in: waksman g, caparon mg, hultgren s (eds), structural basis of bacterial pathogenesis, asm press, washington, dc, pp 241-263, 2005. vasselon t, detmers pa. toll receptors: a central element in innate immune responses. infect. immun. 70: 1033-1041, 2002. vivier e, tomasello e, paul p. lymphocyte activation via nkg2d: towards a new paradigm in immune recognition? curr. opin. immunol. 14: 306-311, 2002. vizzini a, arizza v, cervello m, cammarata m, gambino r, parrinello n. cloning and expression of a type ix-like collagen in tissues s56 of the ascidian ciona intestinalis. biochim. biophys. acta 1577: 38-44, 2002. structure and the evolutionary implication of the triplicated complement factor b genes of a urochordate ascidian, ciona intestinalis. immunogenetics 56: 930-942, 2005. vizzini a, pergolizzi m, vazzana m, salerno g, di sano c., macaluso p, et al. facit collagen (1α chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian ciona intestinalis. dev. comp. immunol. 32: 682-692, 2008. yu c, dong m, wu x, li s, huang s, su j, et al. genese “waiting” for recruitment by the adaptive immune system: the insights from amphioxus. j. immunol. 174: 3493-3500, 2005. wakoh t, ikeda m, uchino r, azumi k, nonaka m, kohara y, et al. identification of transcripts expressed preferentially in hemocyte of ciona intestinalis that can be used as molecular markers. dna res. 11: 345-352, 2004 zeng l, swalla bj. molecular phylogeny of the protochordates: chordate evolution. can. j. zool. 83: 24-33, 2005. zhang sm, adema cm, kepler tb, loker es. diversification of ig superfamily genes in an invertebrate. science 305: 251-254, 2004. watson fl, püttmann-holgado r, thomas f, lamar dl, hughes m, kondo m, et al. extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 18741878, 2005. zhong j, kyriakis jm. dissection of a signaling pathway by which pathogen-associated molecular patterns recruit the jnk and p38 mapks and trigger cytokine release. j. biol. chem. 282: 24246-24254, 2007. yokobori y, watanabe s, oshima t. mitochondrial genome of ciona savignyi (urochordata, ascidiacea, enterogona): comparison of gene arrangement and trna genes with halocynthia roretzi mitochondrial genome, j. mol. evol. 57: 574-587, 2003. zucchetti i, marino r, pinto mr, lambris jd, du pasquier, l, de santis r. cicd94-1, an ascidian multipurpose c-type lectin-like receptor expressed in ciona intestinalis hemocytes and larval neural structures differentiation 76: 267-282, 2008. yoshizaki fy, ikawa s, satake m, satoh n, nonaka m. s57 isj 4: 51-54, 2007 55 isj 4: 55-64, 2007 issn 1824-307x research report separated hemocyte populations from the ascidian ciona intestinalis contain and release in vitro opsonizing ca2+-independent and β-galactoside specific lectins n parrinello, v arizza, m vazzana, m cammarata, ft giaramita, ml di bella, a vizzini, d parrinello marine immunobiology laboratory, department of animal biology, university of palermo, italy accepted june 14, 2007 abstract cytosolic lectins, ca2+-independent and β-galactoside-specific, were determined to be contained in hemocyte and pharynx lysate supernatants of ciona intestinalis, as revealed by hemagglutination assay with trypsinized rabbit erythrocytes. ca2+-independence and decreasing β-galactosides inhibitory capacity (tdg > lacnac ≥ lactose > galactose) have been considered properties typical of galectins. these lectins can be promptly released by hemocytes maintained in vitro suggesting their involvement in defense responses including inflammatory reactions. both cell lysate supernatants and hemocyte culture medium presented β-galactoside-inhibitable opsonizing activity versus yeast. although a percoll density gradient separation method showed that several hemocyte types contain and release β-galactoside-specific molecules, results suggest that hyaline and granular amoebocytes are the primary source of these molecules. key words: hemocyte lectins; hemagglutinins: β-galactosides; phagocytosis; opsonization; hemocytes; tunicates; ciona intestinalis introduction animal lectins, usually revealed by their hemagglutinating activity, are components of a wellconserved protein-carbohydrate recognition mechanism that function in a variety of biological systems (feizi, 2000; sharon and lis, 2003, 2004). functions include intraand extracellular transport processes, sensor branches of innate immunity and recognition of foreign glycans, induction and suppression of effector release, regulation of cellcell/cell-matrix adhesion or migration, positive/negative growth control with implication for differentiation and malignancy (kilpatrick, 2000, 2002; gobius et al., 2002; sharon and lis, 2003). matching glycan diversity with the lectin presence and various glycan epitopes identified as ligands, immune functions appear to be based on the sugar code. in vertebrates and invertebrates lectin families ___________________________________________________________________________ corresponding author: nicolò parrinello marine immunobiology laboratory department of animal biology university of palermo via archirafi 18 palermo, italy e-mail address: nicpar@unipa.it have been established (cooper and barondes, 1999; cooper 2002; kilpatrick, 2002; vasta et al., 1999, 2004a), and, among them, ca2+-independent soluble lectins, generally characterized for a typical carbohydrate recognition domain (s-crd) with affinity for β-galactosides (s-type/galectins), can be pro-inflammatory (brewer, 2002; rabinovich et al., 2002; vasta et al., 2004b). of critical importance for galectin characterization is the binding specificity of the basic unit of recognition as shown by the relative inhibitory efficiency of key oligosaccharides αlactose, n-acetyl-d-lactosamine (lac-nac) and thiodi-galactoside (tdg) (cooper and barondes, 1999; dodd and drickamer, 2001; sharon and lis, 2003). in invertebrates, the defence responses are mainly based on hemocyte types that release humoral factors, including lectins, lysins, clotting and antibacterial proteins (loker et al., 2004), or display cell-linked activities (parrinello, 1996; parrinello et al., 2003). cellular recognition has been attributed to a protein-carbohydrate molecular mechanism located at the cell surface. sugar-binding proteins have been found on hemocyte surface (amirante et al., 1978; vasta et al., 1984; parrinello and arizza, 1988) and they are present in the hemolymph of all mailto:nicpar@unipa.it 56 the examined species, probably involved in nonadaptive immune recognition. in many cases, an opsonic function has been demonstrated (cheng et al., 1984; renwrantz and stahmer, 1983). in ascidians, considered a key group in chordate phylogenesis (hori and osawa, 1987; field et al., 1988; swalla et al., 2000; zeng and swalla, 2005), multiple naturally occurring or inducible galectins have been found in cell-free hemolymph or bound to hemocyte surface (parrinello 1995; nair et al., 2001; vasta et al., 2001; green et al., 2003; quesemberry et al. 2003; vasta et al., 2004a). in the colonial ascidian botryllus schlosseri, ballarin et al. (1999, 2000) purified from the hemocyte lysate supernatant a dgalactose specific humoral opsonin with galectin properties. in styela clava a c-type humoral lectin with opsonin properties has been purified from the hemolymph (kelly et al., 1992). in addition, lectins could be released from hemocytes cultured in vitro (arizza et al., 1991; cammarata et al., 1993) and were contained and released from pharynx explants (raftos et al., 1990; arizza et al., 1991, 1997). previous papers reported that ciona intestinalis serum hemolymph contains ca2+-dependent (ctype) and ca2+-independent lectins (wright, 1974; parrinello and patricolo, 1975). a recent report showed that serum galectins could be enhanced in inflammatory responses (parrinello et al., 2007), whereas c-type lectins may be responsible of complement activation (pinto et al., 2003). in this respect c. intestinalis genome-wide analysis revealed that several c-type lectin and galectin genes have been annotated in the genome (hori and hosawa, 1987; cooper and barondes 1999; dehal et al., 2002). however, few data exist on the functional role and tissue distribution of lectins of this ascidian. to ascertain the lectin defence role, tissue localization compatible with internal defence are additional requirements to support the involvement of these molecules in immune protection. in this sense, circulating hemocytes assume particular interest as well as pharynx that represents the main route of pathogen entry. in c. intestinalis, several hemocyte types have been described (de leo, 1992), including stem cells, hyaline and granular amoebocytes, unilocular refractile granulocytes, signet ring cells, morula cells, small and large compartment cells. of these cell types, only hyaline and granular amoebocytes are capable of phagocytosis in vitro (rowley, 1981), and several granular and vacuolated hemocytes appeared to be mainly responsible of the inflammatory responses in vivo (parrinello, 1981; parrinello et al., 1984; parrinello and patricolo, 1984; parrinello et al., 1990). in the present paper we show that soluble lectins are contained in c. intestinalis hemocyte and pharynx lysate supernatants, as revealed by β-dgalactoside inhibition of rabbit erythrocytes agglutination. in addition, these lectins are promptly released by hemocytes in vitro. both cell lysate supernatants and hemocyte short-term culture medium presented opsonizing activity versus yeast, revealing the involvement of β-galactoside specific lectins as opsonins. a percoll density gradient separation method displayed that several hemocyte types, that have been shown to be involved in distinct phase of the inflammatory response, contain and release these lectins, whereas hyaline and granular amoebocytes, that posses in vitro phagocytic activity, appear to be rich in cytosolic lectins and are the main source of their release. materials and methods tunicates, hemolymph collection ascidians (7-10 cm long) were collected from mazara del vallo harbor (italy), held in refrigerated (18 °c) and aerated sea water (60 l aquaria) and fed every second day with a marine invertebrate filter feeding diet (kent marine inc. wi usa). the animals were blotted dry to remove any excess of seawater, and bled by removal of the tunic and puncture of the heart. to collect hemocytes, hemolymph was harvested into a fourfold excess of ice cold sterile artificial sea water without cacl2 and mgcl2 (fsw: 9 mm kcl, 29 mm na2so4, 2 mm nahco3, 0.5 m nacl, ph 7.4) containing 10 mm ethylenediaminetetracetic acid (fsw-edta) as an anticoagulant. after centrifugation at 850xg (10 min, 4 °c), pooled hemocytes (10 ascidians/experiment) were washed twice with fsw-edta and, finally, suspended in sterile fsw adjusted for osmolarity with the hemolymph (1,090 mosm kg-1). hemocyte mortality, estimated by trypan blue (0.05 % in fsw) exclusion test, was lower than 5 %. pharynx explants were surgically excised with sterile scissors and washed three times with sterilized fsw-edta. the same amount of tissue (about 1 gr) was used for every preparation. all media were sterilized through a 0.22 µm filter (millipore, millex). preparation of hemocyte and pharynx lysate supernatants (hls, phls) hemocytes from pooled hemolymph (15 ascidians for every preparation) were pelleted by centrifuging at 850 xg for 10 min at 4 °c. after two washings in fsw, hemocytes (10 x 106 cells/ml) were suspended in diluted ice-cold medium (1:5 in d.w.) to be sonicated at 4 °c for 60 seconds (branson, model b15, danbury, ct, usa). the cell lysate was spun (27,000 xg, 20 min, 4 °c), and the resulting supernatant (designed hls) was dialyzed against tbs (tris hcl 50 mm, nacl 0.15 m, ph 7.4), and used for the hemagglutination assay. pharynx explants (1 gr tissue) dried with filter paper, frozen at -80 °c and homogenized on ice, were sonicated at 4 °c for 60 seconds. after centrifuging at 27,000xg for 20 min at 4 °c, the supernatant (designed phls 0.6 – 0.7 mg/ml protein content) was extensively dialyzed against tbs. for hemagglutination and opsonization assays, samples were 100 times diluted. to examine the possible effect of cytosolic proteases, in previous experiments, a protease inhibitor cocktail (sigma, st. louis, usa) was added (0.1 % final concentration) into the medium just before hls preparation. 57 hemocyte culture and supernatant preparation details of the method have been reported elsewhere (cammarata et al., 1993). unfractionated or enriched hemocyte populations were suspended in sterile isosmotic artificial sea water (sw) (fsw containing 12 mm caci2.6 h2o and 26 mm mgci2.6 h2o). osmolarity was adjusted to 1090 mosm kg -1. hemocytes (3x106 in 200 µl medium) were put into each well of sterile flat-bottomed culture plates (nunc, denmark) and maintained at 4, 10, or 18 °c. in each experiment, cell-free medium from 10 cultures (designed hes) was pooled and dialyzed against tbs prior to be assayed. percentage of dead cells was evaluated with the trypan blue exclusion test. cell viability of hemocytes cultured for 1.5 h at 10 °c in sw was evaluated with neutral red vital stain (borenfreund and puerner, 1984). values lesser than 3 ± 0.5 % dead cells, and more than 97 ± 1.1 % viable cells were found. preparation of rabbit and sheep erythrocyte suspensions and hemagglutination assay rabbit erythrocytes (re) and sheep erythrocytes (se) were obtained from “istituto zooprofilattico della sicilia” (palermo). the erythrocyte pellet was washed with pbs (pbs: 6 mm kh2po4, 30 mm na2hpo4, 0,11 m nacl, ph 7.4) and centrifuged at 500xg for 10 min at 4 °c, then resuspended in tbs to obtain a 1 % suspension. as previously reported (parrinello and canicattì, 1982), hemagglutinating activity (designed ha) was determined in 96-well round bottom microtiter plates using tbs containing 0.1 % gelatin as a dilution medium (serial two-fold dilutions), and an equal volume of 1 % re or se in tbs. the microplate was incubated at 37 °c for 1 h, and 2 h at 4 °c. to increase the erythrocyte sensitivity to the hemagglutination assay, trypsin-treated erythrocytes (try-re, try-se) were prepared by suspending erythrocyte pellet, from 1.0 ml blood, into 6 ml tbs containing 300 µg trypsin (stock solution prepared in 10 mm hcl). the reaction mixture was incubated at 37 °c for 15 min, and, trypsinized erythrocytes, washed with tbs, were resuspended (1 %) in the same medium. to verify the role of ca2+, the hemagglutination assay was carried out in the presence of 20 mm edta or 10 mm cacl2. the titre of hemagglutinating activity (designed ht) was expressed as the reciprocal of the highest dilution giving unequivocal agglutination judged by eye or with a low power binocular microscope. the ht values, expressed as log2, were recorded as the average ± sd of 10 different assays. controls consisted of tbs samples in which serum was not added. to verify the role of ca2+, lysate supernatants were dialyzed against tbs in the presence of 20 mm edta or 10 mm cacl2, and erythrocytes were suspended in this medium for hemagglutination assay. formaldehyde-fixed rabbit erythrocytes (f-re) were prepared according to the csizmas’s method (1960), and suspended in tbs. yeast preparation, opsonization, and in vitro phagocytosis assay a saccharomyces cerevisiae (baker's yeast, type ii) suspension was prepared in distilled water at 0.25 % w/v (approx. 1x10 8 yeast cells/ml), autoclaved for 15 min, washed twice by centrifuging at 2,000xg (5 min, 4 °c), and finally incubated for 2 h at 20 °c with a solution of eosin-y at 0.05 % final concentration (cammarata and arizza, 1994). after repeated washing, yeast were suspended at 0.125 % final concentration in sterile calciumand magnesium-free sw (fsw), and used immediately. for opsonization, yeast were incubated with lysate supernatant (0.125 % w/v) for 1.5 h at 20 °c, washed in fsw (3 times), and finally suspended in the same medium at the initial concentration. after this treatment, yeast appeared to be agglutinated, forming small clumps, but they were easily resuspended by washing with fsw. to verify the role of divalent cations in opsoninyeast binding, opsonization was carried out in the presence of 20 mm edta. furthermore, the effect of added ca2+ or mg2+ was estimated with fsw containing 10 mm cacl2 or mgcl2. for the phagocytosis assay, 200 µl hemocyte suspension (1x10 6 cells in sw, indicated as he) was mixed with 100 µl of yeast preparation (10:1 yeast:hemocyte ratio), and incubated in 1 ml test plastic tubes with gentle stirring for 90 min at 20 °c. then, 50 µl of a quenching solution (2 mg/ml trypan blue, 2 mg/ml crystal violet in 0.02 citrate buffer, ph 4.4, containing 33 mg/ml nacl) was added. a drop of this suspension was smeared onto slides and examined under a light microscope equipped with a nomarsky differential interference contrast optic (diaplan, leica, wetzlar, germany). hemocytes (about 1000 for every assay, and at least 200/slide) were counted at 800x magnification. lysate supernatant opsonizing capacity was expressed as percent hemocytes showing ingested yeasts. results were compared to percent phagocytes in a reaction mixture in which hemocytes from ascidians were mixed with non-opsonized yeasts. the phagocytic index was calculated according to the following formula: total number of ingested yeasts/total number of counted phagocytes absorption with erythrocytes to absorb agglutinins or opsonins, fre or fse were used in the reaction mixture containing packed f-erythrocytes and lysate supernatant or culture medium (v/v). the mixture was incubated for 1 h at room temperature and overnight at 4 °c with occasional shaking, centrifuged at 800xg, and the supernatant assayed with try-re and try-se. to control the effect of experimental conditions on the hemagglutinating activity, no absorbed samples were treated as the absorbed ones. inhibition of hemagglutinating and opsonizing activities the supernatants (hls, phls, hes) were incubated for 60 min at 20 °c with decreasing sugar concentrations (starting from 100 mm final concentration), avoiding sample dilution. the 58 treated sample was then assayed for hemagglutinating activity. for inhibiting the opsonizing activity, yeasts were maintained for 1.5 h in lysate or culture supernatant preparations containing decreasing sugar concentrations, and then washed (2 times) with fsw before the phagocytosis assay. the last sugar concentration (mm) giving an unequivocal inhibitory activity was recorded. dgalactose, α-lactose, d-mannose, l-rhamnose, dglucose, l-fucose, n-acetyllactosamine (lacnac) and thio-digalactoside (tdg) were assayed. to examine the effect of sugar added to the yeast suspension, 25 µl of opsonizing serum containing 100 mm sugar was mixed on a slide with 25 µl yeast suspension and observed after 1.5 h incubation in a wet chamber. observations with a microscope equipped with nomarski differential interference contrast optics (leica) did not show any yeast clamps. to verify changes in yeast sensitivity to phagocyte, sugar treated yeasts were assayed, after washing, in a phagocytosis assay. identification and separation of the hemocytes through a percoll discontinuous density gradient the hemocytes were classified according to the most popular terminology (wright, 1981; de leo, 1992). in the hemolymph several hemocyte types have been recognized. lymphocyte-like cells are small stem cells. hyaline amoebocytes contain in their cytoplasm granules of uniform size; granular amoebocytes contain small or large granules; signet ring cells present a single large vacuole; compartment cells contain a variable number of large round and angular vacuoles distributed at the periphery of the cell; morula cells that, when allowed to stand, may assume a berry-like or morular appearance; unilocular refractile granulocyte (urg), characterized by an unique large granule that occupies the cytoplasm and appears to be refractile when observed under a light microscopy. the hemocyte populations were separated using the method described by parrinello et al. (1996). briefly, freshly collected hemocyte suspension (approximately 6x107/ml in 4 ml) diluted with fswedta was spun through a discontinuous gradient of equilibrated percoll (pharmacia fine chemicals uppsala, sweden) (dialyzed against hemocyte isosmotic fsw-edta). a gradient was performed with isosmotic medium to obtain decreasing densities (1.105, 1.098, 1.090, 1.079, 1.071, and 1.060 g/ml) into a 10 ml tube. the tube was centrifuged in a swing-out rotor (850xg, 15 min, 7 °c). bands of cells were gently removed by aspiration from the gradients and washed twice before suspension in fsw. the total population of hemocytes was portioned into six distinct, discrete bands (b1-b6). dead cells lower than 5 % were found, and viable cells were higher than 95 %. although each band was mainly enriched for certain hemocyte types, homogeneous populations could not be separated. for microscopy observations, the cells were removed from the gradients, washed twice in fsw. to check for the hemocyte types contained in each band, 200 µl of the cell suspension was layered on a slide soaked with the poly-l lysine, fixed (30 min) with 1 % saccharose and 1 % glutaraldeyde in fsw, and stained with hematoxilin-eosin (5 min). differential count of the hemocytes from each band was performed (at least 200 cells/slide). to prepare hls the correspondent bands from different percoll gradients were pooled to reach 1.0x107 cells/ml. protein content determination protein content was measured by the bradford method (1976). bovine serum albumin was used as standard. statistical analysis data were from five distinct experiments, and each assay repeated three times. hemagglutinin titres, recorded as log2 ± sd, were examined by the student t-test. differences were considered significant at p < 0.05. 2.9 chemicals unless otherwise reported, all chemicals were purchased from sigma. table 1 hemagglutinating activity of hemocyte lysate supernatant (hls), pharynx lysate supernatant (phls) and supernatant from cultured hemocytes (hes) cultured for 1.5 h in sw at 18 °c, assayed against trypsinized rabbit (try-re) or sheep erythrocytes (try-se), in the presence or absence of divalent cations hemagglutinating activity (log2 ± sd, n=5) hls phls hes medium try-re try-se try-re try-se try-re try-se tbs 5.2 ± 0.4 -4.8 ± 0.8 -3.8 ± 0.4 - tbs-10 mm cacl2 5.4 ± 0.5 -4.6 ± 0.8 -3.8 ± 0.8 - tbs-10 mm mgcl2 4.8 ± 0.8 -4.8 ± 0.4 -3.8 ± 1.0 - tbs-20 mm edta 4.8 ± 1.0 -4.4 ± 0.5 -3.6 ± 0.8 - sw 4.6 ± 0.8 -5.6 ± 0.5 -3.6 ± 0.8 - f-sw-edta 4.6 ± 0.8 -5.6 ± 0.8 -3.6 ± 0.5 - 59 results hls and phls agglutinated rabbit erythrocytes in the absence of ca2+ preliminary hemagglutination assays with re of five distinct hls (20-25 µg/ml protein content), and phls in 100 times diluted samples preparations (20-25 µg/ml) showed 3.0 ± 0.8 ht, whereas higher titres (ht: 5.0 ± 0.5 and 4.8 ± 0.5, respectively) were found with try-re as targets. consequently, trypsinized erythrocytes were used for the next agglutinin titration. no activity was found against se or try-se (table 1). the presence of a protease inhibitor cocktail in lysate preparations did not enhance the hemagglutinating titre compared to that of inhibitor-free samples (data not shown). the addition of 10 mm (final concentration) cacl2, mgcl2 or 20 mm edta into the medium did not affect the hemagglutinating activity of hls and phls samples (table 1). hemocytes release in vitro ca2+-independent hemagglutinins supernatant from hemocyte cultures (hes) agglutinated try-re but not try-se. the hemocytes cultured at 4 °c for 1.5 h released a low amount of lectins into the cell-free medium (ht: 2.2-2.4), whereas highest levels (ht: 3.6-3.8) were found at 10 °c (table 1). higher temperatures (up to 18 °c) did not significantly enhance the agglutinin release. the presence of 20 mm edta as well as the addition of cacl2 or mgcl2 in the hemagglutination medium (tbs) did not affect the activity vs both the erythrocyte types (table 1). to compare the activity of supernatants from unseparated hemocyte culture with hls, 15x106 hemocytes/ml were divided in two groups, one of the two was homogenized, and the other one was cultured (1.5 h, 10 °c). in four distinct experiments, the hlss presented the highest activity (ht: 6.7 ± 0.9) compared with the hemocyte culture supernatants (ht: 4.0 ± 0.15). protein content of unseparated hemocyte culture supernatants from twelve distinct experiments ranged fig. 1 a ciona intestinalis phagocyte with ingested eosin-y treated yeasts, as shown by nomarski contrast interference observation (a), or uv–light observation (b). y: eosin-y treated yeast. bar = 5 µm from 8 µg/ml to 20 µg/ml. a significant proportionality between protein content and hemagglutinin titre was not observed. the culture medium composition did not affect the agglutinin-release. in preliminary experiments the same hts were recorded by assaying supernatants from hemocytes cultured in m199 enriched medium or sw. therefore, in the next experiments hemocytes were cultured in sw. erythrocyte specificity absorption experiments with fre or fse showed that the hemagglutinating activity of hls and phls was lost after absorption with fre, whereas it was maintained after treatment of hls and phls with fse (ht: 4.6-5.6, respectively). the same effect was exerted by fre on hes. hls, phls and hes opsonize yeast table 2 shows that 15 % (mean value) of hemocytes spontaneously ingested non-opsonized yeast (fig. 1). when the targets were opsonized with hls, phls or hes, a greater number of hemocytes ingested yeast, and the percentage significantly increased up to 20-24 % (p < 0.05). the phagocytic index significantly increased as an effect of the opsonization. table 2 opsonizing activity, in the absence or presence of edta (20 mm), of hemocyte lysate supernatant (hls), pharynx lysate supernatant (phls), and supernatant from hemocytes (hes) cultured for 1.5 h in sw at 18 °c, assayed against yeast cells. percent (%) phagocytes with ingested opsonized yeasts were compared with % phagocytes assayed with non opsonized (sw) targets. values are expressed as mean percentage of hemocyte containing yeasts ± sd (n=5). phagocytic index = total number of ingested yeasts/total number of counted phagocytes. ** p < 0.01; ***p<0.001 10 mm edta yeasts treated with phagocytes (%) phagocytic index phagocytes (%) phagocytic index sw 15.3 ± 1.2 1.9 ± 0.2 14.6 ± 0.9 1.7 ± 0.3 hls 24.2 ± 3.2 (**) 2.6 ± 0.3 (**) 22.1 ± 2.3 (**) 2.9 ± 0.3 (**) phls 23.9 ± 1.2 (**) 2.4 ± 0.2 (**) 22.8 ± 1.4 (**) 2.6 ± 0.3 (**) hes 20.2 ± 2.1 (**) 3.3 ± 0.3 (***) 19.7 ± 1.8 (*) 3.2 ± 0.2 (***) 60 table 3 sugar inhibition of the hemagglutinating and the opsonizing activity of hemocyte lysate supernatant (hls), pharynx lysate supernatant (phls) and supernatant from hemocytes (hes) cultured for 1.5 h in sw at 18 °c, assayed against trypsinized rabbit erythrocytes and yeasts respectively. the lowest sugar concentration (mm) that abolished the hemagglutinating activity of the sample against rabbit erythrocytes, or giving significant inhibitory activity of yeast opsonization was recorded. 100 mm starting sugar concentration sugar inhibitory concentration (mm ± sd, n=5) hemagglutinating activity (1) opsonizing activity (2) compound hls phls hes hls phls hes d-galactose 21.6 ± 9,8 10.6 ± 4,6 5.25 ± 1.7 25.0 ± 5.6 25.0 ± 8.8 25.0 ± 6.3 α-lactose 21.6 ± 9,8 16.0 ± 0 12.5 ± 1.0 50.0 ± 12.9 50.0 ± 11.7 50.0 ± 11.3 lactulose 44.0 ± 19,0 27.3 ± 9,8 25.0 ± 1.5 50.0 ± 10.4 50.0 ± 7.5 25.0 ± 10.9 lacnac 25.0 ± 6.8 25.0 ± 4.8 1.5 ± 1.0 50.0 ± 11.7 25.0 ± 3.8 25.0 ± 3.2 tdg 1.5 ± 0.5 1.5 ± 0.5 0.5 ± 1.0 50.0 ± 10.1 25.0 ± 5.1 12.5 ± 9.7 l-fucose n.i. n.i. n.i. n.i. n.i. n.i. d-mannose n.i. n.i. n.i. 25.0 ± 3.7 25.0 ± 6.3 25.0 ± 8.7 d-glucose n.i n.i. n.i. n.i. n.i. n.i. nana n.i. n.i. n.i. n.i. n.i. n.i. nag n.i. n.i. n.i. n.i. n.i. n.i. laminarin (%) 0.006 ± 0.002 0.012 ± 0.04 0.015 ± 0.001 0.5 ± 0.09 0.05 ± 0.001 0.5 ± 0.05 (1) hemagglutination assay against trypsinized-rabbit erythrocytes. (2) supernatant opsonising activity vs yeast examined in a hemocyte phagocytosis assay. n.i.: no inhibition the ca2+-independence of the opsonizing activity was shown by treating yeast with hls, phls or hes in the presence of 20 mm edta, 10 mm cacl2, or mgcl2. no differences were observed as an effect of opsonization medium composition (table 2). finally, opsonins were absorbed by treating samples with packed fre whereas was unchanged after fse absorption. galactosides inhibit the hemagglutinating and opsonizing activities the sugar-lectin binding of both agglutinins and opsonins was shown by sugar inhibition assay. the hemagglutinating activity of hls and phls was abolished by d-galactose, α-lactose, lactulose, lacnac, at various mm concentrations (table 3). except lactulose, lower concentrations (ranging from 1.5 mm lacnac to 12.5 mm lactose) of these sugars inhibited hemocyte culture supernatant. tdg was the most effective saccharides in inhibiting hemagglutination activity of hls, phls and hes (ranging from 0.5 to 1.5 mm). in all cases, the hemagglutinating activity was not affected by 100 mm d-glucose, l-fucose, d-mannose (table 2). the above reported active sugars inhibited the opsonizing activity of hls, phls and hes (table 3) even if higher concentrations were needed, whereas mannose inhibited the yeast opsonization. the presence of high sugar concentration in the medium used for preparing yeast did not affect their sensitivity to phagocytes, and, after washing, they were phagocytosed as the untreated ones. hemagglutinating activity of hls and culture supernatants from hemocyte populations enriched through a percoll discontinuous density gradient the cells removed from density gradient separated bands were examined and identified for their morphology. differential count of the hemocytes from each band was performed (at least 200 cells/slide). b1 mainly contained hyaline amoebocytes (~ 76 %) and, to a lesser extent, stem cells (~ 9 %) and signet ring cells (~ 11 %); b2 was mainly enriched in hyaline amoebocytes (~57 %), but also contained lymphocyte-like cells (~ 5 %), granular amoebocytes (~ 20 %), signetring cells (~ 9 %); b3 consisted primarily of ~ 71 % granular amoebocytes, ~ 22 % hyaline amoebocytes and ~ 8 % signet ring cells; b4 was composed of granular amoebocytes (~ 45 %) and morula cells (~ 52 %); b5 contained morula cells (~ 59 %) and univacuolar refractile granulocytes (~ 41 %); finally, b6 was largely (~ 84 %) made up of morula cells. hemocyte types present in each band at very low percentage were not reported. 61 fig. 2 ciona intestinalis main hemocyte types in b2, b3 and b6 bands separated through a discontinous percoll density gradient. hyaline amoebocytes (ha), granulocytes (g); morula cells (mc). bar = 10 µm in fig. 2 hyaline amoebocytes, granular amoebocyte, univacuolar refringent granulocytes and morula cells contained in the separated bands are shown. to identify the hemocytes that contained antire lectins, hlss from percoll-gradient separated hemocytes were assayed for their activity. as shown in fig. 3, hls from enriched hemocyte populations, standardized at a same hemocyte number (10x106/ml), presented various levels of hemagglutinating activity revealing that b2-hls and b3-hls reached titres higher (ht: 8.2 and 5.9, respectively; p < 0.01) than hls from the whole hemocyte preparations (4.2 ht). lower levels were associated with b1 and b6 (ht: 4.0 and 3.6, respectively), whereas the lowest ones (ht: 1.4-2.0) were recorded in b4 and b5 hlss. to identify the lectin-releasing hemocytes, the density-gradient enriched hemocyte populations were cultured at 10 °c for three hours, and, then, cell-free medium from pools of each separated band was assayed. the hemagglutination titres of b2 cellfree culture medium, compared with the culture medium of the remaining bands or unseparated hemocytes (ht: 1.3), showed the highest hemagglutinating titre (ht: 3.4), whereas 1.7 ht was found for b1 (fig. 3). very low hts were recorded for b3-b6 cell free culture medium. discussion the serum hemolymph of c. intestinalis contains naturally occurring lectins that agglutinated rabbit and sheep erythrocytes (wright, 1974; parrinello and patricolo, 1975). in the present paper we show that hemocytes and pharynx lysate supernatants agglutinated rabbit erythrocytes, whereas they were inactive against sheep erythrocytes revealing a certain range of specificity in discriminating target membrane sugar components. accordingly, erythrocyte trypsinization of sheep erythrocytes that exposed glycosylated components of the membrane outer layer did not affect the hemagglutination of sheep erythrocytes whereas increased rabbit erythrocytes sensitiveness, and higher hemagglutinin titres were recorded. the anti-tryre agglutinin titres (ht: 4-5) revealed in the hls and phls were higher than those (ht: 1-3) reported by parrinello and patricolo (1975) for the hemolymph serum. hemocytes and pharynx tissues cannot be directly compared in their agglutinin titres, in fact pharynx blood vessels can contain various amounts of hemocytes. these agglutinins, are cytosolic being identified in water-soluble extracts from hemocyte and pharynx preparations. the sample preparation method, carried out on ice followed by a prompt separation of the supernatant at 4 °c, presumably avoided any effect of cytosolic proteases as shown by the unchanged hemagglutinin titres after the addition of an anti-protease inhibitor cocktail. hemagglutinins can be promptly released by hemocytes maintained viable in vitro for a short time (1-3 h). such a release was temperature-dependent as it decreased with low temperature and reached the highest values at 10-18 °c. we do not know if lectins are secreted or a non classical secretory mechanism may externalize lectins confined to cytoplasm as reported for some galectins (sato et al.,1993; sato and hughes, 1994). the anti-re agglutinins contained in hemocytes and pharynx as well as released by hemocytes are ca2+-independent, d-galactoside specific lectins that showed a relative inhibitory activity. tdg, an analog of lactose, and lacnac, an analog of lactose with a hydroxyl substituted with an acetamide group, were more active than lactose in inhibiting the agglutination. tdg, lacnac, lactose and galactose were effective at higher concentrations. a such lectin-binding affinity of β-galactosides (tdg > lacnac ≥ lactose > galactose) and the ca2+independence have been considered properties typical of galectins (vasta et al., 2004b). accordingly to the defence role, these βgalactoside-specific lectins showed opsonic properties vs yeast, and hemocytes could release them in vitro presumably as an effect of cell activation due to the experimental procedures. a low percentage of phagocytes internalized non opsonized yeasts, presumably due to mannose receptors on the phagocyte membrane, whereas the opsonization of the targets with supernatants from hemocyte and pharynx lysates, and hemocyte culture medium enable a significantly major number 62 fig. 3 ( ) hemagglutinating activity of hemocyte lysate supernatant from non fractioned hemocytes (nf), or enriched hemocyte populations separated through a discontinous percoll gradient as discrete bands b1, b2, b3, b4, b5 and b6. ( ) hemagglutinating activity of supernatant from cultured nf (1.5 h at 18 °c), or enriched hemocyte populations separated through a discontinous percoll gradient as discrete bands b1, b2, b3, b4, b5 and b6 and supernatant from 1.5 h hemocyte colture from cell populations enriched in bands b1-b6 through a discontinous percoll density gradient. inset: student t-test comparison between the various groups. *** p<0.001, **p<0.01, *p<0.05 of phagocytes to engulf opsonized targets, also showing an increased phagocytic index. the possibility exists that, as reported for mammalian galectin 10 (swaminthan et al., 1999), some crd variants could be contained in c. intestinalis β-galactosidespecific lectins that could interact with mannose. although pure hemocyte populations could not be separated, in an attempt of correlating lectin content and release with hemocyte types we examined cell populations separated on percoll discontinuous density gradient. lysate supernatants from the separated hemocyte bands, prepared from a constant cell number, presented a various degree of hemagglutination titres as compared with the unfractionated hemocytes. hemocytes from the separated bands appeared to be mixed cell populations therefore it is not possible to assign the content in cytosolic lectin molecules to discrete hemocytes. the highest hemagglutinating titres were found in b2-hls that mainly contained hyaline amoebocytes (~ 57 %) and a lower proportion of granulocytes (~ 20 %). likewise the high titre that characterized b1-hls, composed with 76 % hyaline amoebocytes in the absence of granular amoebocytes, emphasized the effect of the enrichment in hyaline amoebocytes as lectincontaining cells. on the other hand, also granular amoebocytes enriched in b3 (~ 71 % granular amoebocytes, and 22 % hyaline amoebocytes) could be responsible of the high titre registered in their lysate supernatant. finally, a low level was found in the lysate from morula cells enriched (85 %) in b6. cytosolic lectins appeared to be contained in several hemocyte types, supporting their role in multiple cell functions. in addition, the high content of lectins in inflammatory hemocytes, like hyaline and granular amoebocytes, suggested their involvement in inflammatory reactions. accordingly, a similar hemagglutin titre profile was observed by examining the activity of supernatant from b1-b6 hemocyte cultures even if the titres were lower than those observed in the hemocyte 63 lysate supernatants, probably due to a lesser number of hemocytes, as yielded from separated bands, cultured for a short time. the highest activity was found by culturing hemocytes from b1 and b2 mainly enriched in hyaline amoebocytes that have a role as phagocytes and presumably release lectins to be involved in opsonization. however, the low hemagglutinating activity of supernatants from enriched granular amoebocyte population (b3) does not exclude their involvement in lectin-dependent immune functions. in fact, the experimental conditions and the short time we used for preparing culture supernatants could affect the lectin release from this hemocyte type. further research is required to uncover the molecular structure of hemocyte β-galactosidespecific lectins and ascertain whether they belong to the galectin family as identified in the c. intestinalis genome. aknowledgements this work was supported by research grants from the italian ministry of education (prin 2004 4057000) to n parrinello co-funded by the university of palermo. tanks are also due to mr g miceli for collecting ascidians. references amirante ga, mazzalai fg. synthesis and localization of hemoagglutinins in hemocytes of the cockroach leucophaea maderae l. dev. comp. immunol. 2:735-40,1978. arizza v, cooper el, parrinello n. circulating hemocytes and pharyngeal explants of styela clava release hemagglutinin in vitro. j. mar. biotech. 5: 31-35, 1997. arizza v, parrinello n, schimmenti s. in vitro release of lectins by phallusia mamillata hemocytes. dev. comp. immunol. 15: 219-226, 1991. ballarin l, tonello c, guidolin l, sabbadin a. purification and characterization of a humoral opsonin, with specificity for d-galactose, in the colonial ascidian botryllus schlosseri. comp. biochem. physiol. 123b: 115-123, 1999. ballarin l, tonello c, sabbadin a. humoral opsonin from the colonial ascidian botryllus schlosseri as a member of the galectin familiy. mar. biol. 136: 823-827, 2000. borenfreund e, puerner ja, a simple quantitative procedure using monolayer cultures for cytotoxicity assays (htd/nr-90). methods in cell science, 9 :7-9, 1984 bradford mm. a rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. anal. biochem. 72: 248-54. 1976. brewer cf. binding and cross-linking properties of galectins. biochim. biophys. acta 1572: 255262, 2002. cammarata m, arizza v. method for phagocytosis fluorescence quencing in vitro assay for hemocytes in tunicates. anim. biol. 3: 173-174, 1994. cammarata m, parrinello n, arizza v. in vitro release of lectins from phallusia mamillata hemocytes after their fractionation on a density gradient. j. exp. zool. 266: 319-27, 1993. cheng tc, marchalonis jj, vasta gr. role of molluscan lectins in recognition processes. prog. clin. biol. res.157: 1-15, 1984. cooper dnw, barondes sh. god must love galectins; he made so many of them. glycobiology 9: 979-984, 1999. cooper dnw. galectinomics: finding themes in complexity. biochim. biophys. acta 1572: 209231, 2002. csizmas cc, preparation of formalinized erythrocytes. proc. soc. exp. biol. 103 :157, 1960 de leo g. ascidian hemocytes and their involvement in defense reaction. boll. zool. 59: 195-213, 1992. dehal p, satou y, campbell rk, chapman j, degnan b, de tommaso a, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157-2167, 2002. dodd rb, drickamer k. lectin-like proteins in model organisms: implications for evolution of carbohydrates binding activity. glycobiology 11: 71-79, 2001. field kg, olsen gj, giovannoni sj, ghiselin mt, raff ec, pace nr, et al. molecular phylogeny of the animal kingdom. science 239: 748-753, 1988. feizi t. carbohydrate-mediated recognition systems in innate immunity. immunol. rev. 173: 79-88, 2000. gobius h-j, andré s, kaltner h. siebert h-c. the sugar code: functional lectinomies. biochim. biophys. acta 1572: 165-177, 2002. green pl, nair sv, raftos da. secretion of a collectin-like protein in tunicates is enhanced during inflammatory responses. dev. comp. immunol. 27: 3-9, 2003. hori h, osawa s. origin and evolution of organisms as deduced from 5s ribosomal rna sequences. mol. biol. evol. 5: 445-72, 1987. kelly kl, cooper el. raftos da. purification and characterization of a humoral opsonin from the solitary urochordate styela clava. comp. biochem. physiol. 103b: 749-753, 1992. kilpatrick dc. animal lectins: a historical introduction and overview. biochim. biophys. acta 1572: 187-197, 2002. kilpatrick dc. handbook of animal lectins. wiley j & sons, ltd, chichester, 2000 loker es, adema cm, zhang sm, kepler tb. invertebrate immune systems not homogeneous, not simple, not well understood. immunol. rev. 198: 10–24, 2004. nair s, burandt m, hutchinson a, raison r, raftos d. a c-type lectin from the tunicate, styela plicata, that modulates cellular activity. comp. biochem. physiol. 129c: 11-24, 2001. parrinello n. the reaction of ciona intestinalis l. (tunicata) to subcuticular erythrocyte and protein injection. dev. comp. immunol. 5 (suppl.1): 105-110, 1981. parrinello n. humoral and cellular lectins of ascidians. j. mar. biotech. 3: 29-34, 1995. http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=showdetailview&termtosearch=6483870&ordinalpos=5&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum 64 parrinello n. cytotoxic activity of tunicate hemocytes. prog. mol. subcell. biol. 15: 190217, 1996. parrinello n. arizza v. d-galactose binding lectins from the tunicate ascidia malaca: subunit haracterization and hemocyte surface distribution; dev. comp. immunol. 12:495-507, 1988. parrinello n, arizza v, cammarata m, giaramita f, pergolizzi m, vazzana m, et al. inducible lectins with galectin properties and human il1α epitopes opsonize yeasts in the ascidian ciona intestinalis inflammatory response. cell tissue res. 2007 (in press). parrinello n, cammarata m, arizza v. univacuolar refractile hemocytes from the tunicate ciona intestinalis are cytotoxic for mammalian erythrocytes in vitro. biol. bull. 190: 418-425, 1996. parrinello n, cammarata m, arizza v, vazzana m, cooper el. how the cell of the invertebrate immune system kill the other cells?. in: legakis a, sfenthourakis s, polymeni r, thessaloulegaki m. (eds), the new panorama of animal evolution. proc. 18th int. congr. zoology, pp 167-175, 2003. parrinello n. canicattì c. carbohydrate binding specificity and purification by biospecific affinity chromatography of ascidia malaca traust. hemagglutinins. dev comp immunol. 6:53-64, 1982. parrinello n, de leo g, di bella ma. fine structural observations of the granulocytes involved in the tunic inflammatory-like reaction of ciona intestinalis (tunicata). j. inv. pathol. 56: 181189, 1990. parrinello n, patricolo e. erythrocyte agglutinins in the blood of certain ascidians. experientia 31: 1092-1093, 1975. parrinello n., patricolo e. inflammatory-like reaction in the tunic of ciona intestinalis (tunicata). ii capsule components. biol. bull. 167: 238-250, 1984. parrinello n, patricolo e, canicattì c. inflammatorylike reaction in the tunic of ciona intestinalis (tunicata). i encapsulation and tunic injury. biol. bull. 167: 229-237, 1984. pinto mr, chinnici cm, kimura y, melillo d, marino r, spruce la, et al. cic3-1a mediated chemotaxis in the deuterostome invertebrate ciona intestinalis (urochordata). j. immunol. 171: 5521–5528, 2003. quenseberry mh, ahmed h, elola mt, o’leary n, vasta gr. diverse lectin repertoires in tunicates mediate broad recognition and effector innate immune responses. integr. comp. biol. 43: 323-330, 2003. rabinovich ga, rubinstain n, toscano ma. role of galectins in inflammatory and immunomodulatory processes biochim. biophys. acta 1572: 274-284, 2002. raftos da, stillman dl, cooper el. in vitro culture of tissue from the tunicate styela clava. in vitro cell dev. biol. 26: 962-70, 1990. renwrantz l, stahmer a. opsonizing properties of an isolated hemolymph agglutinin and demonstration of lectin-like molecules at the surface of hemocytes from mytilus edulis. j. comp. physiol. 149: 535–546,1983. rowley a.f. the blood cells of the sea squirt, ciona intestinalis: morphology, differential counts, and in vitro phagocytic activity. j. inv. pathol. 37: 91-100, 1981. sato s, burdett i, hughes rc. secretion of the baby hamster kidney 30-kda galactose-binding lectin from polarized and nonpolarized cells: a pathway independent of the endoplasmic reticulum-golgi complex exp. cell res. 207: 818, 1993. sato s, hughes rc. regulation of secretion and surface expression of mac-2, a galactosidebinding protein of macrophages. j. biol. chem. 269: 4424–4430, 1994. sharon n, lis h. lectins, 2nd edition, kluwer academic publishers, london, 2003. sharon n, lis h. history of lectins: from hemagglutinins to biological recognition molecules. glycobiology 14: 53-62, 2004. swalla bj, cameron cb, corley ls, garey jr. urochordates are monophyletyc within the deuterostomes. syst. biol. 49: 52-64, 2000. swaminathan gj, leonidas dd, savage mp, ackerman sj, achayra kr. selective recognition of mannose by the human eosinophil charcit-leydon crystal protein (galectin 10): a crystallographic study at 1.8 å resolution. biochemistry 38: 13837-13843, 1999. vasta gr, ahmed h, odom ew. structural and functional diversity of lectin reperoires in invertebrates, protochordates and ectothermic vertebrates. curr. opin. struct. biol. 14: 617630, 2004a. vasta gr, ahmed h, du s-j, henrikson d. galectins in teleost fish: zebrafish (danio rerio) as a model species to address their biological roles in development and innate immunity. glycoconjugate j. 21: 503-521, 2004b. vasta gr, cheng tc, marchalonis jj. a lectin on the hemocyte membrane of the oyster (crassostrea virginica). cell immunol. 88:47588, 1984. vasta gr, quesenberry m, ahmed h, o'leary n. ctype lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. dev. comp. immunol. 23: 401-20, 1999. vasta gr, quesenberry ms, ahmed h, o’leary n. lectins from tunicates: structure-function relationships in innate immunity. adv. exp. med. biol. 484: 275-287, 2001. wright rk. protochordate immunity. i. primary immune response of the tunicate ciona intestinalis to vertebrate erythrocytes. j. invertebr. pathol. 24: 29-36, 1974. wright, rk. in: ratcliffe na, rowley af (eds), urochordata, invertebrate blood cells, vol. 2, academic press, london, pp 565-625, 1981. zeng l, swalla bj. molecular phylogenesis of the protochordates: chordate evolution. can. j. zool. 83: 24-33, 2005. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=retrieve&dopt=abstractplus&list_uids=2243059&query_hl=16&itool=pubmed_docsum http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22vasta+gr%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22quesenberry+m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22ahmed+h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22o%27leary+n%22%5bauthor%5d 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj11x.pdf 1 isj 3: 1-3, 2006 issn 1824-307x short communication monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005 d malagoli, l casarini, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted january 13, 2006 abstract the monthly evaluation of the cytotoxicity of hemolymph from the mussel mytilus galloprovincialis revealed some variations in the percentage of cytotoxic animals during the year. cytotoxicity is confirmed to be a dynamic parameter that can be used as an indicator of immune efficiency and, therefore, of the state of health of the animals. key words: mytilus galloprovincialis; cytotoxicity assay; cytotoxic activity; adriatic sea mussel farms introduction the state of health of mytilus galloprovincialis mussels was evaluated in terms of cytotoxic activity in the hemolymph. it is well-known that molluscs, as all invertebrates, possess only innate or natural immunity and recognize and eliminate non-self material mainly through cellular and humoral components. among the latter, cytotoxicity is one of the most important immune functions (franceschi et al., 1991). this function has been well conserved during evolution and described both in invertebrates and vertebrates (ratcliffe et al., 1985; savary and lotzová, 1986). wittke and renwrantz (1984) have demonstrated that circulating cells (immunocytes) from mytilus edulis are able to produce cytotoxic substances, which lyse human erythrocytes. a cytotoxic protein complex has also been found in m. galloprovincialis hemolymph (hubert et al., 1997). recently, we have designed an easy-to-use and lowcost cytotoxicity test (malagoli and ottaviani, 2005) that has been used here to evaluate the cytotoxic activity of m. galloprovincialis collected each month during 2005 from adriatic sea mussel farms. corresponding author: enzo ottaviani department of animal biology via campi 213/d 41100 modena, italy e-mail: ottaviani.enzo@unimore.it materials and methods animals specimens of the mussel mytilus galloprovincialis were obtained each month from local fishermen in the cesenatico area (fc, italy) immediately after being caught. forty animals were sacrificed to collect hemolymph on the spot, while the remaining samples were taken to the department of animal biology (modena, italy) and maintained in the laboratory aquarium (artificial seawater, temperature 19 ± 1 °c, ph 8.0 ± 0.2, salinity 35 ± 2 psu) for at least 10 days before control experiments. no animals could be obtained in february because of adverse weather conditions. hemolymph preparation and cytotoxicity assay the detailed procedure for the cytotoxicity assay has been described elsewhere (malagoli and ottaviani, 2005). briefly, the hemolymph was collected from the posterior adductor muscle using a sterile syringe, filtered into sterile tubes with 0.2 µm filters, aliquoted, immediately frozen and maintained at –80 °c for at least 12 h before use. the cytotoxic activity was assessed by checking the cytolysis of human a positive erythrocytes. in order to eliminate damaged erythrocytes, the whole blood sample was washed at least three times in 9 vol. of sterile nacl 0.9 %, and the erythrocytes were then resuspended in sterile tbs (50 mm tris-hcl, 200 mm nacl, 10 mm cacl2, ph 8.5) at a final concentration of 2x109 cells/ml. 500 µl of filtered hemolymph were added 2 fig. 1 mussel cytotoxicity during 2005 in the cesenatico sea area. the red line represents the mean percentage. to 500 µl of erythrocyte suspension and incubated for 1h at 25 °c (hubert et al., 1997; malagoli and ottaviani, 2005). after incubation, samples were centrifuged at 3.000xg for 5 min at 4 °c, and the optical density (od) of supernatants was measured by evaluating the absorbance at 541 nm with a helios β spectrophotometer (spectronic unicam, cambidge, uk). it should be noted here that a threshold od level of 0,5 was fixed as the minimum for a positive test, while samples that clearly exceeded this value were considered positive without further measurement (malagoli and ottaviani, 2005). the experiment was repeated in duplicate two times for each animal. statistical analysis statistical analysis was performed using the χ2 test with p< 0.05 taken as significant. results and discussion the periodic evaluation of the cytotoxicity of hemolymph from the mussel m. galloprovincialis reveals variations in the percentage of cytotoxic animals during the year (fig. 1). the mean percentage of mussels positive to the cytotoxicity assay was around 53 %, but there were various peaks. it is interesting to note that the minimum and the maximum values were during the periods in which the water temperature reached its minimum and maximum levels, respectively, as indicated in the annual report on inshore water conditions of the italian region emilia-romagna in 2003. in a previous study, we observed that rapid changes in water temperature, salinity and ph resulted in a significant decrease in the mean cytotoxic activity. a rapid increase in water temperature resulted in a significant drop in the number of animals positive to the cytotoxicity assay (malagoli and ottaviani, 2005). conversely, the observations reported here seem to indicate a positive correlation between water temperature and the percentage of cytotoxic molluscs. however, no sudden fig. 2 comparison between mussel cytotoxicity immediately after being caught (a) or after a period of maintenance in the aquarium (b). the mean values of six independent experiments ± sd are shown. modifications in environmental parameters were seen in the present study. it should be underlined that the mean percentages of cytotoxic animals did not differ among mussels maintained in the laboratory aquarium for at least 10 days prior to control experiments (fig. 2). as far as the peak registered in september is concerned, it should be noted that the samples were collected after a period in which many animals had been died as a result of a loss of byssus. this pathology is still of uncertain origin, but a fungal infection has been observed to be involved in serious damage to the byssus apparatus (franchini et al., 2005). even if more detailed studies are needed to clarify the question, the large number of cytotoxic specimens in the survivor population could suggest a link between cytotoxic activity and the animal's ability to overcome epidemics. overall, cytotoxicity is confirmed as a dynamic parameter that can be used as an indicator of immune efficiency and, therefore, of the good state of health of the mussels. 0 10 20 30 40 50 60 70 80 90 100 jan feb mar apr may jun jul aug sep oct nov dec month % o f c y to to x ic a n im a ls % o f c y to to x ic a n im a ls month 0 10 20 30 40 50 60 70 80 90 100 % o f c y to to x ic a n im a ls a b % o f c y to to x ic a n im a ls 4 acknowledgement we are grateful to the centro di ricerche marine (cesenatico, fc, italy) for financial support for this study. the authors also wish to thank mr m marangoni who kindly provided the mussels and the centro trasfusionale policlinico (modena, italy) for providing the blood. references franceschi c, cossarizza a, ortolani c, monti d, ottaviani e. natural cytotoxicity in a freshwater polmonate mollusc: an unotorthodox comparative approach. adv. neuroimmunol. 1: 99–113, 1991. franchini a, malagoli d, ottaviani e. investigation of the loss of byssus in mytilus galloprovincialis from mussel farms in the adriatic sea. cell biol. int. 29: 857-860, 2005. hubert f, cooper el, roch ph. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloprovincialis. biochim. biophys. acta 1361: 29–41, 1997. malagoli d, ottaviani e. cytotoxicity as a marker of mussel health status. j. mar. biol. ass. uk 85: 359-362, 2005. ratcliffe na, rowley af, fitzgerald sw, rhodes cp. invertebrate immunity: basic concepts and recent advances. int. rev. cytol. 97: 183–350, 1985. savary ca, lotzová e. phylogeny and ontogeny of nk cells. in: lotzová e, heberman rb (eds), immunobiology of natural killer cells, vol 1, fla: crc press, boca raton, pp 45–61, 1986. wittke m, renwrantz l. quantification of cytotoxic hemocytes of mytilus edulis using a cytotoxicity assay in agar. j. invertebr. pathol. 43: 248-253, 1984. 3 isj 6: 32-43, 2009 isj 6: 32-43, 2009 issn 1824-307x review signaling pathways in invertebrate immune and stress response r hatanaka, y sekine, t hayakawa, k takeda, h ichijo laboratory of cell signaling, graduate school of pharmaceutical sciences, strategic approach to drug discovery and development in pharmaceutical sciences, global center of excellence (gcoe) program, and core research for evolutional science and technology (crest), japan science and technology corporation, the university of tokyo, japan accepted march 4, 2009 abstract a wide variety of signaling pathways regulate immune and stress response in invertebrates and vertebrates. the fruit fly drosophila melanogaster and the nematode caenorhabditis elegans are extensively utilized model organisms for studies of such signaling pathways in invertebrates. intriguingly, major signaling pathways in immune response in drosophila and c. elegans, as represented by the toll and imd pathways and the dbl-1 and daf-2/daf-16 pathways, respectively, are different from each other. on the other hand, the mitogen-activated protein kinase (mapk) pathways function in common in these organisms not only in immune response but also in response to various abiotic stressors such as heat shock, ultraviolet (uv) irradiation, oxidative stress and osmotic shock. given that all of the above pathways are highly conserved and play diverse roles in vertebrates, particularly in mammals, drosophila and c. elegans are important invertebrate models that facilitate the elucidation of evolutionarily conserved mechanisms of immune and stress response. we therefore focus on signaling pathways that regulate immune and stress response in drosophila and c. elegans in this review. key words: innate immunity; stress; map kinase; toll; imd; daf-2 introduction to cope with pathogenic microorganisms, invertebrates rely solely on innate immunity because they do not have adaptive immunity. the well-characterized strategy against pathogens in invertebrate innate immunity is the induction of antimicrobial peptide (amp) genes, the regulatory mechanisms of which have been extensively explored using model organisms such as drosophila melanogaster and caenorhabditis elegans. in vertebrates, on the other hand, it has long been recognized that adaptive immunity is the main strategy used in the fight against various pathogens. however, a large body of recent evidence has demonstrated that innate immunity also plays a critical role in vertebrate immunity. therefore, in order to understand the highly complex immune systems in vertebrates, and in mammals in particular, it will be of great importance to elucidate the regulatory mechanisms of innate immunity in invertebrate models, ___________________________________________________________________________ corresponding author: hidenori ichijo laboratory of cell signaling graduate school of pharmaceutical sciences the university of tokyo, 7-3-1 hongo, bunkyo-ku tokyo 113-0033, japan e-mail: ichijo@mol.f.u-tokyo.ac.jp with a particular focus on signaling molecules that are conserved among species and involved in such mechanisms. invertebrate models also provide important information on the mechanisms that regulate the response to various abiotic stressors, such as heat shock, ultraviolet (uv) irradiation, oxidative stress and osmotic shock. the mitogen-activated protein kinase (mapk) pathways, especially those converging on two subgroups of stress-responsive mapks, jnk and p38, are the major players in a wide variety of response to these stressors (widmann et al., 1999; kyriakis and avruch, 2001). although these pathways are highly conserved from invertebrates to mammals and many physiological functions of these pathways have been revealed using mammalian models such as gene targeting mice, a great deal has also been revealed by studying these pathways as a primitive and efficient defense system in drosophila and c. elegans (stronach and perrimon, 1999; sakaguchi et al., 2004). in fact, the genetic evidence that the stress-responsive mapk pathways play pivotal roles not only in stress response but also in innate immunity was first revealed in analyses using these more rudimentary animals models (boutros et al., 2002; kim et al., 2002). 32 fig. 1 the toll pathway. cell wall components of gram-positive bacteria and fungi (lysine-type peptidoglycans and glucans, respectively) are recognized by pattern recognition proteins and activate protease cascades including such proteases as the serine protease grass. proteases secreted from fungi and gram-positive bacteria activate the serine protease persephone and its downstream protease cascade. these cascades finally activate spe, which cleaves pro-spätzle to form mature spätzle. upon the binding of spätzle to toll, dmmyd88, tube and pelle are recruited to toll. following the phosphorylation and degradation of cactus, dif and dorsal are released from cactus, translocate to the nucleus and induce amp genes such as drosomycin. in the first and second parts of this review, we focus on major signaling pathways in immune response in drosophila and c. elegans, as represented by the toll and imd pathways and the dbl-1 and daf-2/daf-16 pathways, respectively. in the last part we examine the functions of the stress-responsive mapk pathways as a common signaling system regulating immune and stress response in drosophila and c. elegans. signaling pathways in immune response in drosophila the toll and imd pathways have been extensively investigated as major signaling pathways in immune response in drosophila. these pathways are known to be functionally and molecularly conserved among other insects, such as the mosquitoes aedes aegypti and anopheles gambiae (christophides et al., 2002; waterhouse et al., 2007), the beetles tribolium castaneum (zou et al., 2007), the honey bees apis mellifera (evans et al., 2006) and the silkworms bombyx mori (tanaka et al., 2008), indicating that these pathways play central roles in immune response in insects. the toll pathway when flies are infected with gram-positive bacteria or fungi, amp gene expression is enhanced via the toll pathway in the fat body cells (lemaitre et al., 1996). the toll gene was first found to be required for the establishment of dorsal-ventral polarity in drosophila embryos (anderson et al., 1985). toll is a founder member of the highly conserved toll-like receptor (tlr) family, which is composed of type i transmembrane proteins with an ectodomain characterized by several repeats of leucine-rich motifs. although most members of this family recognize and directly bind to pathogen-associated molecular patterns (pamps) as host pattern recognition proteins, toll does not directly bind to pathogens or pamps but instead 33 binds to the mature form of the extracellular protein spätzle as an endogenous ligand (weber et al., 2003). upon infection with gram-positive bacteria, drosophila recognizes a bacterial cell wall component, lysine-type peptidoglycan (pgn), by means of pattern recognition proteins such as peptidoglycan-recognition protein (pgrp) short-form a (pgrp-sa), pgrp short-form d (pgrp-sd) and gram-negative binding protein 1 (gnbp1) in the hemolymph (werner et al., 2000; michel et al., 2001; gobert et al., 2003; bischoff et al., 2004) (fig. 1). results from genetic and biochemical analyses suggest that the ternary complex of all these proteins and the complex formed between pgrp-sa and gnbp1 differentially bind to pgns in a manner dependent on the bacterial strains from which pgns are purified (wang et al., 2008). these recognition proteins thereafter activate serine protease cascades, resulting in cleavage of pro-spätzle by the spätzle processing enzyme (spe) (jang et al., 2006). although the proteases that are engaged in spe activation have not been fully revealed, a recent study has clarified that the serine protease grass functions upstream of spe (el chamy et al., 2008). fungal infection also activates the toll pathway via protease cascades, and is dually detected by a pattern recognition protein gnbp3 and a serine protease persephone (psh) (gottar et al., 2006). fungal cell wall components such as β-(1,3)-glucans are recognized by gnbp3. proteases and chitinases secreted from fungi, which perforate the cuticle barrier and allow entry of fungi into the body cavity (clarkson and charnley, 1996), are sensed by psh. these recognition systems activate spe through protease cascades, leading to cleavage of pro-spätzle. recently, psh has also been shown to be required for sensing gram-positive bacterial proteases (el chamy et al., 2008). upon binding of spätzle to toll, dmmyd88, the drosophila ortholog of mammalian myd88, and the death domain proteins tube and pelle are recruited to the intracellular domain of toll, where they form a complex (horng and medzhitov, 2001; tauszig-delamasure et al., 2002). pelle is the drosophila ortholog of il-1 receptor-associated kinase (irak), while the counterpart of tube has not been found in mammals. this protein complex formation induces phosphorylation of cactus, the drosophila homolog of mammalian iκb (nicolas et al., 1998), although it has not yet been revealed whether the kinase pelle directly phosphorylates cactus. under unstimulated conditions, cactus retains and thus inhibits the nf-κb-like transcription factors dif and dorsal in the cytoplasm. once phosphorylated, cactus is degraded by the ubiquitin-proteasome system, and thereby dif and dorsal are released from cactus and translocate to the nucleus (ip et al., 1993), where they eventually induce the amp genes such as drosomycin (engström, 1999). the imd pathway another major immune control system in drosophila is the imd pathway (fig. 2). the imd gene was originally discovered in a survey of immune deficient mutant flies that exhibited lower viability than wild type flies with reduced expression of some amp genes in response to bacterial challenges (lemaitre et al., 1995). much as in the toll pathway, pgn recognition by pgrps is the initial step in the imd pathway. in this pathway, pgrp-lc recognizes diaminopimelic acid (dap)-type pgns, which are components of gram-negative bacteria and some gram-positive bacteria (choe et al., 2002; gottar et al., 2002; rämet et al., 2002). three alternatively spliced isoforms of pgrp-lc, lca, lcx and lcy, have been identified, and all of them are putative type ii transmembrane proteins with common n-terminal cytoplasmic and transmembrane domains but different extracellular domains (werner et al., 2003; kaneko et al., 2004). among these isoforms, pgrp-lca and pgrp-lcx have been shown to form the lca-lcx heterodimer and lcx-lcx homodimer, which function in the signal transduction induced by monomeric and polymeric pgns, respectively (mellroth et al., 2005). recent analyses have revealed that pgrp-le is another player in the recognition of dap-type pgns and functions synergistically with pgrp-lc (takehana et al., 2004; kaneko et al., 2006). consistent with the fact that pgrp-le possesses no predicted transmembrane domain or signal sequence, pgrp-le is detected in the cytosol and functions as an intracellular receptor for the monomeric dap-type pgn tracheal cytotoxin (tct). on the other hand, a version of pgrp-le (pgrp-lepg) containing only the pgrp domain that is conserved among all pgrps is found outside the cell and enhances pgrp-lc-mediated pgn recognition on the cell surface. these findings suggest that drosophila also employs conserved mechanisms for pamps recognition using both extracellular and intracellular receptors as in mammals (sansonetti, 2006). after the recognition of pgns, pgrp-lc and pgrp-le activate intracellular signaling. imd, a protein with a death domain similar to that of mammalian receptor interacting protein (rip), appears to be most proximal to the pgrps, since imd has been shown to physically interact with pgrp-lc and to a lesser extent with pgrp-le (georgel et al., 2001; choe et al., 2005; kaneko et al., 2006). although it remains elusive whether these interactions are indeed required for downstream signaling, imd subsequently binds to the drosophila fas-associated death domain-containing protein (dfadd). the imd-dfadd complex further recruits dredd, the drosophila ortholog of mammalian caspase-8, and elicits caspase activity of dredd (hu and yang, 2000; leulier et al., 2000; leulier et al., 2002; naitza et al., 2002). downstream of the formation of this active protein complex, drosophila tgf-β-activated kinase 1 (dtak1) is activated by a mechanism not fully understood but involving the drosophila homologs of human ubiquitin-conjugating enzymes ubc13 and uev1a (zhou et al., 2005). dtak1 then activates the drosophila iκb kinase (dmikk) complex (lu et al., 2001; vidal et al., 2001; silverman et al., 2003), which in turn conceivably directly phosphorylates the nf-κb-like transcription factor relish (silverman et al., 2000). relish possesses a dna-binding rel homology domain but 34 fig. 2 the imd pathway. upon the recognition of dap-type peptidoglycans of gram-negative bacteria by pattern recognition proteins, imd binds to dfadd and dredd, and the caspase activity of dredd is induced. dtak1 is also activated, and activated tak1 in turn activates the dmikk complex. subsequent dmikk-induced phosphorylation and dredd-mediated cleavage of relish generate the mature form of relish as a transcription factor, which translocates to the nucleus and induces amp genes such as diptericin. it is masked by an ankyrin repeats-containing iκb-like domain within the same molecule. phosphorylation of relish triggers endoproteolytic cleavage of the linker region between these two domains of relish, probably via the activation of dredd (stoven et al., 2003). finally, relish translocates to the nucleus and induces amp genes such as diptericin (cornwell and kirkpatrick, 2001). signaling pathways in the immune response in c. elegans whereas drosophila toll is a major player in immune response, as described above, the sole c. elegans toll homolog tol-1 has been assumed not to function in immune response. indeed, tol-1 deletion mutants have been reported not to exhibit altered sensitivity to various pathogens (pujol et al., 2001). recently, however, a possible role of tol-1 in combating bacteria has been proposed, because tol-1 mutants were shown to be sensitive to salmonella enterica and escherichia coli, and tol-1 was needed to prevent s. enterica from invading into the pharynx (tenor and aballay, 2008). however, the downstream signaling remains unknown mainly due to a lack of functionally conserved nf-κb-like factors in c. elegans. in this section, therefore, we focus on the dbl-1 and daf-2/daf-16 pathways as the major immune control systems in c. elegans. although these pathways are highly conserved from invertebrates to vertebrates, their direct immune functions have not been extensively investigated in other invertebrates than c. elegans, except for anopheles mosquitoes, in which these pathways have been shown to be critically involved in innate immunity to malaria parasite infection (lieber and luckhart, 2004; luckhart and riehle, 2007). the dbl-1 pathway in drosophila, induction of the amp genes upon infection is the most prominent mechanism of defense against pathogens, as described above. until recently, however, it was not understood whether such inducible anti-pathogen defense systems are also employed in c. elegans. mallo et al. clearly addressed this issue (mallo et al., 2002). by using a high-density cdna microarray, they showed that infection of c. elegans by serratia marcescens induced an upregulation of the expression of many genes–including those encoding lectins and lysozymes, which are known to be involved in immune response in other organisms, thereby demonstrating 35 fig. 3 functional interaction between the daf-2/daf-16 and jnk pathways. the daf-2/daf16 pathway starts with the binding of insulin-like peptides, which triggers activation of the pi3 kinase-like complex formed between age-1 and aap-1. age-1/aap-1 then catalyzes the conversion of pi-4,5-p2 (pip2) to pi-3,4,5-p3 (pip3). pdk-1 activated by pip3 phosphorylates and thus activates the akt-1–akt-2–sgk-1 kinase complex. finally, this complex phosphorylates and retains daf-16 in the cytoplasm. in contrast, jnk-1 promotes the translocation of daf-16 into the nucleus in response to environmental stressors, leading to the expression of numerous target genes to prevent damage to the cell. for the first time the existence of inducible antibacterial defenses in c. elegans. these amp genes in c. elegans have recently been shown to be preferentially expressed in the intestine, which is directly exposed to microbes (alper et al., 2007). intriguingly, some of the infection-inducible genes found in this study overlapped with genes under control of the dbl-1 signaling pathway (mochii et al., 1999). dbl-1 is a transforming growth factor (tgf)-β-related ligand that was first found in the context of body size regulation and male tail patterning (morita et al., 1999; suzuki et al., 1999), in accordance with the functions of the tgf-β-family members in body patterning in both invertebrates and vertebrates. consistent with these gene expression analyses, dbl-1 mutants have been shown to exhibit increased susceptibility to s. marcescens (mallo et al., 2002). recently, it has been demonstrated that neuronal expression of dbl-1 promotes expression of the caenacin family antimicrobial peptides in the epidermis probably in a paracrine manner after infection with the fungus drechmeria coniospora, suggesting the tissue-specific role of dbl-1 in immune response (zugasti and ewbank, 2009). although it remains to be clarified how the dbl-1 ligand is regulated in response to bacterial infection, it has been speculated that dbl-1 activates the following signaling pathway in its target cells (nicholas and hodgkin, 2004). in a manner analogous to the mammalian tgf-β receptor-smad system (kawabata and miyazono, 1999), sma-6 (type i receptor) and daf-4 (type ii receptor) form a heterodimer in response to dbl-1 stimulation, which induces phosphorylation of sma-2 and sma-3, the orthologs of the receptor-regulated smad proteins, mammalian smad1 and smad5, respectively. then sma-2 and sma-3 form a complex with sma-4, the ortholog of the mammalian common-mediator smad4, and translocate to the nucleus, leading to the expression of antimicrobial factors (savage et al., 1996; krishna et al., 1999). however, it has recently been shown that sma-3 alone, but neither sma-2 nor sma-4, is required for amp expression in response to d. coniospora infection, implying the existence of a non-canonical tgf-β signaling pathway in c. elegans (zugasti and ewbank, 2009). 36 fig. 4 the stress-responsive mapk pathways. the stress-responsive mapk pathways that converge on jnk and p38 in mammalian, drosophila and c. elegans are shown. each pathway consists of three classes of protein kinases: mapk, map2k and map3k. map3k phosphorylates and thereby activates map2k, and activated map2k in turn phosphorylates and activates mapk. the daf-2/daf-16 pathway daf-2 and daf-16 are c. elegans orthologs of the mammalian insulin/insulin-like growth factor (igf)-i receptor and the foxo family transcription factor, respectively (kimura et al., 1997; lin et al., 1997; ogg et al., 1997). the daf-2/daf-16 pathway has been investigated mainly from the viewpoint of metabolism, longevity, and dauer formation, which is an alternate larval stage that worms enter under unfavorable environmental conditions. the best-known phenotype of daf-2 mutants is the marked increase in life span, but they also exhibit resistance to pathogenic bacteria such as enterococcus faecalis, staphylococcus aureus, pseudomonas aeruginosa and bacillus subtilis (garsin et al., 2003). the daf-2/daf-16 pathway comprises signaling components highly homologous to those in well-characterized mammalian insulin/igf-i signaling. this pathway starts with the binding of insulin-like peptides to daf-2, which triggers the activation of age-1 and aap-1, the orthologs of the p110 catalytic and p55 regulatory subunits of mammalian pi3 kinase, respectively (morris et al., 1996; wolkow et al., 2002) (fig. 3). age-1/aap-1 then catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (pi-4,5-p2) to phosphatidyl-3,4,5-trisphosphate (pip3) (engelman et al., 2006). pip3 activates the 3-phosphoinositide-dependent kinase-1 homolog pdk-1 (paradis et al., 1999), which in turn phosphorylates and thus activates the akt-1–akt-2–sgk-1 kinase complex (hertweck et al., 2004). finally, this akt complex phosphorylates and retains daf-16 in the cytoplasm (paradis and ruvkun, 1998; lin et al., 2001). in the presence of daf-2 antagonists or perturbation of daf-2 signaling, daf-16 stays in the nucleus, which is a prerequisite for the induction of a wide variety of genes, including those regulating metabolism and cellular stress response (murphy et al., 2003). importantly, antimicrobial genes, such as the antibacterial lysozyme genes lys-7 and lys-8, which have been shown to be induced upon infection with s. marcescens (mallo et al., 2002), and the saposin-like gene spp-1, which has been shown to have antibacterial activity (bányai and patthy, 1998), are also the targets of daf-16. this finding suggests that insulin-like signaling in c. elegans counteracts the antibacterial activity through daf-16-dependent gene induction, and provides a reasonable explanation for the previous finding that daf-2 mutants exhibited increased resistance to pathogenic bacteria (garsin et al., 2003). consistent with these findings, p. aeruginosa was found to suppress daf-16-dependent immune response by activating the daf-2 pathway, probably through the induction of the insulin-like peptide ins-7 (evans et 37 al., 2008), further demonstrating the importance of the daf-2/daf-16 pathway in the immune response in c. elegans. in most literature, daf-16 regulation is mainly observed in the intestinal cells where it is feasible to detect nuclear translocation of daf-16, whereas regulation in other tissues remains to be elucidated. the mapk pathways the mapk pathways are signal transduction systems evolutionarily conserved in all eukaryotic organisms, and consist of three classes of protein kinases: mapk, mapk kinase (map2k) and map2k kinase (map3k) (widmann et al., 1999; kyriakis and avruch, 2001). map3k phosphorylates and thereby activates map2k, and activated map2k in turn phosphorylates and activates mapk. through these sequential biochemical events, the signals are amplified and diversified in a step-by-step manner (fig. 4). the stress-responsive mapk pathways that converge on the mapks, jnk and p38, are activated by various environmental stressors such as heat shock, uv irradiation, oxidative stress and osmotic shock. in response to these stressors, these pathways induce a wide variety of cellular responses such as dna repair, cell cycle arrest and apoptosis. these pathways are also activated in response to pathogen challenges and mediate immune responses. in this section, the roles of the stress-responsive mapk pathways in immune and stress response in drosophila and c. elegans are reviewed, although such roles have been assigned to highly conserved mapk pathways. the mapk pathways in immune response dtak1, which is the activator of the ikk complex in the drosophila imd pathway as mentioned above, is a member of the drosophila map3k family and activates the drosophila jnk (djnk) pathway (boutros et al., 2002; silverman et al., 2003). when lipopolysaccharides (lps), the principal cell wall components of gram-negative bacteria, are applied to adult flies and cultured cells, djnk is activated downstream of dtak1 and induces immune-related genes such as those encoding cytoskeletal regulators and pro-apoptotic signaling (sluss et al., 1996; boutros et al., 2002). however, it is still controversial whether the dtak1-djnk axis directly induces the amp genes (silverman et al., 2003; kallio et al., 2005; delaney et al., 2006). in addition to this transcriptional control, it has recently been proposed that the djnk pathway negatively controls the transcription of a group of genes mediated by the ikk-relish axis, another branch downstream of dtak1 (kim et al., 2005). conversely, the djnk pathway has been shown to be negatively regulated by relish-induced proteasomal degradation of dtak1 (park et al., 2004). this cross talk between two branches downstream of dtak1 appears to be critical to determine the duration, i.e., transient versus sustained, of gene induction in the immune response. thus, djnk is directly or indirectly involved in the control of a wide variety of genes, including the amp genes, and therefore is a critical regulator of immune response in drosophila. the drosophila p38 (dp38) pathway is also involved in immune response. among the three isoforms of dp38 identified so far-dp38a, dp38b and dp38c-dp38a and dp38b have mainly been examined in their role as mediators of immune and stress response in drosophila. treatment of drosophila cultured cells with the p38 inhibitor sb203580, which inhibits both dp38a and dp38b, has been shown to enhance lps-induced amp gene expression, while overexpression of dp38a reduced this expression in flies infected with bacteria (han et al., 1998b). however, the recent analysis of mutant flies deficient in dp38a has revealed that dp38a is not required for survival or for amp gene induction following infection with several bacteria (craig et al., 2004). nevertheless, the latter finding does not necessarily exclude the relevance of the dp38 pathway in immune response, because dp38b and/or dp38c may compensate for the deficiency of dp38a. consistent with this hypothesis, a possible immune function of dp38c was recently proposed. in response to bacterial septic injury, dp38c has been shown to mediate the induction of the dopa decarboxylase (ddc) gene. ddc catalyses the production of dopamine, which is metabolized to produce reactive quinones that kill invading microorganisms, and thus dp38c may upregulate quinones via the ddc gene (davis et al., 2008). in c. elegans, it remains unknown whether the jnk pathway is directly involved in innate immune response. on the other hand, the p38 ortholog pmk-1 has received much attention as a regulator in innate immunity. pmk-1 has been characterized as a mapk functioning downstream of the map2k sek-1 and the map3k nsy-1, orthologs of mammalian mkk3/mkk6 and ask1, respectively. nsy-1 and sek-1 were first identified as critical regulators of cell fate determination in a subset of olfactory neurons (sagasti et al., 2001; tanaka-hino et al., 2002). the nsy-1-sek-1-pmk-1 pathway was later found to play a crucial role in innate immune response (kim et al., 2002). loss-of-function mutation of or rnai against these genes clearly increases susceptibility to p. aeruginosa. however, mutation of unc-43, which encodes a ca2+/calmodulin-dependent protein kinase acting upstream of nsy-1 in cell fate determination in neurons (sagasti et al., 2001), did not result in enhanced susceptibility. instead, tir-1, the c. elegans ortholog of mammalian toll-interleukin 1 receptor (tir) domain protein sarm, appears to regulate the nsy-1-sek-1-pmk-1 pathway and to be required for survival and for the expression of amp genes following bacterial infection (couillault et al., 2004; liberati et al., 2004). moreover, tir-1, nsy-1 and sek-1 were found to physically interact with each other (tanaka-hino et al., 2002; chuang and bargmann, 2005). these findings suggest that tir-1 plays a pivotal role in innate immune response through activation of the nsy-1-sek-1-pmk-1 pathway. whereas the immune function of this pathway has been proposed to exert in the intestine (sakaguchi et al., 2004), this pathway has recently been shown to play a critical role in response to wounding as well as infection in the epidermis (pujol et al., 2008). 38 the mapk pathways in stress response apart from biotic stressors such as the bacterial components, various abiotic stressors also activate the jnk and p38 pathways. in drosophila, dp38 is activated by heat shock, uv irradiation, osmotic shock and oxidative stress in the cultured cells, and dp38a mutant flies are consistently susceptible to some of these stressors, indicating that dp38 confers stress resistance to flies (han et al., 1998a; han et al., 1998b; craig et al., 2004; zhuang et al., 2006). djnk is also activated by stressors similar to those that activate dp38 in the cultured cells (botella et al., 2001; chen et al., 2002; ryabinina et al., 2006). it has been shown using genetically engineered flies that djnk activity alleviates the toxic effects of reactive oxygen species (ros), probably through the induction of various categories of protective genes, thereby reducing the accumulation of oxidative damage and prolonging the lifespan of flies (wang et al., 2003). recently, it has been shown that djnk suppresses the insulin/igf-i signaling and activates dfoxo, the counterpart of c. elegans daf-16, in response to oxidative stress. djnk suppressed expression of insulin-like peptides in neuroendocrine cells in the brain, called the insulin-producing cells (ipcs), whereas djnk induced expression of target genes of dfoxo in the cells of peripheral tissues. this functional interaction between djnk and the insulin/igf-i signaling appears to contribute to the protective role of djnk against oxidative stress (wang et al., 2005). also in c. elegans, daf-16 is an important target molecule of jnk-1, an isoform among three jnk-like kinases in c. elegans (fig. 3). in response to environmental stressors such as heat shock, uv irradiation and excess ros, jnk-1 was found to directly interact with and phosphorylate daf-16 and promote the translocation of daf-16 into the nucleus, leading to the expression of numerous target genes to prevent damage from harmful stressors (oh et al., 2005; wolf et al., 2008). intriguingly, the jnk pathway acted in parallel with the daf-2 pathway to regulate lifespan, and both pathways converged on daf-16. moreover, in mammals, the nuclear translocation and transcriptional activation of foxo4, a member of the mammalian foxo family, in response to low levels of oxidative stress have been shown to be mediated through phosphorylation of foxo4 by jnk (essers et al., 2004). thus, this interaction between the jnk and insulin/igf-i pathways is functionally conserved among species and suggests that signaling pathways for stress response and lifespan are closely regulated by each other. heavy metals are well-studied stressors used in the analysis of stress response in c. elegans. mutants of the components of the jnk pathway have been shown to display elevated sensitivity to copper and cadmium, although the tissue(s) where the jnk pathway functions is unclear (koga et al., 2000; villanueva et al., 2001; mizuno et al., 2004). importantly, a daf-2 mutant and an age-1 mutant gained resistance to these metals, suggesting that the functional interaction between the jnk and daf-2 pathways is critical for the response to heavy metals as well (barsyte et al., 2001). perspectives and conclusions to take an overview of signaling pathways in invertebrate immune and stress response, we here focused on drosophila and c. elegans as suitable model organisms. however, we must note that a large body of evidence from other useful invertebrate models supports the important findings obtained using drosophila and c. elegans. as the present review has shown, these two organisms rely on different signaling pathways for their immune response, i.e., the toll and imd pathways versus the dbl-1 and daf-2/daf-16 pathways. this appears to depend at least in part on the existence or absence of the nf-κb-like factors and their regulatory systems. at the same time, the stress-responsive mapk pathways are utilized by both drosophila and c. elegans to regulate immune and stress response. taken together with the highly conserved functional interaction between the mapk and insulin/igf-i pathways, this suggests that the mapk pathways might play pivotal roles in integrating the other diverged and specialized signaling pathways with reference to the external and internal stress conditions. further investigation of such signal networks in invertebrate models will surely shed new light on the highly complex systems regulating immune and stress response in mammals. acknowledgements we thank all of the members of the laboratory of cell signaling for their valuable input. references alper s, mcbride sj, lackford b, freedman jh, schwartz da. specificity and complexity of the caenorhabditis elegans innate immune response. mol. cell. biol. 27: 5544-5553, 2007. anderson kv, bokla l, nüsslein-volhard c. establishment of dorsal-ventral polarity in the drosophila embryo: the induction of polarity by the toll gene product. cell 42: 791-798, 1985. bányai l, patthy l. amoebapore homologs of caenorhabditis elegans. biochim. biophys. acta 1429: 259-264, 1998. barsyte d, lovejoy da, lithgow gj. longevity and heavy metal resistance in daf-2 and age-1 long-lived mutants of caenorhabditis elegans. faseb j. 15: 627-634, 2001. bischoff v, vignal c, boneca ig, michel t, hoffmann ja, royet j. function of the drosophila pattern-recognition receptor pgrp-sd in the detection of gram-positive bacteria. nat. immunol. 5: 1175-1180, 2004. botella ja, baines ia, williams dd, goberdhan dc, proud cg, wilson c. the drosophila cell shape regulator c-jun n-terminal kinase also functions as a stress-activated protein kinase. insect biochem. mol. biol. 31: 839-847, 2001. boutros m, agaisse h, perrimon n. sequential activation of signaling pathways during innate immune responses in drosophila. dev. cell 3: 711-722, 2002. chen w, white ma, cobb mh. stimulus-specific requirements for map3 kinases in activating the jnk pathway. j. biol. chem. 277: 49105-49110, 2002. 39 choe km, lee h, anderson kv. drosophila peptidoglycan recognition protein lc (pgrp-lc) acts as a signal-transducing innate immune receptor. proc. natl. acad. sci. usa 102: 1122-1126, 2005. choe km, werner t, stöven s, hultmark d, anderson kv. requirement for a peptidoglycan recognition protein (pgrp) in relish activation and antibacterial immune responses in drosophila. science 296: 359-362, 2002. christophides gk, zdobnov e, barillas-mury c, birney e, blandin s, blass c, et al. immunity-related genes and gene families in anopheles gambiae. science 298: 159-165, 2002. chuang cf, bargmann ci. a toll-interleukin 1 repeat protein at the synapse specifies asymmetric odorant receptor expression via ask1 mapkkk signaling. genes dev. 19: 270-281, 2005. clarkson jm, charnley ak. new insights into the mechanisms of fungal pathogenesis in insects. trends microbiol. 4: 197-203, 1996. cornwell wd, kirkpatrick rb. cactus-independent nuclear translocation of drosophila relish. j. cell. biochem. 82: 22-37, 2001. couillault c, pujol n, reboul j, sabatier l, guichou jf, kohara y, et al. tlr-independent control of innate immunity in caenorhabditis elegans by the tir domain adaptor protein tir-1, an ortholog of human sarm. nat. immunol. 5: 488-494, 2004. craig cr, fink jl, yagi y, ip yt, cagan rl. a drosophila p38 orthologue is required for environmental stress responses. embo rep. 5: 1058-1063, 2004. davis mm, primrose da, hodgetts rb. a member of the p38 mitogen-activated protein kinase family is responsible for transcriptional induction of dopa decarboxylase in the epidermis of drosophila melanogaster during the innate immune response. mol. cell. biol. 28: 4883-4895, 2008. delaney jr, stoven s, uvell h, anderson kv, engstrom y, mlodzik m. cooperative control of drosophila immune responses by the jnk and nf-κb signaling pathways. embo j. 25: 3068-3077, 2006. el chamy l, leclerc v, caldelari i, reichhart jm. sensing of 'danger signals' and pathogen-associated molecular patterns defines binary signaling pathways 'upstream' of toll. nat. immunol. 9: 1165-1170, 2008. engelman ja, luo j, cantley lc. the evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. nat. rev. genet. 7: 606-619, 2006. engström y. induction and regulation of antimicrobial peptides in drosophila. dev. comp. immunol. 23: 345-358, 1999. essers ma, weijzen s, de vries-smits am, saarloos i, de ruiter nd, bos jl, et al. foxo transcription factor activation by oxidative stress mediated by the small gtpase ral and jnk. embo j. 23: 4802-4812, 2004. evans ea, kawli t, tan mw. pseudomonas aeruginosa suppresses host immunity by activating the daf-2 insulin-like signaling pathway in caenorhabditis elegans. plos pathog 4: e1000175, 2008. evans jd, aronstein k, chen yp, hetru c, imler jl, jiang h, et al. immune pathways and defence mechanisms in honey bees apis mellifera. insect mol. biol. 15: 645-656, 2006. garsin da, villanueva jm, begun j, kim dh, sifri cd, calderwood sb, et al. long-lived c. elegans daf-2 mutants are resistant to bacterial pathogens. science 300: 1921, 2003. georgel p, naitza s, kappler c, ferrandon d, zachary d, swimmer c, et al. drosophila immune deficiency (imd) is a death domain protein that activates antibacterial defense and can promote apoptosis. dev. cell 1: 503-514, 2001. gobert v, gottar m, matskevich aa, rutschmann s, royet j, belvin m, et al. dual activation of the drosophila toll pathway by two pattern recognition receptors. science 302: 2126-2130, 2003. gottar m, gobert v, matskevich aa, reichhart jm, wang c, butt tm, et al. dual detection of fungal infections in drosophila via recognition of glucans and sensing of virulence factors. cell 127: 1425-1437, 2006. gottar m, gobert v, michel t, belvin m, duyk g, hoffmann ja, et al. the drosophila immune response against gram-negative bacteria is mediated by a peptidoglycan recognition protein. nature 416: 640-644, 2002. han sj, choi ky, brey pt, lee wj. molecular cloning and characterization of a drosophila p38 mitogen-activated protein kinase. j. biol. chem. 273: 369-374, 1998a. han zs, enslen h, hu x, meng x, wu ih, barrett t, et al. a conserved p38 mitogen-activated protein kinase pathway regulates drosophila immunity gene expression. mol. cell. biol. 18: 3527-3539, 1998b. hertweck m, göbel c, baumeister r. c. elegans sgk-1 is the critical component in the akt/pkb kinase complex to control stress response and life span. dev. cell 6: 577-588, 2004. horng t, medzhitov r. drosophila myd88 is an adapter in the toll signaling pathway. proc. natl. acad. sci. usa 98: 12654-12658, 2001. hu s, yang x. dfadd, a novel death domain-containing adapter protein for the drosophila caspase dredd. j. biol. chem. 275: 30761-30764, 2000. ip yt, reach m, engstrom y, kadalayil l, cai h, gonzález-crespo s, et al. dif, a dorsal-related gene that mediates an immune response in drosophila. cell 75: 753-763, 1993. jang ih, chosa n, kim sh, nam hj, lemaitre b, ochiai m, et al. a spätzle-processing enzyme required for toll signaling activation in drosophila innate immunity. dev. cell 10: 45-55, 2006. kallio j, leinonen a, ulvila j, valanne s, ezekowitz ra, rämet m. functional analysis of immune response genes in drosophila identifies jnk pathway as a regulator of antimicrobial peptide gene expression in s2 cells. microbes infect. 7: 811-819, 2005. kaneko t, goldman we, mellroth p, steiner h, fukase k, kusumoto s, et al. monomeric and 40 polymeric gram-negative peptidoglycan but not purified lps stimulate the drosophila imd pathway. immunity 20: 637-649, 2004. kaneko t, yano t, aggarwal k, lim jh, ueda k, oshima y, et al. pgrp-lc and pgrp-le have essential yet distinct functions in the drosophila immune response to monomeric dap-type peptidoglycan. nat. immunol. 7: 715-723, 2006. kawabata m, miyazono k. signal transduction of the tgf-β superfamily by smad proteins. j. biochem. 125: 9-16, 1999. kim dh, feinbaum r, alloing g, emerson fe, garsin da, inoue h, et al. a conserved p38 map kinase pathway in caenorhabditis elegans innate immunity. science 297: 623-626, 2002. kim t, yoon j, cho h, lee wb, kim j, song yh, et al. downregulation of lipopolysaccharide response in drosophila by negative crosstalk between the ap1 and nf-κb signaling modules. nat. immunol. 6: 211-218, 2005. kimura kd, tissenbaum ha, liu y, ruvkun g. daf-2, an insulin receptor-like gene that regulates longevity and diapause in caenorhabditis elegans. science 277: 942-946, 1997. koga m, zwaal r, guan kl, avery l, ohshima y. a caenorhabditis elegans map kinase kinase, mek-1, is involved in stress responses. embo j. 19: 5148-5156, 2000. krishna s, maduzia ll, padgett rw. specificity of tgfβ signaling is conferred by distinct type i receptors and their associated smad proteins in caenorhabditis elegans. development 126: 251-260, 1999. kyriakis jm, avruch j. mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. physiol. rev. 81: 807-869, 2001. lemaitre b, kromer-metzger e, michaut l, nicolas e, meister m, georgel p, et al. a recessive mutation, immune deficiency (imd), defines two distinct control pathways in the drosophila host defense. proc. natl. acad. sci. usa 92: 9465-9469, 1995. lemaitre b, nicolas e, michaut l, reichhart jm, hoffmann ja. the dorsoventral regulatory gene cassette spätzle/toll/cactus controls the potent antifungal response in drosophila adults. cell 86: 973-983, 1996. leulier f, rodriguez a, khush rs, abrams jm, lemaitre b. the drosophila caspase dredd is required to resist gram-negative bacterial infection. embo rep. 1: 353-358, 2000. leulier f, vidal s, saigo k, ueda r, lemaitre b. inducible expression of double-stranded rna reveals a role for dfadd in the regulation of the antibacterial response in drosophila adults. curr. biol. 12: 996-1000, 2002. liberati nt, fitzgerald ka, kim dh, feinbaum r, golenbock dt, ausubel fm. requirement for a conserved toll/interleukin-1 resistance domain protein in the caenorhabditis elegans immune response. proc. natl. acad. sci. usa 101: 6593-6598, 2004. lieber mj, luckhart s. transforming growth factor-βs and related gene products in mosquito vectors of human malaria parasites: signaling architecture for immunological crosstalk. mol. immunol. 41: 965-977, 2004. lin k, dorman jb, rodan a, kenyon c. daf-16: an hnf-3/forkhead family member that can function to double the life-span of caenorhabditis elegans. science 278: 1319-1322, 1997. lin k, hsin h, libina n, kenyon c. regulation of the caenorhabditis elegans longevity protein daf-16 by insulin/igf-1 and germline signaling. nat. genet. 28: 139-145, 2001. lu y, wu lp, anderson kv. the antibacterial arm of the drosophila innate immune response requires an iκb kinase. genes dev. 15: 104-110, 2001. luckhart s, riehle ma. the insulin signaling cascade from nematodes to mammals: insights into innate immunity of anopheles mosquitoes to malaria parasite infection. dev. comp. immunol. 31: 647-656, 2007. mallo gv, kurz cl, couillault c, pujol n, granjeaud s, kohara y, et al. inducible antibacterial defense system in c. elegans. curr. biol. 12: 1209-1214, 2002. mellroth p, karlsson j, häkansson j, schultz n, goldman we, steiner h. ligand-induced dimerization of drosophila peptidoglycan recognition proteins in vitro. proc. natl. acad. sci. usa 102: 6455-6460, 2005. michel t, reichhart jm, hoffmann ja, royet j. drosophila toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. nature 414: 756-759, 2001. mizuno t, hisamoto n, terada t, kondo t, adachi m, nishida e, et al. the caenorhabditis elegans mapk phosphatase vhp-1 mediates a novel jnk-like signaling pathway in stress response. embo j. 23: 2226-2234, 2004. mochii m, yoshida s, morita k, kohara y, ueno n. identification of transforming growth factor-β-regulated genes in caenorhabditis elegans by differential hybridization of arrayed cdnas. proc. natl. acad. sci. usa 96: 15020-15025, 1999. morita k, chow kl, ueno n. regulation of body length and male tail ray pattern formation of caenorhabditis elegans by a member of tgf-β family. development 126: 1337-1347, 1999. morris jz, tissenbaum ha, ruvkun g. a phosphatidylinositol-3-oh kinase family member regulating longevity and diapause in caenorhabditis elegans. nature 382: 536-539, 1996. murphy ct, mccarroll sa, bargmann ci, fraser a, kamath rs, ahringer j, et al. genes that act downstream of daf-16 to influence the lifespan of caenorhabditis elegans. nature 424: 277-283, 2003. naitza s, rosse c, kappler c, georgel p, belvin m, gubb d, et al. the drosophila immune defense against gram-negative infection requires the death protein dfadd. immunity 17: 575-581, 2002. nicholas hr, hodgkin j. responses to infection and possible recognition strategies in the innate immune system of caenorhabditis elegans. mol. immunol. 41: 479-493, 2004. 41 nicolas e, reichhart jm, hoffmann ja, lemaitre b. in vivo regulation of the iκb homologue cactus during the immune response of drosophila. j. biol. chem. 273: 10463-10469, 1998. ogg s, paradis s, gottlieb s, patterson gi, lee l, tissenbaum ha, et al. the fork head transcription factor daf-16 transduces insulin-like metabolic and longevity signals in c. elegans. nature 389: 994-999, 1997. oh sw, mukhopadhyay a, svrzikapa n, jiang f, davis rj, tissenbaum ha. jnk regulates lifespan in caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/daf-16. proc. natl. acad. sci. usa 102: 4494-4499, 2005. paradis s, ailion m, toker a, thomas jh, ruvkun g. a pdk1 homolog is necessary and sufficient to transduce age-1 pi3 kinase signals that regulate diapause in caenorhabditis elegans. genes dev. 13: 1438-1452, 1999. paradis s, ruvkun g. caenorhabditis elegans akt/pkb transduces insulin receptor-like signals from age-1 pi3 kinase to the daf-16 transcription factor. genes dev. 12: 2488-2498, 1998. park jm, brady h, ruocco mg, sun h, williams d, lee sj, et al. targeting of tak1 by the nf-κb protein relish regulates the jnk-mediated immune response in drosophila. genes dev. 18: 584-594, 2004. pujol n, cypowyj s, ziegler k, millet a, astrain a, goncharov a, et al. distinct innate immune responses to infection and wounding in the c. elegans epidermis. curr. biol. 18: 481-489, 2008. pujol n, link em, liu lx, kurz cl, alloing g, tan mw, et al. a reverse genetic analysis of components of the toll signaling pathway in caenorhabditis elegans. curr. biol. 11: 809-821, 2001. rämet m, manfruelli p, pearson a, mathey-prevot b, ezekowitz ra. functional genomic analysis of phagocytosis and identification of a drosophila receptor for e. coli. nature 416: 644-648, 2002. ryabinina op, subbian e, iordanov ms. d-mekk1, the drosophila orthologue of mammalian mekk4/mtk1, and hemipterous/d-mkk7 mediate the activation of d-jnk by cadmium and arsenite in schneider cells. bmc cell biol. 7: 7, 2006. sagasti a, hisamoto n, hyodo j, tanaka-hino m, matsumoto k, bargmann ci. the camkii unc-43 activates the mapkkk nsy-1 to execute a lateral signaling decision required for asymmetric olfactory neuron fates. cell 105: 221-232, 2001. sakaguchi a, matsumoto k, hisamoto n. roles of map kinase cascades in caenorhabditis elegans. j. biochem. 136: 7-11, 2004. sansonetti pj. the innate signaling of dangers and the dangers of innate signaling. nat. immunol. 7: 1237-1242, 2006. savage c, das p, finelli al, townsend sr, sun cy, baird se, et al. caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor β pathway components. proc. natl. acad. sci. usa 93: 790-794, 1996. silverman n, zhou r, erlich rl, hunter m, bernstein e, schneider d, et al. immune activation of nf-κb and jnk requires drosophila tak1. j. biol. chem. 278: 48928-48934, 2003. silverman n, zhou r, stöven s, pandey n, hultmark d, maniatis t. a drosophila iκb kinase complex required for relish cleavage and antibacterial immunity. genes dev. 14: 2461-2471, 2000. sluss hk, han z, barrett t, goberdhan dc, wilson c, davis rj, et al. a jnk signal transduction pathway that mediates morphogenesis and an immune response in drosophila. genes dev. 10: 2745-2758, 1996. stoven s, silverman n, junell a, hedengren-olcott m, erturk d, engstrom y, et al. caspase-mediated processing of the drosophila nf-κb factor relish. proc. natl. acad. sci. usa 100: 5991-5996, 2003. stronach be, perrimon n. stress signaling in drosophila. oncogene 18: 6172-6182, 1999. suzuki y, yandell md, roy pj, krishna s, savage-dunn c, ross rm, et al. a bmp homolog acts as a dose-dependent regulator of body size and male tail patterning in caenorhabditis elegans. development 126: 241-250, 1999. takehana a, yano t, mita s, kotani a, oshima y, kurata s. peptidoglycan recognition protein (pgrp)-le and pgrp-lc act synergistically in drosophila immunity. embo j. 23: 4690-4700, 2004. tanaka h, ishibashi j, fujita k, nakajima y, sagisaka a, tomimoto k, et al. a genome-wide analysis of genes and gene families involved in innate immunity of bombyx mori. insect biochem. mol. biol. 38: 1087-1110, 2008. tanaka-hino m, sagasti a, hisamoto n, kawasaki m, nakano s, ninomiya-tsuji j, et al. sek-1 mapkk mediates ca2+ signaling to determine neuronal asymmetric development in caenorhabditis elegans. embo rep. 3: 56-62, 2002. tauszig-delamasure s, bilak h, capovilla m, hoffmann ja, imler jl. drosophila myd88 is required for the response to fungal and gram-positive bacterial infections. nat. immunol. 3: 91-97, 2002. tenor jl, aballay a. a conserved toll-like receptor is required for caenorhabditis elegans innate immunity. embo rep. 9: 103-109, 2008. vidal s, khush rs, leulier f, tzou p, nakamura m, lemaitre b. mutations in the drosophila dtak1 gene reveal a conserved function for mapkkks in the control of rel/nf-κb-dependent innate immune responses. genes dev. 15: 1900-1912, 2001. villanueva a, lozano j, morales a, lin x, deng x, hengartner mo, et al. jkk-1 and mek-1 regulate body movement coordination and response to heavy metals through jnk-1 in caenorhabditis elegans. embo j. 20: 5114-5128, 2001. wang l, gilbert rj, atilano ml, filipe sr, gay nj, ligoxygakis p. peptidoglycan recognition protein-sd provides versatility of receptor formation in drosophila immunity. proc. natl. acad. sci. usa 105: 11881-11886, 2008. 42 wang mc, bohmann d, jasper h. jnk signaling confers tolerance to oxidative stress and extends lifespan in drosophila. dev. cell 5: 811-816, 2003. of a three-kinase module from yeast to human. physiol. rev. 79: 143-180, 1999. wolf m, nunes f, henkel a, heinick a, paul rj. the map kinase jnk-1 of caenorhabditis elegans: location, activation, and influences over temperature-dependent insulin-like signaling, stress responses, and fitness. j. cell. physiol. 214: 721-729, 2008. wang mc, bohmann d, jasper h. jnk extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. cell 121: 115-125, 2005. waterhouse rm, kriventseva ev, meister s, xi z, alvarez ks, bartholomay lc, et al. evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. science 316: 1738-1743, 2007. wolkow ca, munoz mj, riddle dl, ruvkun g. insulin receptor substrate and p55 orthologous adaptor proteins function in the caenorhabditis elegans daf-2/insulin-like signaling pathway. j. biol. chem. 277: 49591-49597, 2002. weber an, tauszig-delamasure s, hoffmann ja, lelièvre e, gascan h, ray kp, et al. binding of the drosophila cytokine spätzle to toll is direct and establishes signaling. nat. immunol. 4: 794-800, 2003. zhou r, silverman n, hong m, liao ds, chung y, chen zj, et al. the role of ubiquitination in drosophila innate immunity. j. biol. chem. 280: 34048-34055, 2005. zhuang zh, zhou y, yu mc, silverman n, ge bx. regulation of drosophila p38 activation by specific map2 kinase and map3 kinase in response to different stimuli. cell signal. 18: 441-448, 2006. werner t, borge-renberg k, mellroth p, steiner h, hultmark d. functional diversity of the drosophila pgrp-lc gene cluster in the response to lipopolysaccharide and peptidoglycan. j. biol. chem. 278: 26319-26322, 2003. zou z, evans jd, lu z, zhao p, williams m, sumathipala n, et al. comparative genomic analysis of the tribolium immune system. genome biol. 8: r177, 2007. werner t, liu g, kang d, ekengren s, steiner h, hultmark d. a family of peptidoglycan recognition proteins in the fruit fly drosophila melanogaster. proc. natl. acad. sci. usa 97: 13772-13777, 2000. zugasti o, ewbank jj. neuroimmune regulation of antimicrobial peptide expression by a noncanonical tgf-β signaling pathway in caenorhabditis elegans epidermis. nat. immunol. 10: 249-256, 2009. widmann c, gibson s, jarpe mb, johnson gl. mitogen-activated protein kinase: conservation 43 table 1 isj 6: 144-153, 2009 issn 1824-307x research report an investigation on the disruptive effect of pollution in coldand warmadapted clam populations f gagné1, c andré1, c blaise1 2, j pellerin , j sherry3, a talbot1,3 1fluvial ecosystem research section, environment canada, 105 mcgill, montréal, québec, canada, h2y 2e7 2ismer-université du québec à rimouski, rimouski, 310 grande allée, rimouski, québec, canada, g5l 3a1 3environment canada, national water research institute, 867 lakeshore rd., po box 5050 burlington, ontario, canada, l7r 4a6 accepted october 27, 2009 abstract the purpose of this study was to examine the relationship between mitochondrial activity and gonad lipid stores in clams exposed to anthropogenic pollution at coldand warm-water sites. the balance between energy expenses and energy reserves was measured by mitochondrial electron transport (met) activity and lipid content in the gonad. the activity of malate dehydrogenase (mdh) was measured as an intermediary between energy production and the production of lipids in gonadal tissues. the results revealed that intertidal clam populations at warm-water sites under no source of pollution had less heavy metal content (ag, as, cr, hg and ni), lower mdh activity and temperaturedependent met than clams from cold-water sites. however, mdh activity measured at 6 oc was higher at the warm-water sites. lipid peroxidation in the gonad was higher in clams from the coldwater sites. the impacts of pollution differed among the study sites, clams from cold-water sites having increased mdh activity, temperature-dependent met activity, higher lipid content and dna strand breaks; clams from the warm-water sites had increased temperature-dependent mdh activity and lower gonadal lipid reserves. a multiple regression analysis revealed that gonad lipid reserves were positively correlated with mdh activity and negatively correlated with its temperature-dependent activity, suggesting that increased temperature sensitivity was negatively related to gonad energy reserves. the data show that pollution increases temperature sensitivity at the met level in clams in cold water, while temperature sensitivity in mdh activity was observed in clams from warm-water sites. discriminate function analysis revealed that pollution stress shows a tendency to be closer to clams adapted to warmer temperatures. in conclusion, pollution could increase mdh activity in cold-adapted clams which can lead to increased lipid stores in the gonad, oxidative stress and genotoxicity while pollution seems to increase the temperature dependence in met. in warm-adapted clams, temperature dependent mdh activity was higher by pollution with decreased lipid content in the gonad tissues which was independent of gonad maturation and size. key words: mitochondrial electron transport; malate dehydrogenase; gonad lipid; clam health introduction the st. lawrence river is subjected to various sources of chemical pollution that are historically linked to a variety of industrial (aluminum plants and pulp and paper mills), municipal effluents and agricultural runoffs from adjacent basins and harbors. clams are particularly at risk to these types of pollution since they are sessile, relatively long-lived (12-15 years for mya arenaria), and in close contact with sediments, which are well-known sinks for substances such as tributyltin (tbt) and industrial contaminants such as polycyclic aromatic hydrocarbons (pahs), polychlorinated biphenyls (pcbs) and heavy metals (gagnon et al., 2004; yang et al., 2006). the ecotoxicological impacts of pollution in this intertidal bivalve were extensively examined in the st. lawrence and one of its largest tributaries, the saguenay fjord (blaise et al., 2003; gagné et al., 2008a, b; pellerin et al., 2009). in these studies, it was revealed that the density of intertidal clam beds was closely related to immunocompetence (hemocyte concentration and phagocytic activity) and mitochondrial electron ___________________________________________________________________________ corresponding author: françois gagné fluvial ecosystem research section environment canada 105 mcgill st., montreal, quebec, canada h2y 2e7 e-mail:francois.gagne@ec.gc.ca 144 transport activity (met), a measure of respiration and energy expenditure. moreover, pollution increased clam temperature dependence or sensitivity to electron transport activity in mitochondria (gagné et al., 2008b). increased energy expenditure could occur at the cost of energy reserves in the form of lipids, sugars and proteins to a lesser extent (smolders et al., 2004). the balance between energy expenditure and reserves was introduced as the concept of cellular energy allocation, which proved a very predictive and sensitive endpoint of impacts at higher levels of biological effects such as survival, growth and reproduction in zebra mussels and daphnids (de coen and janssen, 2003). in a context of global warming and given the cumulative effects of environmental stressors, a recent study revealed that met activity was sensitive to temperature changes and pollution readily increased this temperature sensitivity (gagné et al., 2007). indeed, this study revealed that clams under pollution stress were more sensitive (i.e., spend more energy) to temperature increments than clams from lesscontaminated sites. to further investigate the interaction of pollution with thermal adaptation and stress, we undertook a spatial survey of intertidal clams at cold-water sites (st. lawrence estuary) and from areas of warmer water in the saguenay fjord. the balance between energy expenditure (met) and reserves in the gonad (gonad size and lipid content) was closely examined by tracking the transfer of precursors from the mitochondria to the cytoplasm to assist lipid synthesis. in cells, oxaloacetate is transferred in the cytosol to support lipid and amino acid synthesis through the malate shunt pathway, which involves nadh-dependent malate dehydrogenase (mdh). mdh catalyzes the reversible reduction of oxaloacetate to malate in the presence of the cofactor nadh in the mitochondria (stryer, 1995): oxaloacetate + nadh malate + nad+. malate diffuses out the mitochondria and is re-oxidized into oxaloacetate in the cytosol by a cytosolic mdh. during this process, nadh is formed which, in turn, can be used to support lipid synthesis. adaptation to cold or warm temperatures brings about changes in allelic mdh expression, selecting towards isoforms with various temperature dependence (fields et al., 2006). indeed, adaptation to colder temperatures was associated with lower enzyme affinity for nadh, but the activity was more sensitive to temperature. lower enzyme affinity for nadh will displace the reaction in favor of the oxidation of malate into oxaloacetate and nadh, hence is consistent with lipid production in organisms acclimated to colder temperatures. the production of oxaloacetate and malate will also depend on mitochondria respiration rate i.e. aerobic metabolism and the cytosolic activity of mdh. in another study, the affinity constant km for nadh was less sensitive to high temperatures in bivalve species adapted to warmer temperatures (l. scabra and l. gigantea) than in those adapted to colder temperatures such as l. scutum or l. pelta (dong and somero, 2009). this change in enzyme affinity was associated with a single amino acid substitution of glycine to serine at position 291 in cytosolic mdh, a change that favors hydrogen bonding and reduced conformational entropy, hence less affected to temperature variations. these studies suggest that warm/cold adaptation calls for changes in protein/enzyme kinetic activity and sensitivity to temperature changes. intertidal clams have to cope with air-temperature fluctuations during low tide which bring about shifts between anaerobic and aerobic metabolism and how clams handle these stresses in either coldor warm-water habitats is not fully understood. moreover, the interaction of pollution in these adaptation processes complicates the outcome of clam survival. there is a need to further study into how pollution stress and temperature interacts in feral clam populations. the purpose of this study was therefore to examine the impacts of pollution on the relationship between met, cytosolic mdh activity, lipid reserves in the gonad and the gonado-somatic index (gsi) of intertidal clam populations adapted to cold and warm water considering their relative distance from the shore (i.e., emersion time to air). moreover, toxic tissue damage was concurrently investigated by tracking changes in lipid peroxidation (lpo) and the formation of dna strand breaks. an attempt was made to relate the impacts of pollution on met and mdh activity, including their temperature-sensitive properties and gonad lipid reserves in clams obtained from coldand warm-water. materials and methods spatial survey and clam collection mya arenaria soft-shell clams were collected by hand on mud flats at morning low tide from two sites in the st. lawrence estuary and two sites in the saguenay fjord (fig. 1). sampling was made in later-half june of 2006 which corresponds to the pre-spawning period with gravid gametes and high gsi. clams from the former area thrive in the colder water of the st. lawrence (typically 3-6 oc compared to water temperatures of 5-12 oc at the fjord site) owing to the inputs of warmer river water draining nearby basins and adjacent tributaries (small streams and rivers, drainage of rainfall). both areas are influenced by tidal rhythms and they are interconnected and relatively close to each other; no significant ph and conductivity differences were observed for the surface waters at the site of collection. temperature measurements of the surface waters at the shore line were also determined during clam collection. among the st. lawrence estuary sites, baie sainte-catherine (bsc) is influenced by heavy traffic from sightseeing and whale-watching boats which is considered the cold temperature and polluted sites (ct+p); baiedu-moulin à baude (bau) was considered the coldtemperature reference site (ct) because of the absence of any direct source of pollution. as for the saguenay fjord sites, anse étienne site (ase), located on the south shore 20 km upstream of the estuary, is under no direct source of anthropogenic activity and was thus considered as the reference site for the fjord for the warm water temperature site (wt); the anse saint-jean (asj) site is exposed to the minimally treated (screening) urban wastewater of a population of about 2000 residents, located 40 145 fig. 1 clams were collected at four sites (identified by stars) in june 2006. the sites anse de saint-étienne (ase) and baie du moulin à baude (bau) were considered as the reference ct and wt sites respectively, while anse saint-jean (asj) and baie sainte-catherine (bsc) were the wt+p and ct+p sites respectively. clams from ase and asj sites were located in the warmer water temperatures of the saguenay river; clams from bau and bsc sites were located in the colder waters of the st. lawrence estuary. km upstream of the estuary and was considered the warm-temperature polluted site (wt-p). a total of 20 clams were collected at each site for the biomarker analyses. clam weights, soft tissue mass and shell length were determined on site and in the laboratory for gsi (gonad wet weight /soft tissues wet weight), condition factor (clam weight/shell length), and growth index (shell length/age ratio) determinations. age was determined by counting the number of major grooves on the shell. sex and gonad maturity were determined by microscopic analysis of gonad smears at 400x. parasitism was rarely observed but there were some occurrences of trematode infection; clams so infected were discarded. the clams retained for analysis were frozen under dry ice for transport to the laboratory and stored at -85 oc until the analyses. after thawing on ice, the visceral mass containing the gonad tissues were removed and homogenized with a teflon pestle tissue grinder apparatus in 25 mm hepes-naoh buffer, ph 7.4, containing 145 mm nacl, 10 µg/ml apoprotinin and 0.1 mm dithiothreitol at a 1:5 volume/volume ratio (five passes). samples (100 µl) of each homogenate were collected for total protein determinations (bradford, 1976), lipid peroxidation (lpo), total lipid content and dna damage. the remaining homogenate was centrifuged at 1,500xg (20 min at 2 oc) to remove cell debris and nuclei (pellet). the supernatant was centrifuged at 10,000xg (20 min at 2 oc) to isolate the mitochondria (pellet) for mitochondrial electron transport (met) activity, and the remaining supernatant was kept for the post-mitochrondrial malate deshydrogenase (mdh) activity evaluations. the mitochondria pellet was resuspended in 2 volumes of ice-cold homogenization buffer before measuring met activity during the same day. energy status cellular energy allocation was determined by measuring mitochondrial electron transport (met) activity and lipid content in gonad. met activity was determined in the mitochondrial fraction (10,000xg pellet) using the p-iodonitrotetrazolium dye method (king and packard, 1975; smolders et al., 2004). briefly, 100 µl of the supernatant was mixed with 100 mm tris-hcl, ph 8.5, containing 100 µm mgso4, 146 table 1 heavy metal content in m. arenaria clams and concordance analysis with the measured biomarkers metals bsc bau asj ase (µg/g) 0.58±0.25 0.14±0.02* 0.09±0.02 0.03±0.05 ag 0.91±0.05* 0.84±0.04 as 0.30±0.01 0.48±0.03 cd 0.06±0.01 0.04±0.02 0.09±0.01 0.11±0.01 7±1.4* 4±0.8 1.3 ±2* cr 4 ±0.9 1.3±0.26 1.5±0.03 cu 1.35±0.3 1.28±0.26 0.03±0.005* 0.03±0.003* hg 0.008±0.002 nd 4±0.8* ni 0.8±0.2 1.7±0.3 1.6±0.3 pb 0.04±0.003 0.06±0.1 0.09±0.02 0.065±0.01 0.12±0.05* sn 0.03±0.01 -- zn (x10) 1.1±0.2 1±0.2 1.7±0.1 1.3±0.3 sum 24.65 16.6 27.6 17.9 * indicates significance at p<0.05 0.1 % triton x-100 and 5 % polyvinylpyrrolidone for 1 min on ice. the reaction mixture was mixed with 1 mm and 0.2 mm nadh and nadph, respectively, on ice and then divided into two portions, one being equilibrated at 6 biomarkers of toxic effects toxic effects were determined by tracking lpo and dna strand breaks in gonad. lpo was determined in gonad homogenates using the thiobarbituric acid method (wills, 1987). standard solutions of tetramethoxypropane were used for calibration and fluorescence was measured at 520 nm excitation and 600 nm emission (chameleon-ii, multireader, bioscan, usa). given that the reagent could react with other aldehydes, results were expressed as µg of thiobarbituric acid reactants (tbars)/mg of homogenate protein. dna damage was determined by the alkaline precipitation assay developed by olive (1988), with fluorescence quantitation of dna strand breaks in the presence of detergents and an alkaline ph (bester et al., 1994). the assay principle is based on the potassiumdetergent (sds)-assisted precipitation of proteinlinked genomic dna, which leaves protein-free dna strand breaks in the supernatant. the number of dna strand breaks results from the dna repair of dna adducts and alkali-labile sites. the results were expressed as µg of dna/mg proteins. calibration was achieved with salmon sperm dna (sigma chemical company, usa). o oc and the other at 25 c. the reaction was initiated with the addition of 50 µl of 5 mm p-iodonitrotetrazolium for 30 min. absorbance readings were measured at 15-min intervals at 520 nm. the data were expressed as the loss of absorbance at 6 o oc and 25 c/30 min/mg total proteins in the corresponding supernatant. temperature-dependent met (mett) was determined and the rate difference in met at 25 oc and 4 oc was divided by the temperature change (25-6 o oc = 19 c) as determined previously (gagné et al., 2007). the data were expressed as the rate change (normalized against total proteins)/unit temperature in oc. gonad lipid levels were determined in gonad homogenates following the phosphovanillin method (frings et al., 1972). the detergent triton x-100 was used for calibration. the data were expressed as μg lipid equivalents/mg protein. malate dehydrogenase (mdh) activity in the mitochondrial fraction (s3) was determined by tracking the formation of nadh in the presence of 0.2 mm malate and nad+ o for 30 min at 20 c. the reaction buffer was 50 mm hepes-naoh, ph 7.4, containing 100 mm nacl and 0.1 mm edta. the appearance of nadh was measured by fluorescence (excitation 360 nm; emission 450 nm). temperature-dependent mdh was also determined as described with met data analysis a total of 20 clams were analyzed for each study site. the homogeneity of variances was determined using bartlett’s test. where the data proved heterogeneous, they were log-transformed. the data were subjected to a two-way anova, with gender and site of collection as the main factors. a discriminant analysis was performed to identify if the sites could be correctly classified using the biomarker t. the kinetic constants for mdh (km and vmax) were also determined at both temperatures using the classic lineweaver_burk plot method (stryer, 1995). 147 table 2 clam morphological characteristics clam weight/shell length growth index gonado-somatic index site age (shell length/age) saguenay fjord 10±0.31 0.49±0.022 6.04±0.25 0.12±0.01 ase (reference) asj 11.5±0.33 0.48±0.01 5.6±0.17 0.12±0.005 st. lawrence estuary b b b0.57±0.02 5.62±0.12 0.06±0.005bau (reference) 12±0.28 bsc 9.2±0.38 a,c a a,c a,c0.47±0.02 6.43±0.22 0.04±0.003 a significance at p<0.05 between the polluted site relative to the corresponding reference site b significant difference between the reference sites in coldand warm-water sectors c significant difference between polluted sites in coldand warm-water sectors test battery and which measurements contributed the most to site discrimination. correlation (pearson-moment) and multiple regression analyses were also performed. significance was set at p < 0.05 but marginal effects (0.1 < p < 0.05) were also provided. all statistical tests were performed using the statistica version 8 software package. results clams contamination and morphological characteristics the surface water ph, conductivity and temperature were measured in surface waters from the saguenay fjord and the st-lawrence estuary (fig. 1). clams collected at the st-lawrence area were exposed to colder surface water temperatures while those collected in the saguenay fjord were exposed to warmer surface water temperatures. indeed, the water temperature was significantly (p = 0.02) higher at the saguenay fjord (10 ± 4 oc) than the estuary (5 ± 1 oc) confirming the temperature differences between these two sectors. the ph and water conductivity did not significantly changed albeit a marginal (p = 0.07) increase in conductivity for the st-lawrence estuary sites. moreover, clams were analyzed for heavy metal content to confirm that they were indeed exposed to pollution stress (table 1). the data revealed that clams at the heavy-traffic harbor were more contaminated in arsenic (as), silver (ag), chromium (cr), mercury (hg), nickel (ni) and total tin (sn). clams exposed to urban wastewaters from a township of 2,000 inhabitants had the highest levels on ag with higher amounts of as, cr, and hg. these data corroborates previous findings (gagnon et al., 2004; yang et al., 2006) and confirms that the organisms were significantly more contaminated with some metals at least. the morphological characteristics of clams collected at two impacted sites, and their corresponding reference sites, were examined (table 2). globally, no significant changes in the sex ratio were observed throughout the study sites (kruskal-wallis rank anova, p = 0.51). the condition factor (clam weight/shell length) changed significantly according to site of collection. condition factor was significantly reduced at ct+p site compared to its corresponding reference ct site. the condition factor was significantly lower at wt than at ct reference sites. the growth index (shell length/age) significantly changed across the sites but gender was not significant. the growth index was significantly reduced at the ct+p sites. growth was significantly lower at wt than at ct sites. correlation analysis revealed that cf was significantly correlated with growth index (r = 0.26; p = 0.005) and shell length (r = 0.2; p = 0.03). gametogenic activity was determined by measuring the gsi. it was significantly affected by site location with no significant effects with clam’s gender. moreover, gsi increased significantly with clam age. the gsi was significantly reduced at site wt+p site and somewhat increased at site ct+p only when corrected for age differences. clams from the wt reference site had higher gsis than those from the ct reference site. a correlation analysis revealed that gsi was positively correlated with shell length (r = 0.19; p = 0.04), and age (r = 0.21; p = 0.03). energy status clam energy status in the gonad was determined by measuring temperature-dependent met at 25 and 6 o oc, mdh at 25 and 6 c), and gonad lipid content (figs 2a, b, c). met at 25 oc was significantly increased at ct+p site, with no change in mdh activity at the same temperature (fig. 2a). however, mdh activity at 25 oc was significantly higher at the wt reference site compared to the ct site. no significant differences in met activity at 25 oc were observed between the warmand cold-water sites. at colder temperatures (6 oc), there was no significant difference between the clean and polluted sites and both reference sites (fig. 2b). the temperature sensitivity of met activity (met ), but not mdh activity (mdh ), was readily t t 148 wt wt +p ct ct +p sites 0 1 2 3 4 5 6 7 m e t o r m d h a ct iv ity 25 o c met mdh a b a wt wt +p ct ct +p sites 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 m e t o r m d h a ct iv ity 6o c met mdh b wt wt+p ct ct+p sites 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 0,22 0,24 0,26 0,28 0,30 t em pe ra tu re -d ep en de nt m e t a nd m d h a ct iv ity d met/dt d mdh/dt c a b a wt wt+p ct ct+p site nu 2 4 6 8 10 12 14 16 18 li pi ds in g on ad (u g/ m g pr ot ei ns ) mean stand. error a ad fig. 2 temperature-dependent energy status in selected clam populations. the effects of clam length (size) and site location of m. arenaria clams are shown for met and mdh at 25 o oc (a), met and mdh at 6 c (b), temperature-dependent met and mdh (c) and gonad lipid reserves (d). met activity was expressed as the increase in dye reduction (520 nm)/min/mg proteins and mdh activity as the increase in nadh fluorescence/min/mg proteins. the letter ‘a’ indicates significance relative to each control sites (ase or bau) from the saguenay fjord and st. lawrence estuary. the letter ‘b’ denotes the significance between the cold-water and warm-water reference sites. increased at the ct+p site relative to the corresponding ct reference site (fig. 2c). mett but not mdht was significantly higher at the wt site compared to the ct site. lipid levels in gonad were significantly reduced at the wt+p site while they were increased at the ct+p site (fig. 2d). no significant change in lipid content was observed between the coldand warm-water reference sites (i.e., ct and wt). correlation analysis revealed that met at 25 oc was correlated with growth index (r = 0.3; p = 0.001), met at 6 oc (r = 0.59; p < 0.001), gonad lipids levels (r = -0.24; p = 0.011) and mett (r = 0.77; p < 0.001). met at 6 oc was significantly correlated with cf (r = -0.21; p = 0.02), growth index (r = -0.34; p < 0.001), mdh at 6 oc (r = -0.25; p = 0.01) and mett (r=0.53; p<0.001). mdh at 25 oc was significantly correlated with gsi (r=0.3; p=0.005) and mdht (r = 0.84; p < 0.001). mdh at 6 oc was significantly correlated with met at 6 o c (r = -0.25; p = 0.01), and mett (r = -0.40; p = 0.002). mdh was significantly correlated with gsi (r = 0.38; p = 0.003), cf (albeit marginally; r = 0.23; p = 0.08), gonad lipids (r = -0.7; p < 0.001) and mdh at 25 oc (r = 0.84; p < 0.001). because of the relationships between mdh activity and met (energy expenditure) and gonadal lipids (energy reserves), the kinetic properties of this enzyme complex were characterized at cold and warm temperatures (table 3). the enzyme has a higher affinity for nad+ than for malate, regardless of the water quality and thermal history of the site. the enzyme affinity at 25 oc for malate remains the same for both reference sites (wt and ct), but affinity increases in clams taken from the ct+p site. no changes were observed for nad+ when determined at 25 oc. however, at 6 oc, the enzyme affinity for both malate and nad+ increased at the ct site, whereas this was not found in clams at the ct+p site. temperature had more influence on the maximum rate of reaction (vmax) for malate and nad+. at cold temperatures (6 oc), vmax was increased at the ct+p site but this was lost at t 149 table 3 mdh* kinetic characteristics in m. arenaria clams enzyme turnover 25 enzyme turnover 6 temperature temperature vmax km vmax km sensitivity for enzyme affinity** sensitivity for reaction rate** substrate 6oc 6o o oc 25 c 25 c o oc c (vmax/km) (vmax/km) wt malate 9.7 0.2 28 0.6 0.02 0.02 18 0.4 0.19 0.11 1.3 0.4 nad 1.7 0.2 3 0.6 wt+p malate 0.02 0.07 17 0.06 7.8 0.52 25 0.6 0.6 0.12 0.5 0.2 nad 2.4 0.3 2.9 0.4 ct malate 5.4 0.2 27 0.5 0.02 0.03 22 0.3 0.58 1 1.1 0.6 nad 0.1 0.1 1.2 0.7 ct+p malate 8.2 0.5 12 0.4 0.03 0.06 4 -0.1 0.34 0.11 -1.1 0,2 nad 2.6 0.3 1.5 0.5 +* mdh activity: malate + nad oxaloacetate + nadh ** km or vmax at 25 oc km or vmax at 6 oc ohigher temperatures (25 c). the turnover number (vmax/km) at 6 oc was readily increased in clams from the ct+p site. a concordance analysis revealed that lipid gonad levels were significantly correlated with km and vmax, turnover number for nad+ at 6 oc (p < 0.001), km for nad + at 25 oc (p = 0.02), km and vmax, turnover number for malate at 6 oc (p < 0.001) and the turnover number at 25 oc (p < 0.001). the gsi was significantly correlated with km at 6 oc and 25 oc, vmax at 25 oc and temperature-dependent km and vmax for malate. for nad+, the gsi was significantly correlated with km and vmax at 25 oc (p = 0.02), turnover number at 6 oc (p = 0.02), temperature-dependent km and vmax (p = 0.02). it appears therefore that gonad lipids levels are more dependent on the respective activities at 6 oc (which is a function of clam immersion in cold water), while gsi is related more to temperature dependence, which is favored at the warm-water sites (and exposed to surface air temperature). the temperature-dependent mdh activity and mdh activity were correlated (as expected) with temperature-dependent vmax and vmax at 25 oc, respectively. tissue damage was investigated by measuring the level of dna strand breaks and lpo in gonad (figs 3a, b). lpo tended to be higher at the polluted sites but the increase was only significant for wt+p site (anova p < 0.01, least squares difference test). lpo at the ct site was significantly higher than the reference wt site in the saguenay fjord. the levels of dna strand breaks in the gonad were significantly higher at site wt+p site, reaching 1.9-fold the levels of the reference wt site. in an attempt to determine the interaction between pollution and energy production and transfers to lipid reserves and gonadal mass in gonad tissues, a discriminate function analysis was performed (fig. 4). the analysis revealed that sites were correctly classified in increasing order: ct+p site (68 %), wt site (68 %), wt+p site (70 %) and ct site (94 %). the following biomarkers were significantly correlated with the root 1 function of the x-axis, in descending order: gsi, gonad lipid, mdht and lpo in gonad. for the second function of the yaxis, the following biomarkers were significantly correlated, in descending order: mett, gonad lipid, condition factor and mdht. this indicates that temperature sensitivities of mdh and met activity are key variables of site discrimination. at the colder sites (ct and ct+p), the effects of pollution displaced the data towards the warmer site (ase). the effects of pollution at the warmer sites (ase, asj) displaced the data away from the colder sites, indicating that pollution has “warming” effects but not the reverse (i.e., no cooling effects). discussion increased met activity leads to increased activity in the malate shunt pathway, where malate is transferred to the cytosol and converted back to oxaloacetate by a cytosolic mdh. the process forms nadh, which can be used to support lipid synthesis. indeed, a multiple regression analysis revealed that lipid gonad levels were positively related with mdh activity but temperature dependence was negatively correlated with gonad lipid levels. in fact, lower-temperature dependence (from cold-adapted organisms) of mdh, but with increased temperature sensitivity for met, was related to increased lipid stores in gonad tissues. this suggests that increased mitochondrial energy production with a more temperature invariant mdh could favor the accumulation of lipids in gonad tissues. in turn, lipid synthesis calls for temperatureindependent mechanisms (hence, the production of lipid precursors is maintained at cold temperatures). mdh activity was shown to be less sensitive to high temperature changes in cold-adapted bivalves (fields 150 wt wt+p ct ct+p sites 0 5 10 15 20 25 30 35 40 li pi d pe ro xi da tio n (u g t b a r s /m g pr ot ei ns ) mean stand. error a b a 1000 1500 2000 2500 3000 3500 4000 4500 5000 d n a s tra nd b re ak s (u g d n a /m g pr ot ei ns ) mean stand. error b a wt wt+p ct ct+p sites fig. 3 tissue damage in m. arenaria clams collected at polluted sites. the levels of lpo (a) and dna damage (b) were measured in the gonad. the letter ‘a’ indicates significance at each reference site (ase or bau). et al., 2006). the present study corroborated the pollution-induced increase in temperature sensitivity at the warmer site (wt+p) than at the colder site (ct+p). increased sensitivity to temperature for mdh was associated with lower gonad lipids. it is noteworthy that these clams were fully ripped with no clear indication that spawning occurred. at the polluted cold-water site (ct+p), met activity was more sensitive to temperature than mdh activity. the relative distance from the shore differed from the various study sites indicating that the clams were exposed to different emersion times. however, the relative distance from the shore was not significantly related with neither mdh activity (25 in this study, increased met activity and lipid stores in gonad were observed at the ct+p site. in intertidal clams subject to large variations in temperature (i.e., eurythermal), oxygen radical formation was positively related with temperaturecontrolled respiration rates (state 3 and 4) and negatively correlated with mitochondrial coupling (abele et al., 2002). thus, wide temperature variations in clams from cold-adapted sites could lead to the formation of reactive oxygen species and lpo. however, no significant correlations were obtained with lpo in gonads and met activity at low and high temperatures, suggesting that antioxidant mechanisms such as superoxide dismutase, glutathione peroxidase and catalase were able to scavenge the oxygen radicals resulting from mitochondrial uncoupling. mitochondrial uncoupling from an increased temperature could be oc) nor with mdht. however, the influence from the distance from the shore should be considered since it was shown to have an important influence on oxidative stress in clams (gagné et al., 2009). 151 -4 -3 -2 -1 0 1 2 3 4 root 1 (gsi> lipid>mdht>lpo) -5 -4 -3 -2 -1 0 1 2 3 4 5 6 r oo t 2 (m e t t >l ip id e> c on d fa ct or >m d h t ) wt: 68 % ct+p: 62 % ct: 94 % wt+p: 70 % ct ct+p wt wt+p fig. 4 discriminate function analysis of clam morphological status, energy status and tissue damage. a discriminate function analysis revealed that the sites were correctly assigned at > 62 %. the site names shown in bold correspond to the centre of gravity of the discriminate function. the dotted arrows indicate departures from the reference sites to the corresponding polluted sites (within the cold-water or polluted-water sites. the result of changes in protein phosphorylation states in mitochondria of the clam mercenaria mercenaria (ulrich and marsh, 2009). the investigators identified three proteins that followed temperature-specific phosphorylation patterns and suggested that a suite of protein kinases and phosphatases regulate mitochondrial physiology in response to temperature increases. changes in protein phosphorylation in mitochondria show promise for use in investigating the interaction of temperature with pollution in the production of reactive oxygen species and oxidative stress. the data in the present study show that pollution could contribute to temperature sensitivity in mdh activity, which is related to lower lipid content in the gonad and dna strand breaks. this was consistent with discriminate function analysis of the biomarker data, which showed a shift in the centre of gravity of the discriminate functions of the polluted cold-water site toward the warmer sites (ase and asj). it is noteworthy that the principal biomarkers with the highest factorial weights were temperature sensitivity in mdh and met activities, gonad weight and lipid content. the physiological equivalency between low temperature dependence and cold adaptation observed here could perhaps favor the formation of dna strand breaks in the gonad, rendering the organisms more susceptible to genotoxic compounds. this is consistent with the observation that dna strand breaks were much higher at the polluted site in the cold-water sector (ct+p). however, this is difficult to ascertain at present since the genotoxicity could have been the result of relatively more contamination occurring at site ct+p relative to wt+p. in conclusion, clams from cold-water sites display less sensitivity to temperature but pollution increases temperature sensitivity in cytosolic mdh activity. temperaturedependent met at the polluted and cold-water site rises to levels identical to those of clams from the warmer sites, indicating that pollution alleviates cold-adaptation processes. although clams had lower gsis at the cold-water and polluted site, gonad lipid levels were higher at the polluted site than at the corresponding cold-water reference site indicating altered energy allocation towards gamete development. the impacts of pollution in clams from cold water site increased mdh activity at 25 oc and met temperature-dependence which would favor the formation of nadh in the cytosol for the production of lipids and steroidogenesis. the increase of mdh activity was related to a 2fold increase of the enzyme’s affinity constant towards malate at 25 oc while the affinity at 6 oc was somewhat lower. these changes were consistent with increased lipids stores at the ct-p site which were related with oxidative stress and genotoxicity. the impacts of pollution in clams from warm water sites seems to intervene rather at the mitochondria level (increased met activity) which was related to decreased lipid stores in the gonad and this was independent from changes in gsi or 152 gonad maturity in the clams. however, temperature-dependent mdh activity was higher at the polluted site. acknowledgements the authors are grateful for the technical assistance of s trépanier, b walker, s trottier and c boyko of the aquatic ecosystem protection research division, environment canada and to pascal rioux from uqar-ismer. this project was funded by environment canada under the st. lawrence action plan and by the nserc discovery grant attributed to j pellerin. references abele d, heise k, pörtner ho, puntarulo s. temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam mya arenaria. j. exp. biol. 205: 1831-1841, 2002. bester mj, potgieter hc, vermaak wjh. cholate and ph reduce interference by sds in the determination of dna with hoescht. anal. biochem. 223: 299-305, 1994. blaise c, gagné f, pellerin j. bivalve population status and biomarker responses in mya arenaria clams (saguenay fjord, québec, canada). fresenius environ. bull. 12: 956-960, 2003. de coen w, janssen cr. the missing biomarker link: relationships between effects on the cellular energy allocation biomarker of toxicantstressed daphnia magna and corresponding population characteristics. environ. toxicol. chem. 22: 1632-1641, 2003. dong y, somero gn. temperature adaptation of cytosolic malate dehydrogenases of limpets (genus lottia): differences in stability and function due to minor changes in sequence correlate with biogeographic and vertical distributions. j. exp. biol. 212: 169-177, 2009. fields pa, rudomin el, somero gn. temperature sensitivities of cytosolic malate dehydrogenases from native and invasive species of marine mussels (genus mytilus): sequence-function linkages and correlations with biogeographic distribution. j. exp. biol. 209: 656-667, 2006. gagné f, blaise c, pellerin j, fournier m, gagnon c, sherry j, talbot a. impacts of pollution in feral mya arenaria populations: the effects of clam bed distance from the shore. sci total environ. 407, 5844-5854, 2009. gagné f, blaise c, andré c, pellerin j. implication of site quality on mitochondrial electron transport activity and its interaction with temperature in feral mya arenaria clams from the saguenay fjord. environ. res. 103: 238246, 2007. gagné f, blaise c, pellerin j, fournier m. biomarker studies of the soft-shell clam (mya arenaria) in the saguenay fjord: research results (1997-2006). revue science de l’eau, 22 : 253-259, 2008a. gagné f, blaise c, pellerin j, fournier m, durand mj, talbot a. relationships between intertidal clam population and health status of the softshell clam mya arenaria in the st. lawrence estuary and saguenay fjord (québec, canada). environ. internat. 34: 30-42, 2008b. gagnon f, tremblay t, rouette j, cartier jf. chemical risks associated with consumption of shellfish harvested on the north shore of the st. lawrence river's lower estuary. environ. health perspect. 112: 883-888, 2004. king f, packard tt. respiration and the activity of the respiratory electron transport system in marine zooplankton. limnol. oceanogr. 20: 849-854, 1975. olive pl. dna precipitation assay: a rapid and simple method for detecting dna damage in mammalian cells. environ. mol. mutagen. 11: 487-495, 1988. pellerin j, fournier m, gauthier-clerc s, blaise c, florent g, amiard j-c, gagné, f. how is the health status of mya arenaria in the saguenay fjord? a statetement from a 14-year survey. rev. sci. de l'eau 22 : 271-289, 2009. smolders r, bervoet l, de coen w, blust r, 2004. cellular energy allocation in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization. environ. poll. 129: 99-112, 2004. stryer, l. pentose phosphate pathway and gluconeogeneis. in: biochemistry 4th edition, chap. 22, edited by wh freeman and company, new york, usa, pp 559-579, 1995. ulrich pn, marsh ag. thermal sensitivity of mitochondrial respiration efficiency and protein phosphorylation in the clam mercenaria mercenaria. mar. biotechnol. (ny) 11: 608618, 2009. wills ed. evaluation of lipid peroxidation in lipids and biological membranes. in: snell k, mullock b (eds), biochemical toxicology: a practical approach. irl press, washington, usa, pp 127-150, 1987. yang r, zhou q, jiang g. butyltin accumulation in the marine clam mya arenaria: an evaluation of its suitability for monitoring butyltin pollution. chemosphere 63: 1-8, 2006. 153 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22abele%20d%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22heise%20k%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22p%c3%b6rtner%20ho%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22puntarulo%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22dong%20y%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22somero%20gn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22rudomin%20el%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22somero%20gn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/pubmed/19698974?ordinalpos=1&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/19698974?ordinalpos=1&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/19698974?ordinalpos=1&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22gagnon%20f%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22tremblay%20t%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22rouette%20j%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22ulrich%20pn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22marsh%20ag%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'mar%20biotechnol%20(ny).'); arasite-host relationship: a lesson from a professional killer 41 isj 2: 41-53, 2005 issn 1824-307x review parasite-host relationship: a lesson from a professional killer mf brivio, m mastore, m pagani  dept of structural and functional biology (dbsf), university of insubria, varese, italy accepted april 18, 2005 abstract this paper outlines some of the main features of parasites immunoevasion/depression strategies. insects humoral and cellular responses are briefly discussed and correlated to the active and passive strategies of insects parasites, with particular emphasis on nematocomplexes used as biological insecticides. we have reviewed data on the interaction, at immunological level, of the parasite steinernema feltiae (rhabditidae) with the host model galleria mellonella (lepidoptera, pyralidae); the putative role of the parasite body-surface in active and passive evasion mechanisms has been evaluated and discussed. key words: insect; immunity; parasite; prrs-pamps; immunodepression; immunoevasion introduction invertebrates, particularly insects, act as vectors of important diseases such as malaria, chagas’ disease, sleeping sickness, filariases, dengue fever, yellow fever, etc. moreover many insect species, usually named insect pests, have a strong impact on the environment since they are phytophagous and harmful for both crops and urban areas. as a consequence of the widespread diffusion of insect species (reflecting the great success of this group) occupying almost all the habitats on earth, many insects live in environmental conditions infested by parasites and pathogens. insects can survive mainly because of the extreme efficacy of their immune system; any foreign parasite must then counteract these defenses to survive into its host. many parasites reproduce, develop and survive in invertebrate hosts that possess an immune system devoted to self-integrity and to discriminate self from not self. invertebrates lack finely tuned immunorecognition receptors but they possess instead useful patternrecognition molecules (prrs); these factors are able to interact specifically with a broad range of foreign corresponding author: maurizio f brivio dbsf, university of insubria, via jh dunant, 21100 varese, italy e-mail: maurizio.brivio@uninsubria.it  in memory of the late m pagani antigenic surface compounds (commonly named pamps and defined as pathogen-associated molecular patterns). pamps-prrs interaction is a key process among the discriminatory steps of innate immunity that usually precede the effectors-based mechanisms responsible for the elimination of not self (medzhitov, 2001; 2002; kanost et al., 2004 ). pamps consist of various compounds, including oligosaccharides, proteins, glycoproteins, lipids and distinct nucleic acid motifs that are unique to, and essential for, microorganism survival. an important feature of pamps is their strongly conserved structures, which are invariant among organisms of a given class (janeway and medzhitov, 2002; medzhitov and janeway, 2002). in insects, infections with different microorganisms or parasites selectively activate various defense reactions and effector-coding genes. the molecular basis of discrimination between different types of not self and the activation of immune responses is attributed to the specificity of prrs toward pamps, such as lps (lipopolysaccharide), pglc (peptidoglycan) or various glucans (medzhitov and janeway, 2002; dimopoulos, 2003). several proteins both in insect hemolymph or on hemocytes plasma membrane seem to function as prr, as they perform surveillance by binding to molecular patterns (hoffmann et al., 1999; hoffmann, 2003) (fig. 1). in galleria mellonella naïve larvae, two lpsbinding proteins, named lbp-1 (17.2 kd) and lbp-2 (26 kd), have been described by dunphy and halwani (1997); these humoral factors can be considered as prrs. these receptors bind the surface of bacteria and seem to act as detoxifiers, thus protecting 42 fig. 1 foreign stimula and receptors of insect immune system. perceiving of not-self in invertebrate seems to be modulated by the presence of pamps (pathogen-associated molecular patterns) which interact with free or membrane-bound receptors called prrs (pattern-recognizing receptors). these interactions lead to the activation of different cell-mediated and humoral effector immune processes. hemocytes from damage. both the lbps are specific for the lipid a portion of lps, in addition lbp-1 seem to act as an activator of galleria pro-phenoloxidase (propo) system. in the same year, wiesner and colleagues (1997) isolated and described a similar protein, called apolipophorin-iii (apolp-iii) of 17 kd of molecular mass; apolp-iii has been identified as an immune-stimulating molecule and is an exchangeable apolipoprotein, abundant in lepidopteran insects. the immune-stimulating capacity of apolp-iii came as a surprise, since this protein had been previously known to play a main role as lipid carrier in flying insects (niere et al., 1999). on insects hemocytes, cell receptors responsible of toll and imd pathways activation can be considered as the main prrs involved in cellular pamps sensing leading to antimicrobial peptides (amps) synthesis (de gregorio et al, 2002); their importance is not only restricted to the process of amps genes activation but, evolutionary, they also represent a further confirmation of the ancient origin of innate immunity, since they have been identified in several taxa from invertebrates to vertebrates (hoffmann and reichhart, 2002). after detection of not-self by pamps-prrs interactions, the recognition machinery can stimulate defensive humoral and cellular responses: among them, an important humoral defensive process is the melanization (humoral encapsulation) of foreign bodies. the melanization reaction, which is a common response to not self entry in invertebrates, especially arthropods, is due to the activity of an oxidoreductase called po. this enzyme, in the hemolymph, is a component of a complex system of proteases, proteases inhibitors (serpins) and humoral prrs, constituting the so-called propo-activating system (propo-as). propo-as is proposed to be a not self recognition system, because conversion of propo to the enzymatically active form can be induced by foreign pamps, particularly lipopolysaccharides and -1,3-glucans. propo-as, which is physiologically activated by invading micro-organisms or parasites, is a complex enzyme cascade in which the last active enzyme (phenoloxidase) can oxidize phenols into quinones, that in turn will convert into melanin autocatalytically (nappi et al., 2004). this system is a key element in the recognition of foreign bodies and in the production of opsonic factors; moreover, it is now considered to represent an integral component of the insect immune system (ashida, 1990; brivio et al., 1996; söderhall and cerenius, 1998; dimopoulous et al., 2001; cerenius and söderhall, 2004). several hemolymph prrs are involved in the propo system activation pathway: among them, -glucans-binding proteins (-gbp) and lps-binding proteins (lbps) seem to play a key role as receptors, triggering protease cascades that turn on prophenoloxidase enzymatic activity (jomori and natori, 1992; söderhall, 1999). besides, cell-mediated defenses are performed by cellular elements represented by several types of hemocytes that are commonly identified using morphological, histochemical and functional features (gupta, 1985, brehelin and zachary, 1986). hemocytes are immunoreactive cells playing a central role in maintaining host integrity; they are involved in various defense mechanisms such as phagocytosis, nodule formation, encapsulation, melanization, and synthesis of antimicrobial peptides (bulet et al., 1999; kanost et al., 2004). both cellular and humoral factors seem to be involved in the stimulation of cellular defenses, specifically in the early recognition and binding to pamps. several researches have demonstrated that humoral recognition receptors are also needed to stimulate hemocytes aggregation on the target surface during encapsulation processes (bulet et al., 1999, schmidt et al., 2001). the complex relationships between hosts and parasites can be clarified only considering the host 43 defense mechanisms and parasites evasion stategies altogether; the purpose of this paper is to outline some of the main parasite evasion strategies; in particular, the immunological interaction between entomopathogenic nematodes (steinernema feltiae) and the lepidopteran model insect g. mellonella will be described. the chosen examples are focused on insect hosts because of their economical and medical importance and considering their susceptibility to the symbiontic complex steinernema-xenorhabdus commercially available as biological insecticide. the convergence of parasitological and immunological studies also provides valuable knowledge in understanding the evolution of both parasitism and host immune system. parasite evasion strategies: general considerations parasites may successfully colonize their hosts by evading recognition, thus preventing immune defenses; circumvention of the host immune system can be achieved by molecular mimicry (or disguise) strategies or by colonization of young hosts, or host tissues, with low immunocompetence. alternatively (or concurrently) many parasites are able to depress either cell-mediated or humoral effectors mechanisms in a process usually called interference (lie and heyneman, 1976). as pointed out by götz and boman (1985), in order to survive, a parasite must reach an equilibrium with its host; a too efficient parasite may exterminate its hosts, whereas a too permissive parasite could have a low fitness and reproduction efficiency too low to guarantee its survival. in many cases, evolution and selection have finely-tuned host-parasite relationships leading to a long survival of parasitized invertebrate hosts; this process resulted in the production of vector species responsible of the transmission to human and animals of important diseases (richman and kafatos, 1995; ratcliffe and whitten, 2004). molecular mimicry is a strategy by which parasites became antigenically closely related to the host and thus avoid to evoke host immune responses. true molecular mimicry can be defined as the endogenous production of mimicking molecules that are usually exposed on the body-surface (or cell surface) of the parasite. despite the intuitive appeal of molecular mimicry as a mechanism of avoidance, few studies with any invertebrate parasites demonstrate a functionally protective effect of shared antigens (bayne et al., 1987; weston and kemp, 1993). however, antigens such as: -macroglobulin, immunoglobulin receptors, tropomyosin, mhc i and ii antigens, blood group glycolipids and oligosaccharides, related to both hosts and parasites have been identified and characterized (smithers et al., 1969; damian, 1991; vellupilai and harn, 1994). the molecular disguise, another form of mimicry, is described as the acquisition (sequestering) of molecular components from the host (ratcliffe et al., 1985; loker, 1994; strand and pech, 1995). in biomphalaria glabrata, several studies have demonstrated the ability of schistosoma mansoni to acquire host plasma proteins (e.g. hemoglobin, hemagglutinins, etc.) to form a coat of host factors (yoshino and bayne, 1983; dunn and yoshino, 1991; johnston and yoshino, 1996). more recently, kathirithamby and co-workers (2003) have described an alternative disguise mechanism carried out by a strepsiptera (stichotrema dallatorreanum) that is able to manipulate host (segestidea novaeguineae and s. d. defoliaria, orthoptera) epidermal tissues and wraps itself within it. this sort of bag acts thus as a camouflage for the endoparasite which is recognized as self by the host. the stage of development of the host is also essential in determining the outcome of parasitization (khafagi and hegazi, 2004). in general, early instars larvae of insects show a reduced immune activity often due to a lower hemocytes number or to a different array of cell populations (gardiner and strand, 2000; beetz et al., 2004). due to this, parasites penetrating young hosts can find a more favorable environment to overcome host defenses. finally, as a passive strategy, some parasites are able to colonize low-reactivity tissues of the host. a good example is represented by insect parasitoids that lay their eggs, with surgical precision, in nerve ganglia of their hosts into which the hemocytes do not normally circulate, so parasite embryos can develop unmolested within the host (götz and poinar, 1968; salt, 1971). as mentioned above, alternative active strategies are referred as interference; in this case, parasites show an aggressive suppression or alteration of the host immune system defenses. interference can be directed toward host humoral factors that are neutralized by the parasite or, as more commonly proposed, immunocompetent cells could be targeted instead (loker, 1994). another important aspect of host-parasite relationships is host humoral depression. with respect to this, propo system is one of the main target for many parasites or microorganisms; this is probably due to the need to neutralize its drastic and rapid effect when a host is in the presence of not self infections. many parasitic wasps inject maternal factors into the host’s hemocoel to suppress the host immune system and to ensure successful development of their progeny; asgari and colleagues (2003) isolated a 50 kd protein from cotesia rubecula (hymenoptera, braconidae) that blocked melanization in the hemolymph of its host pieris rapae (lepidoptera). the protein, named vn50, is a serine proteinase homolog containing an amino-terminal clip domain; recently the authors also demonstrated that vn50 is stable in the host hemolymph for at least 72 hrs after parasitization (zhang et al, 2004). using m. sexta as a model system, they found that vn50 efficiently downregulated propo system, by significantly reducing its proteolytic activation. this occurred without directly inhibiting and/or damaging the active phenoloxidase. according to the above description, we have obtained similar results in the tobacco budworm heliothis virescens (lepidoptera) larvae infected by toxoneuron nigriceps (hymenoptera). gregorio and ratcliffe (1991) demonstrated that the presence of tripanosoma rangeli in the hemolymph of two insects (rhodnius prolixus and 44 triatoma infestans) significantly reduced the level of propo activation. in their paper, the authors suggested that the susceptibility to tripanosoma infection of both the hosts is strongly dependent on the propo activation intensity. finally, in a recent study, we observed a drastic reduction of po activity in the hemolymph of g. mellonella induced by heat-killed entomoparasite nematodes (s. feltiae) or purified parasites cuticles (brivio et al., 2002). considering that cellular encapsulation is one of the most effective processes directed toward large parasites, it is reasonable to discuss interference mechanisms acting against host immunocompetent cells. with regard to this, parasites might reduce the recognition capability of hemocytes, by damaging surface prrs, by down-regulation of their synthesis, or simply rejecting active cells by means of noxious secretions or refractive body-surface; alternatively, direct damage of host hemocytes could be achieved. a great number of evidences suggest that parasitoid wasps inject factors suppressing host immune system; well-described suppressive factors are venom glands secretions (in braconid wasps), polydna virus and co-injected teratocytes cells (vinson, 1990; summers and dib-hajj, 1995). infection with wasps is known to affect host hemocytes (particularly plasmatocytes) ability to attach to substrates, to aggregate and to spread correctly. moreover, virus-containing calyx fluid from campoletis sonorensis induces a significant reduction in the number of circulating hemocytes, when injected in h. virescens larvae (davies and vinson, 1988). effects on host cells have been broadly described in insects infected (or in in vitro assays) with nematode symbiontic bacteria. the nematocomplex steinernema carpocapsae/xenorhabdus nematophilus seems to be responsible of almost two effects on lepidoptera hemocytes; ribeiro et al. (1999) demonstrated unsticking and cytotoxic effects of two factors released by nematocomplexes in in vitro experiments. however, from this paper is not clear if bacteria themselves, nematodes, or both, produced the above factors. an interesting aspect of the immunodepressive action of xenorhabdus was described by park et al. (2004) in m. sexta; inhibitor(s) released by live bacteria seems to block eicosanoid biosynthesis (that are crucial mediators of insects cellular defense reactions) by reducing phospholipase a2 intracellular activity. finally, processes such as the release of toxic factors from hemocytes, phagocytosis, encapsulation and nodulation are also probably to be targets of parasite-derived interference factors. however, it is reasonable to expect different parasites to adopt different strategies of immunoevasion and that any particular parasite species would employ a variety of evasive tactics. a short profile of the killer (entomopathogen nematocomplex) as described by nathan cobb (1915), nematodes are one of the most abundant type of animals on earth. nematodes, thanks to their small size, to the resistant cuticle and to the ability to adapt to severe environmental changes, have colonized a wide range of habitats including vertebrate and invertebrate bodies. nematodes may be free-living or parasitic; the latter are usually considered pests because they cause important diseases in animals, humans, and for their economic impact on many agricultural products. a small but significant number of parasitic nematodes, called entomopathogenic, are of considerable interest because they possess various features as biological control agents for pest insects (gaugler and kaya, 1990; georgis and manweiler, 1994; poinar, 1998). entomopathogenic nematodes must meet some criteria to be considered good candidates for an overall biological control: they should neutralize agriculture insect pests and, possibly, insect vectors responsible of human and animal diseases; practically, they should be able to kill, sterilize, or hamper the development of their insect targets. insect-parasitic nematodes that possess optimal features as bioinsecticides belong to the families steinernematidae and heterorhabditidae (nematoda, rhabditida). steinernematidae and heterorhabditidae are not closely related phylogenetically but, through convergent evolution, they share similar life histories (poinar, 1993); the main difference between them is the reproductive strategy (steinernematidae are gonochoric, heterorhabditidae are hermaphroditic). these families differ from other rhabditids by having a species-specific mutualistic relationship with bacteria of the genus xenorhabdus (enterobacteriaceae) (poinar, 1979; kaya and gaugler, 1993). x. nematophilus is associated with steinernematidae and photorhabdus luminescens with heterorhabditidae in a specie-specific manner (forst and nealson, 1996; forst et al., 1997; forst and clarke, 2001; silva et al., 2002). the symbiontic bacteria contribute to the mutualistic relationship actively, by killing insect host, by establishing and maintaining suitable conditions for nematode reproduction and by providing nutrients and microbial substances that inhibit growth of a wide range of microorganisms. at the same time the nematode acts as a vector for the symbiontic bacterium. the symbiosis is essential for the efficiency of the biocontrol and it enables nematodes to exploit a diverse array of insect hosts (dunphy and thurston, 1990). the basic life cycle of most entomopathogenic nematodes consists of several stages: an egg stage, four juvenile stages (l1, l2, l3, l4), and a complex adult stage that comprises l5 (early adult stage) and late adult (fig. 2). in general, nematodes moult four times during each life cycle with a moult occurring at the end of each larval stage. therefore, moults separate the first and second larval stages (l1 and l2), the second and third larval stages (l2 and l3), the third and fourth larval stages (l3 and l4) and also the fourth larval stages and immature adults (l4 and l5). the l5 grows up to the size limit of its new cuticle. the third juvenile stage (ij3) of nematodes is known as the “infective juvenile” or “dauer” stage and is the only free-living stage (womersley, 1993). the ij3 is capable to survive in the soil for extended periods until it is able to find a susceptible host; its function is to locate, attack, and infect an insect host (poinar, 1990; akhurst and dunphy, 1993). 45 fig. 2 a (l1) develops inside the egg, hatches (h), grows rapidly, then moults (m1) to l2. the second stage larva also shows a rapid growth followed by a second moult (m2) to third stage larva (l3), the infective juvenile stage 3 for many nematode species (also named ij3). this (l3) grows, then moults (m3) inside the host to a l4 larvae. the final larval stage grows and undertakes a final moult (m4) to an immature adult (l5). host infection consists of various steps (fig. 3); (a) the ij3 parasites find hosts by chemotaxis towards chemical concentration gradients of carbon dioxide, and/or host excretory products. infective juvenile stage enters the host through natural body openings (mouth, anus, spiracles), it reaches the hemocoel of the host, and later on (b) it releases bacterial spores by defecation or regurgitation. symbiontic bacteria live in a monoxenically area or in differentiated vesicles of the anterior part of the infective juvenile intestine modified as a bacterial chamber. after release, bacteria quickly multiply in the hemolymph (c); they are mainly responsible for the host mortality because they produce and release exoand endotoxins to which the insect succumbs by septicemia (d) within 24-48 h of infection; furthermore, bacteria secrete antibiotics that prevent multiplication of the other microflora (khandelwal and banerjee-bhatnagar, 2003). bacterial cells also express and release proteases and lipases that degrade insect host tissues to be utilized by the parasite as a food source. as reported by several authors (wouts, 1984, tanada and kaya, 1993), the parasite itself produces toxins that are lethal to the host, even without its associated bacteria but, in this case, it is unable to reproduce; moreover, without the nematode, bacteria cannot reach and invade the host hemocoel. after mating, the females lay the eggs that hatch as first-stage juveniles that moult successively to second, third and fourth-stage juveniles and then to males and females of the second generation (e). the adults mate and the eggs produced by these secondgeneration females hatch as first-stage juveniles that moult to the second stage. the adult nematodes produce hundreds of thousands of new juveniles. the late second stage juvenile ceases feeding, incorporates a small fresh group of bacteria in the bacterial chamber (f), and moults to the infective juvenile stage (ij3). when the host has been consumed, the infective juveniles (g) emerge from the exoskeleton of the host, move into the soil and begin the search for a new host (h). in nature, under ideal conditions, steinernematidae and heterorhabditidae emerge 6-11 and 12-14 days after the initial infection respectively (kaya and koppenhöfer, 1999). since entomopathogenic nematodes represent an alternative to chemicals for insect pest control, it is fundamental to understand the basis of the infectivity of nemato-bacterial complexes and the interaction with the insect host immune systems. although the immune depressive and lethal effects induced by bacteria (long-term infection phase) are well known (ffrenchconstant et al., 2000), the short-term infection phase, particularly the role of the parasite itself, are not clearly understood (brivio et al., 2002). gun and bullets: a lecture from the killer the efficacy of the nematode s. feltiae in killing its hosts is mainly attributable to the severe effects of its symbiontic bacteria (x. nematophilus) that, by means of multiple factors, cause the death of the host in the later phase of infection. symbiontic bacteria could be viewed as bullets of a gun (the parasite): as it is well known, after a murder the effect and characteristics of the bullets found on the crime scene are easily assessed; but detectives are in trouble when trying to investigate on the missing gun. given that, the bacterial bullets have been well studied; a lot of papers provide in depth descriptions of the immunodepressive effects of bacteria-released factors. host physiological disorder caused by the release and proliferation of the symbiontic bacteria have been investigated (chattopadhyay et al., 2004); particularly, these microorganisms seem to be able to arrange the environment (host’s body) in such a manner to allow the parasite to survive and spawn unmolested. x. nematophilus, upon release into the hemolymph of g. mellonella host, adhere to the surface of hemocytes, proliferate and damage the cells that became vacuolated, unable to adhere to surfaces and finally show positivity to trypan blue (dunphy and webster, 1984, 1986). at the same time, xenorhabdus synthesizes and releases antibiotic compounds within the insect hemocoel that suppress competing microorganisms; in this way they acquire the optimal condition to proliferate, allowing the parasites to complete its development (gaugler and kaya, 1990). a growing number of xenorhabdus-produced factors have been described; recently a cytotoxic pilin subunit (17 kd) of x. nematophilus has been isolated: the protein is expressed on the bacterial surface and also secreted in the extracellular medium; it binds to the surface of larval hemocytes and shows cytotoxic properties against immunocompetent cells of helicoverpa armigera, finally causing agglutination of the cells (khandelwal et al., 2004). ribeiro and colleagues (2003) reported the purification of a cytotoxin of 10 .7 kd of molecular weight from x. nematophilus, nam ed 46 fig. 3 life cycle of entomopathogenic nematodes. alpha-xenorhabdolysin (alphax) peptide; the plasma membrane of spodoptera littoralis hemocytes seems to be the main target of the peptide. alphax peptide induces an increase of monovalent cations permeability that is sensitive to potassium channel blockers, even on mammal macrophages or erythrocytes. as a consequence of alphax binding to the plasma membrane, several events occur intracellularly, such as selective vacuolation of the endoplasmic reticulum, cell swelling and cell death. in insects, bacterial infections usually evoke the activation of the toll/imd pathways that culminate in the synthesis of an array of antibacterial peptides; a well-known property of the parasite symbionts is to interfere with antibacterial responses. xenorhabdus affects antimicrobial activity of lepidoptera by means of two distinct released proteases; caldas and colleagues (2002) demonstrated that one of the two above mentioned proteases (protease ii) destroyed antibacterial activity in the hemolymph of insect larvae (g. mellonella and p. unipuncta) challenged with inoculated bacteria; particularly, the bacteriolytic activity of the inducible antibacterial peptide cecropin a was drastically reduced. furthermore, protease ii did not show toxicity to host hemocytes. finally, symbionts of entomopathogenic nematodes showed inhibitory effects on host propo activating system (yokoo et al., 1992; dunphy et al., 1998). the presence of a large number of studies on bacterial symbionts are mainly due to the economical interests that have led researchers to focus on bacteria-derived patentable molecules valuable in integrated pests management. on the other hand, in order to study the role of the parasite itself, it would be necessary to work with axenic nematodes; unfortunately, it is difficult to obtain nematodes completely deprived of their symbionts and, in this case, parasites might not be in a physiological condition, increasing the risk of obtaining artifacts. it is thus probably that experimental troubles and economical interests could be responsible for the scarcity of literature available on this topic. with the aim of clarifying the involvement of the parasite itself in the relationships with the host, at first we have carried out our experiments in a short period (0-30 min) following s. feltiae infection, when bacteria are not yet released into the g. mellonella hemolymph; besides, we carried out many studies utilizing the isolated body surface (i.e. cuticle and epicuticle) of the parasite. cuticles of steinernema have been obtained without bacterial contamination to a good level of purity (fig. 4) by developing a technique based on sonication, washes and sterilization of parasites. parasite immunoevasion strategies often involve the parasite body surface, which seems to play a key role in the interaction with the host environment (blaxter et al., 1992). nematodes moult several times throughout their developmental cycle, each time changing their body surface with the formation of a new cuticle (cox et al., 1981a, 1981b); although a common model of nematode cuticle has been proposed (maizels et al., 1993), single species may have significant differences in molecular organization and surface properties. this is particularly true for parasitic species (i.e. s. feltiae) that must interact with an unfavorable host environment. furthermore, parasitic nematodes may easily elaborate the composition and organization of the epicuticular external layer, depending upon the particular environment of each species (maizels et al., 1993). together with other surface and secreted molecules (politz and philipp, 1992), the cuticle of parasitic nematodes seems to be involved in immunoevasion and suppression of host’s defenses, as suggested also by akhurst and dunphy (1993); thus, it is likely that nematode body surface plays a crucial role in parasite success. the hypothesis of a key role of the body-surface of parasites was proposed early by vinson (1977); vinson suggested that in absence of active suppression mechanisms the prevention from encapsulation could be achieved by means of: a) the acquisition of a coat composed of host proteins (molecular disguise); b) the possession of heterophilic antigens; c) the presence of a non reactive bodysurface, or molecular mimicry. 47 in 1987 dunphy and webster presented preliminary evidences in favor of a possible role for the epicuticle layer of s. feltiae in cellular immunodepression. the paper pointed out interactions of the body surface of the entomopathogen with g. mellonella hemocytes and suggested its involvement in avoiding cellular encapsulation. the authors described a partial characterization of cuticle sugars by means of lectin specificity but, more interestingly, they assessed the role of the lipidic moiety of the epicuticle of s. feltiae. a simple assay based on lipase treatments determined that surface lipids played a role in escaping from hemocytes recognition; on this basis, they supposed that modifications of the lipidic surface resulted in a changed molecular architecture of the epicuticle, thus exposing discriminable antigens. primarily inspired by these suggestions, we have focused the research on the role of s. feltiae cuticle, with the aim to exclude any contribution from symbiontic bacteria and/or active secretions of the parasite. our preliminary observations (brivio et al., 2002) showed host propo system inhibition in g. mellonella larvae infected with heat-killed nematocomplexes; although these results suggested that factors released from the parasite were not responsible of propo inhibition, they did not completely exclude the involvement of bacteria. however, these data supported the attractive hypothesis that the parasite, after entry into the host hemocoel, exploits its body-surface to immunoevade and/or immunodepress host defenses. a strong confirmation of the above hypothesis came from the assays carried out with isolated cuticles. the suppression of the hemolymph phenoloxidase activity, observed after either in vivo cuticle injection or in vitro co-incubation (cuticles plus cell-free hemolymph), was comparable to that obtained in the experiments performed with killed whole parasites. the integrity of the molecular architecture of the cuticle seems to be essential to retain its immunodepressive properties, since chemical alterations of the structure result in a marked loss of inhibition of the host propo system. moreover, confirming dunphy’s suggestions, the main effect was observable after damage or removal of the lipidic layer obtained with lipase and methanol-chloroform treatments (fig. 5). these data confirmed the first assumption of a key role of the lipids in the host-parasite interaction, although no information concerning the mechanisms by which cuticular lipids may affect the activation of the propo system was provided (brivio et al., 2004). the process by means of which s. feltiae cuticle lipids showed inhibitory effects on the host propo system was further investigated hypothesizing that these molecules might interact with hemolymph factor involved in the activation pathway of host phenoloxidase. a set of experiments based on in vitro interaction of purified parasite cuticle with cell-free host hemolymph (fig. 6), demonstrated a specific binding property of the cuticle: particularly, the lipidic moiety interacts and sequesters three hemolymph proteins (17, 26, 35 kd), named hips (hostinteracting proteins), possibly involved in the propo activation cascade. concerning the identity and functions of hips, on the basis of preliminary characterization based on molecular mass and according to the literature (dettloff et al., 2001), we supposed that, firstly, the 17 kd hip could be identified as the insect lipid-carrier apolipophorin iii. besides reports on its well-known functions in the lipid metabolism (ryan and van der horst, 2000), exhaustive studies have been carried out on the involvement of this protein in immunological responses (wiesner et al., 1997; halwani and dunphy, 1999; zakarian et al., 2002). hip26 has a molecular weight comparable to that of lbp-2 (a lipopolysaccharide-binding protein) described by dunphy and halwani (1997) in g. mellonella. this protein shows a specific affinity for endotoxin lipid-a and seems to be involved both in hemocytes activation and propo system regulation. the third factor of 35 kd has a molecular weight similar to the protease-like molecule scolexin. insect scolexin is well characterized at molecular level, although its biological function is not yet understood (finnerty et al., 1999). the significant quantitative reduction of hips induced by the parasite is responsible for the blockage of the activation pathway of the propo system; when these components, eluted from the parasite body surface, are added during in vitro assays, the normal hemolymph phenoloxidase activity of the host is restored. furthermore, a function of the hips seems to be related to the activation of hemolymph serine proteases, given that their properties of reactivation (fig. 7) are lost if they are assayed in the presence of protease inhibitors. the precise reactivation mechanism of the propo system mediated by hips still has to be determined; to this goal we carried out far western blot experiments by which these proteins showed lps-binding properties (fig. 8). this affinity has been confirmed by their ability to bind to the wall of gram(-) bacteria (fig. 6, panel c, lane b). in addition, cuticle lipids seem to cross-react with anti-lps antibodies suggesting a structural correlation between the parasite lipids and the bacterial lps lipida domain (fig. 9). an intriguing hypothesis could be that surface lipids may act as pathogen-associated molecular patterns-like (pamps-like) but, in this case, their interaction with the host hips (comparable to prrs) would result in the removal of the latter from the insect hemolymph, thus preventing the activation of hemolymph serine proteases required for propo activation and melanotic encapsulation. the interference of the parasite with cell-mediated defenses of the host was investigated by ribeiro and co-workers (1999). the observed hemocyte damages were suggested to be related to factors released in the medium from nematocomplexes; these compounds (not identified) showed unsticking and cytotoxic effects on g. mellonella and m. unipunctata immunocompetent cells. the data presented seem to indicate an active interference process carried up by s. carpocapsae. moreover, the interaction between the parasite body-surface molecules and host hemolymph components could result in a coating effect of the nematode. this coat, composed of host self-proteins, could induce molecular disguise processes. to ascertain the above assumption we have performed various assays clearly showing that galleria hemocytes are unable to recognize the parasites as 48 fig. 4 a: longitudinal section of the parasite body (at tem level) showing the cuticle and epicuticular layer of s. feltiae; in b and c purified cuticular fragments (phase contrast microscopy). fig. 5 effects of the presence of s. feltiae whole individuals or isolated cuticles on propo activity of g. mellonella larvae. in the upper area is shown relative phenoloxidase activity as modulated by: l-n, living parasites; d-n, heat-killed parasites; cut, isolated cuticles; cut-lip, lipase-treated cuticles; cut-mc, methanol-chloroform-treated cuticles; extr-mc, lipidic extracts from cuticles. visualized below is the melanin production in microwells from the above assays. 49 fig. 6 to identify putative specific parasite-bound hips, high-salt elutions from parasite body surface (previously incubated with host hemolymph) were carried out. the supernatants of the elution were analyzed by sds–page (panel b). the parasites were thoroughly washed to eliminate all non-specific hemolymph proteins before elution. the electrophoretic pattern (panel b, lane “elu”) revealed the presence of four main bands with molecular masses of about 80, 35, 26, and 17 kd, respectively. when the assay was carried out with methanol–chloroform or lipasetreated parasites, the lower bands (35, 26, and 17 kd) disappeared (panel b, lanes “mc” and “lip”) and only a reduced amount of the 80 kd band was observed by sds–page. panel c: further interaction assays were carried out with hemolymph (<50 kd) incubated with gram(-) bacteria (e. cloacae). enterobacter was incubated with host hemolymph and 1m nacl eluted proteins were analyzed by sds–page (lane b). three main bands with molecular masses corresponding to the described hips were observed . as a control, high salt eluted bacteria (without prior hemolymph incubation) were analyzed (lane c). fig. 7 reactivation properties of hips directed against parasite-inhibited host propo system are shown (hips). hips activating properties were also assayed in the presence of protease inhibitors: under these conditions host propo system activity was not restored (hips-pi). fig. 8 far-western assay for lps-binding activity of hips. blotted hips were renatured in situ onto nitrocellulose sheets and incubated with lipopolysaccharides. anti-lps was used as primary antibody; the lps-binding to hips was revealed by anti-igg peroxidase-conjugated antibody and luminol. all hips (17, 26, and 35 kd) were positive to the assay (lane b); as a control, the assay was performed without hips in situ renaturation (lane a), without lps incubation (lane c1) or omitting the secondary antibody (lane c2). 50 fig. 9 cross-reactivity of parasite cuticle compounds with anti-lps antibodies. parasites were immunostained with the primary antibody and then with anti-igg tritc-conjugated; as shown, a strong signal is localized at the parasite surface (panel b). panel a shows light micrographs of parasite body section (arrowheads indicate cuticle and epicuticle zone). anti-lps western blot, carried out with samples from methanol–chloroform cuticle extracts (lane nem) shows a positive smeared band observable at the bottom of the gel. the assay was carried out also with bacterial lps, as a positive control (lane c). fig. 10 mimetic properties of s. feltiae. parasites (n1, n2, n3) or cuticles (c1, c2, c3) were incubated with cultured hemocytes to assay for escaping from host cell-mediated encapsulation. n1 and c1 micrographs show the lack of encapsulation of whole parasite and cuticle fragments. in n3 and c3 is seen the formation of capsules on lipase-treated parasites and cuticles. n2 and c2 show the healthy state of hemocytes that, even in the presence of s. feltiae or cuticles, are able to encapsulate beads. not self. the co-incubation experiments with parasites (or isolated cuticles) and abiotic materials provided further evidence that the host cells were healthy and capable of encapsulation (fig. 10). it can thus be supposed that this immunoevasion mechanism may be attributable to achievement of mimetic properties of the body-surface of steinernema rather than to the cells having been damaged by the parasite. finally, as observed in propo system inhibition assays carried out with pre-treated cuticle, upon removal of lipidic compounds parasites become unable to evade cellular encapsulation of the host. based on the data obtained from the relationships between s. feltiae and g. mellonella we propose a possible schematic model (fig. 11) describing some features of host-parasite (galleria-steinernema) immunological interaction. in our lab we have recently undertaken a study to 51 fig. 11 a scheme delineating some parasite immunoevasion/depression processes related to hemolymph prrs removal. ascertain the interference of the parasite with the inducible antibacterial response of g. mellonella; this topic is particularly relevant in the studied model since these parasites, besides escaping host immune defenses, seem to be able to down-regulate amps genes reducing the synthesis of molecules potentially harmful for their symbiontic bacteria (manuscript in preparation). concluding remarks our society increasingly demands alternatives to chemicals for managing insect pests and insect vectors of diseases; even though biological control by means of bioinsecticides holds great promises, no more than 1.5 % of commercial pesticides are represented by biologicals. researches directed towards a better knowledge of nematocomplexesinsects relationships could provide a valuable starting point for the improvement of integrated pest management techniques, with the aim of drastically reducing the use of chemicals. moreover, from studies of model insects immune system is clearly emerging a fascinating picture of the evolution of immune responses common to both invertebrates and vertebrates. acknowledgments we are grateful to magda de eguileor for tem micrograph, to michela curradi for the critical reading of the manuscript and to alberto vianelli for language checking and suggestions. references agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell 5: 441-50, 2003. akhurst, rj, dunphy gb. tripartite interactions between symbiotically associated entomopathogenic bacteria, nematodes, and their insect hosts. in: beckage ne, thompson sn, federici ba. (eds) parasites and pathogens of insects, vol. 2: pathogens, academic press, new york, ny, pp. 1-23, 1993. asgari s, zhang g, zareie r, schmidt o. a serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph. insect biochem. mol. biol. 33: 1017-24, 2003. ashida m. the prophenoloxidase cascade in insect immunity. res. immunol. 90: 908-910, 1990. bayne cj, boswell ca, yui ma. w idespread antigenic crossreactivity between plasma proteins of a gastropod and its trematode parasite. dev. comp. immunol. 11: 321329, 1987. beetz s, brinkmann m, trenczek t. differences between larval and pupal hemocytes of the tobacco hornworm, manduca sexta, determined by monoclonal antibodies and density centrifugation. j. insect physiol. 50: 805819, 2004. blaxter ml, page ap, rudin w , maizels rm. nematode surface coats: actively evading immunity. parasitol. today 8: 243–47, 1992. brehelin m, zachary d. immunity in invertebrates, springer verlag, berlin, 1986. brivio mf, mastore m, moro m. the role of steinernema feltiae body-surface lipids in host–parasite immunological interactions. molec. biochem. parasitol.135: 111-121, 2004. brivio mf, mazzei c, scarì g. propo system of allogamus auricollis (insecta): effects of various compounds on phenoloxidase activity. comp. biochem. physiol. 113b: 281-287, 1996. brivio mf, pagani m, restelli s. immune suppression of galleria mellonella (insecta, lepidoptera) humoral defenses induced by steinernema feltiae (nematoda, rhabditida): involvement of the parasite cuticle. exp. parasitol. 101: 149-156, 2002. bulet p, hetru c, dimarcq jl, hoffmann d. antimicrobial peptides in insects; structure and function. dev. comp. immunol. 23: 329-44, 1999. caldas c, cherqui a, pereira a, simoes n. purification and characterization of an extracellular protease from xenorhabdus nematophila involved in insect immunosuppression. appl. env. microbiol. 68: 12971304, 2002. 52 cerenius l, söderhall k. the prophenoloxidase-activating system in invertebrates. immunol. rev. 198: 116-126, 2004. chattopadhyay a, bhatnagar nb, and bhatnagar r. bacterial insecticidal toxins. crit. rev. microbiol. 30: 33–54, 2004. cobb na. nematodes and their relationships. year book dept. agric. (1914) w ashington, dc: dept. agric., 45790, 1915. cox gn, kusch m, edgar rs. cuticle of caenorhabditis elegans: its isolation and partial characterization. j. cell biol. 90: 7-17, 1981a. cox gn, staprans s, and edgar rs. the cuticle of caenorhabditis elegans. ii. stage-specific changes in ultrastructure and protein composition during postembryonic development. dev. biol. 86: 456-470, 1981b. damian rt. tropomyosin and molecular mimicry. parasitol. today 7: 96, 1991 davies dh, vinson sb. interference with function of plasmatocytes of heliotis virescens in vivo by calyx fluid of the parasitoid campoletis sonorensis. cell. tissue res. 251: 467-475, 1988. de gregorio e, spellman pt, tzou p, rubin gm, lemaitre b. the toll and imd pathways are the major regulators of the immune response in drosophila. embo j. 21: 25682579, 2002. dettloff m, w ittwer d, w eise c, w iesner a. lipophorin of lower density is formed during immune responses in the lepidopteran insect galleria mellonella. cell tissue res. 306: 449–58, 2001. dimopoulos g. insect immunity and its implication in mosquito-malaria interaction. cell. microbiol. 5: 3-14, 2003. dimopoulous g, müller hm, levashina ea., kafatos fc. innate immune defense against malaria infection in the mosquito. curr. opin. immunol.13: 79-88, 2001. dunn ts, yoshino tp. tegumental surface modulation in schistosoma mansoni primary sporocysts in response to ligand binding. parasite immunol. 13: 121-35, 1991. dunphy g and halwani a. haemolymph proteins of larvae of galleria mellonella detoxify endotoxins of the insect pathogenic bacteria xenorhabdus nematophilus (enterobacteriaceae). j. insect physiol. 43: 1023–1029, 1997. dunphy gb, miyamoto cm, meighen ea. generation and properties of a luminescent insect pathogen xenorhabdus nematophilus (enterobacteriaceae). j. gen. appl. microbiol. 44: 259-268, 1998. dunphy g, thurston g. insect immunity. in: gaugler r, kaya hk (eds) entomopathogenic nematodes in biological control, crc press, boca raton, fl, pp. 301-326, 1990. dunphy g, w ebster j. partially characterized components of the epicuticle of dauer juvenile steinernema feltiae and their influence on the hemocyte activity in galleria mellonella. j. parasitol. 73: 584–588, 1987. dunphy gb, w ebster jm. influence of the mexican strain of steinernema feltiae and its associated bacterium xenorhabdus nematophilus on galleria mellonella. j. parasitol. 72: 130-135, 1986. dunphy gb, w ebster jm. interaction of xenorhabdus nematophilus subsp. nematophilus with the hemolymph of galleria mellonella. j. insect physiol. 30: 883-889, 1984. ffrench-constant rh, w aterfield n, burland v, perna nt, daborn pj, bowen d and blattner fr. a genomic sample sequence of the entomopathogenic bacterium photorhabdus luminescens w 14: potential implications for virulence. appl. environ. microbiol. 66: 3310-3329, 2000. finnerty cm, karplus pa, granados r. the insect immune protein scolexin is a novel serine proteinase homolog. protein sci. 8: 242–248, 1999. forst s, clarke d. bacteria-nematode symbiosis. in: gaugler r (ed) entomopathogenic nematology, london, cab international, pp. 57-77, 2001. forst s, nealson k. molecular biology of the symbioticpathogenic bacteria xenorhabdus spp. & photorhabdus spp. microbiol. rev. 60: 21-43, 1996. forst s, dowds b, boemare n and stackebrandt e. xenorhabdus and photorhabdus spp: bugs that kill bugs. ann. rev. microbiol. 51: 47-72, 1997. gardiner em, strand mr. hematopoiesis in larval pseudoplusia includens and spodoptera frugiperda. arch. insect biochem. physiol. 43: 147-164, 2000. gaugler r, kaya hk. entomopathogenic nematodes in biological control. crc press, boca raton, fl, 1990. georgis r, manweiler sa. entomopathogenic nematodes: a developing biological control technology. agric. zool. rev. 6: 63-94,1994. götz p, boman hg. insect immunity. in: kerkut ga, gilbert li (eds) comprehensive insect physiology biochemistry and pharmacology, pergamon press, oxford, pp. 453485, 1985. gotz p, poinar go jr. development of a mermithidae (nematoda) in the nervous system of insects. z. parasitenkd. 31: 10, 1968. gregorio ea, ratcliffe na. the prophenoloxidase system and in vitro interaction of trypanosoma rangeli with rhodnius prolixus and triatoma infestans haemolymph. parasite immunol. 13: 551-564, 1991. gupta ap, kerkut ga, gilbert li. comprehensive insect physiology, biochemistry, and pharmacology vol. 3, pergamon press, oxford, 1985. halwani ae., dunphy gb. apolipophorin-iii in galleria mellonella potentiates hemolymph lytic activity. dev. comp. immunol. 23: 563–570, 1999. hoffmann ja. the immune response of drosophila. nature. 6, 426(6962): 33-38, 2003. hoffmann, ja, reichhart, jm. drosophila innate immunity: an evolutionary perspective. nat. immunol. 3: 121-126, 2002. hoffmann, ja, kafatos fc, janeway ca, ezekowitz rab. phlylogenetic perspectives in innate immunity. science 284: 1313-1318, 1999. janeway ca, medzhitov r. innate immune recognition. annu. rev. immunol. 20: 197-216, 2002. johnston la, yoshino tp. analysis of lectin and snail plasma-binding glycopeptides associated with the tegumental surface of the primary sporocysts of schistosoma mansoni. parasitology 112: 469-79, 1996. jomori t, natori s. function of the lipopolysaccharide-binding protein of periplaneta americana as an opsonin. febs lett. 296: 283–286, 1992. kanost mr, jiang h, qiang yx. innate immune responses of a lepidopteran insect, manduca sexta. immunol. rev. 198: 97-105, 2004. kathirithamby j, ross ld and johnston js. masquerading as self ? endoparasitic strepsiptera (insecta) enclose themselves in host-derived epidermal bag. proc. natl. acad. sci. usa 100: 7655-7659, 2003. kaya hk, koppenhöfer am. biology and ecology of insecticidal nematodes. proc. w orkshop ‘optimal use of insecticidal nematodes in pest management’. new brunswick, nj, 1-8, 1999. kaya hk, gaugler r. entomopathogenic nematodes. ann. rev. entomol. 38: 181-206, 1993. khafagi w e, hegazi em. is the host or the parasitoid in control? effects of host age and temperature on pseudoparasitization by microplitis rufiventris in spodoptera littoralis. j. insect physiol. 50: 577-584, 2004. khandelwal p, banerjee-bhatnagar n. insecticidal activity associated with the outer membrane vesicles of xenorhabdus nematophilus. appl. environ. microbiol. 69: 2032-2037, 2003. khandelwal p, bhatnagar r, choudhur y d, b anerjee n. http://scienceserver.cilea.it/cgi-bin/sciserv.pl?collection=journals&journal=00221910 53 characterization of a cytotoxic pilin subunit of xenorhabdus nematophila. biochem. biophys. res. commun. 314: 943-949, 2004. lie kj and heyneman d. studies on resistance in snails. escape of echinostoma lindoense sporocistis from encapsulation in the snail heart and subsequent loss of the host’s abilty to resist infection by the same parasite. j. parasitol. 62:298-302, 1976. loker es. on being a parasite in an invertebrate host: a short survival course. j. parasitol. 80: 728-747, 1994. maizels rm, blaxter m., selkirk me. forms and functions of nematode surfaces. exp. parasitol. 77: 380-384, 1993. mcguinness dh, dehal pk, pleass rj. pattern recognition molecules and innate immunity to parasites. trends parasitol. 19: 312-319, 2003. medzhitov r, janeway ca. decoding the patterns of self and nonself by the innate immune system. science, 296: 298-300, 2002. medzhitov r. toll-like receptors and innate immunity. nature rev. immunol. 1: 135-45, 2001. nappi aj, kohler l, mastore m. signaling pathways implicated in the cellular innate immune responses of drosophila. inv. surv. j. 1: 5-33, 2004. niere m, meißlitzer c, dettloff m, w eise c, ziegler m, wiesner a. insect immune activation by recombinant galleria mellonella apolipophorin iii. biochem. biophys. acta 1433: 16-26, 1999. park y, kim y, tunaz h, stanley dw . an entomopathogenic bacterium, xenorhabdus nematophila, inhibits hemocytic phospholipase a2 (pla2) in tobacco hornworms manduca sexta. j. invert. pathol. 86: 65-71, 2004. poinar, g. nematode biopesticides fundam. appl. nematol. 21: 733-737, 1998. poinar, go. biology and taxonomy of steinernematidae and heterorhabditidae. in: gaugler r, kaya hk (eds) entomopathogenic nematodes in biological control, crc press, boca raton, fl, pp. 23–61, 1990. poinar, go jr. origins and phylogenetic relationships of the entomophilic rhabditids heterorhabditis and steinernema. fundam. appl. nematol. 16: 333-338, 1993. poinar, go, jr. nematodes for biological control of insects. crc press, boca raton, florida, p. 340, 1979. politz sm, philipp m. caenorhabditis elegans as a model for parasitic nematodes: a focus on the cuticle. parasitol. today 8: 6-12, 1992. ratcliffe na, w hitten mma. vector immunity. sgm symposium 63: microbe–vector interactions in vectorborne diseases, gillespie sh, smith gl, osbourna (eds), cambridge university press, 2004. ratcliffe na, rowley af, fitzgerald sw and rhodes cp. invertebrate immunity: basic concepts and recent advances. int. rev. cytol. 97: 183-350, 1985. ribeiro c, duvic b, oliveira p, givaudan a, palha f, simoes n, brehelin m. insect immunity-effects of factors produced by a nematobacterial complex on immunocompetent cells. j. insect physiol. 45: 677-685, 1999. ribeiro c, vignes m, brehelin m. xenorhabdus nematophila (enterobacteriacea) secretes a cation-selective calciumindependent porin which causes vacuolation of the rough endoplasmic reticulum and cell lysis. j. biol. chem. 278: 3030-3039, 2003. richman a, kafatos fc. immunity to eucaryotic parasites in vector insects. curr. opin. immunol. 8: 14-19, 1995. ryan ro, van der horst dj. lipid transport biochemistry and role in energy production. ann. rev. entomol. 45: 233– 60, 2000. salt g. teratocytes as a means of resistance to cellular defense reactions. nature, 27, 232, (5313): 639, 1971. schmidt o, theopold u, strand m. innate immunity and its evasion and suppression by hymenopteran endoparasitoids. bioessays 23: 344–351, 2001. silva cp, w aterfield nr, daborn pj, dean p, chilver t, au cp, sharma s, potter u, reynolds se, ffrench-constant rh. bacterial infection of a model insect: photorhabdus luminescens and manduca sexta. cell. microbiol. 4: 32939, 2002. smithers sr, terry rj, hockley dj. host antigens in schistosomiasis. proc. r. soc. london b. biol. sci. 171: 483-494, 1969. söderhall k, cerenius l. role of the prophenoloxidaseactivating system in invertebrate immunity. curr. opin. immunol. 10: 23–28, 1998. söderhall k. invertebrate immunity. dev comp immunol. 23: 263–266, 1999. strand mr, pech ll. immunological basis for compatibility in parasitoid-host relationships. annu. rev. entomol. 40: 31-56, 1995. summers md, dib-hajj sd. polydnavirus-facilitated endoparasite protection against host immune defenses. proc. natl. acad. sci. usa. 92: 29-36, 1995. tanada y, kaya hk. insect pathology. academic press, san diego, ca,1993. vellupilai p, harn da. oligosaccharide specific induction of interleukin to production by b 220 cells from schistosome-infected mice: a mechanism for regulation of cd4+ cell subsets. proc. natl. acad. sci. usa 91: 18–22, 1994. vinson sd. insect host responses against parasitoids and the parasitoid’s resistence with emphasis on the lepidoptera-hymenoptera association. in: bulla la, cheng tc (eds.) comparative pathobiology, vol. 3, plenum press, new york, pp. 103-125, 1977. vinson sb. how parasitoid deal with the immune system of their hosts. arch. insect biochem. physiol. 13: 3-28, 1990. w eston ds, kemp w m. schistosoma mansoni comparison of cloned tropomyosin antigens shared between adult parasites and biomphalaria glabrata. exp. parasitol. 76: 358-370, 1993. wiesner a, losen s, kopacek p, gotz p. isolated apolipophorin iii from galleria mellonella stimulates the immune reactions of this insect. j. insect physiol. 43: 383–391, 1997. w omersley cz. factors affecting physiological fitness and modes of survival employed by dauer juveniles and their relationship to pathogenicity. in bedding ra, akhurst rj, kaya hk (eds) nematodes and the biological control of insect pests, csiro publications, australia, pp. 7988, 1993. w outs w m. nematode parasites of lepidopterans. in w . r. nickle (ed.) plant and insect nematodes. m dekker, inc., new york, ny. pp. 655-696, 1984. yokoo s, toja s, ishibashi n. suppression of the prophenoloxidase cascade in the larval hemolymph of the turnip moth, agrotis segetum by an entomopathogenic nematode, steinernema carpocapsae and its symbiotic bacterium. j. insect physiol. 38, 915-924, 1992. yoshino tp, bayne cj. mimicry of snail host antigens by miracidia and primary sporocysts of schistosoma mansoni. parasite immunol. 5: 317-328, 1983. zakarian rj, dunphy gb, albert pj, rau me. apolipophoriniii affects the activity of the hemocytes of galleria mellonella larvae. j. insect physiol. 48: 715–23, 2002. zhang g, lu zq, jiang h, asgari s. negative regulation of prophenoloxidase (propo) activation by a clip-domain serine proteinase homolog (sph) from endoparasitoid venom. insect biochem. mol. biol. 34: 477-483, 2004.1993. differential preference of coloured surface of tribolium castaneum (herbst) isj 3: 84-88, 2006 issn 1824-307x short communication differential preference of colored surface in tribolium castaneum (herbst) ams reza, s parween department of zoology, rajshahi university rajshahi 6205, bangladesh accepted july 28, 2006 ______________________________________________________________________________ abstract insects show color preferences mostly to those which resembles the color of foliage, flower or even their hosts. in the present study observations were made to determine vision towards different colored surfaces in young (second instar), and advanced (fourth instar) larvae, and adults of tribolium castaneum (herbst) (coleoptera: tenebrionidae). the larva and adult beetles showed significant color preferences when given a choice between white (control) and colored surfaces at 24and 48-h exposures. the second instar larvae were more attracted by yellow and pink than green surfaces. the fourth instar larvae did not show any significant preference between white and colored surfaces at 24-h exposure, but avoided red and pink surfaces (p>0.05) and had a marginal choice for black (p<0.05). the adults avoided green significantly at both exposure times and pink at 48-h exposure, but was moderately attracted by black (p<0.05) at both exposure times. key words: color preference; tribolium castaneum _____________________________________________________________________________________________________________________ introduction insect traps are used either for population sampling or for management of pest species. for collecting cropfield insects sticky traps and baited traps are used. the sticky traps are often made attractive by using pheromones in it, or by using different colore and shape traps (hoback et al., 1999). different insect families showed preferences for different trap colores. hoback et al. (1999) provided a list of insect families with their preferences for different color traps. members of the same family may prefer more than one color, for example in case of curculionidae (cross et al., 1976), even color preference may differ at species level (capinera and walmsley, 1978). lobdell et al. (2005) observed that the egg parasitoid trichogramma ostriniae showed differential responses to egg color or the hosts as well as the background color in a petri dish arena while searching for the host. ______________________________________________________________________________ corresponding author: a.m. saleh reza department of zoology, rajshahi university rajshahi 6205, bangladesh e-mail: salehbgd@yahoo.com studies on the cropland insects demonstrated the preferences of many insects for yellow color, as the peak color reflectance of plants is the yellow band at 50-560 nm, so this color may act as a super-normal foliage stimulant to herbivorous insects (prokopy and owens, 1983). sawflies showed least preference to dark colors like blue (anderbrant et al., 1989). it is reported that most hymenoptera have color receptors in the green, blue and ultra-violet range, with a few species also being able to perceive red (peitsch et al., 1992). the previous studies revealed that the insects are attracted to colors, which are close to their food color. numbers of attractant and repellent tests were carried for the stored product insect pests as part of their management program. most of these researches involved addition of different insecticides, hormones, pheromones, plant extracts, etc., to their food. however, color preference in stored product insects remains poorly studied. the objective of this study was to determine whether tribolium castaneum, the red flour beetle can differentiate colors? the differential preferences for colored surfaces were studied for young (2nd instar) and advance (4th instar) larvae and adults of t. castaneum. 84 mailto:salehbgd@yahoo.com materials and methods stages of tribolium castaneum used about 200 adults of t. castaneum (herbst) beetles were collected from the stock culture, which has been maintained in the stored product insect laboratory, department of zoology, rajshahi university, since 1985 without any loss of fitness. the adults were kept in a beaker and provided with 10 g of standard food (park and frank, 1948). after 24 h the subculture was sieved through 500 and 250 µm mesh to separate the adults and the eggs respectively from the food. the newly hatched larvae were collected from these eggs and provided with standard food and reared in beakers, covering the mouth with fine cloth to restrict escape of the larvae. a few similar subculture of the beetles were established during subsequent days. the subcultures were kept at 30 ± 1 °c in an incubator without controlling light and humidity. the food was changed after every three days to avoid contamination by the larvae (park, 1935). the second and fourth instar larvae were normally obtained on the third and ninth days respectively after hatching of the eggs (mondal, 1984). the adults emerged 21-22 days after hatching. in this experiment, 30 larvae of second or fourth instars, or adults of 48-h age were used for each color test. each of these experiments was replicated three times. table 1 distribution of larvae and adults of t. castaneum in different colored surfaces after 24 h exposure (n = 90) percentage distribution (number) life stage colored surface control (white) color χ2-values (df = 2) green 31.11 (28) 68.89 (62) 7.14* red 52.23 (47) 47.77 (43) 0.09 blue 35.55 (32) 64.45 (58) 4.18 chocolate 46.67 (42) 53.33 (48) 0.22 yellow 23.33 (21) 76.67 (69) 14.22*** pink 21.11 (19) 78.89 (71) 16.69*** second instar larvae black 47.77 (43) 52.33 (47) 0.11 green 56.66 (57) 43.34 (39) 0.89 red 50.00 (45) 50.00 (45) 0 blue 38.88 (35) 61.12 (55) 0.50 chocolate 35.55 (32) 64.45 (58) 4.18 yellow 48.88 (44) 51.12 (46) 0.02 pink 62.22 (56) 37.78 (34) 2.99 fourth instar larvae black 43.33 (39) 56.67 (51) 0.89 green 71.11 (64) 28.89 (26) 8.91* red 42.22 (38) 57.88 (52) 1.24 blue 40.00 (36) 60.00 (54) 2.00 chocolate 57.78 (52) 42.22 (38) 1.21 yellow 37.78 (34) 62.22 (56) 2.99 pink 57.78 (52) 42.22 (38) 1.21 adults black 31.11 (28) 68.89 (62) 7.14* *p<0.05; ***p<0.001 85 color choice test these tests were conducted in choice chamber. a choice chamber was made in glass petri dish of 9 cm diameter. a straight line was drawn through the middle of the petri dish. colored poster papers were used for the test, and white paper as the control. the papers of each color were first cut into a circle of 9 cm diameter, and then cut into two equal halves. at the outer surface of the petri dish color paper was pasted on one half and white paper was pasted on the other half. similarly, these choice chambers were made for each color. the color used in this experiment were green, red, blue, chocolate, yellow, pink and black. experimentation at the middle of the each choice chamber, 30 of either second or fourth instar larvae, or adults were released. the petri dish was covered with lid, and kept undisturbed at room temperature (20-22 °c). no food was given to them. after 24 h the number of larvae or adults at colored and white parts of the choice chamber were recorded, and the insects were kept undisturbed for another 24 h. similarly, the number of insects in colored and control halves were recorded after 48 h. table 2 distribution of larvae and adults of t. castaneum in different colored surfaces after 48 h exposure (n = 90) percentage distribution (number) life stage colored surface control (white) color χ2-values (df = 2) green 15.55 (14) 84.45 (76) 23.73*** red 34.44 (31) 65.56 (59) 4.48 blue 33.33 (30) 67.67 (60) 5.56 chocolate 61.11 (55) 38.89 (35) 2.47 yellow 24.44 (22) 75.56 (68) 13.07*** pink 17.78 (16) 82.22 (74) 20.76*** second instar larvae black 45.55 (41) 54.45 (49) 0.39 green 43.33 (39) 56.67 (51) 0.89 red 75.55 (68) 24.45 (22) 13.06*** blue 46.66 (42) 53.33 (48) 0.22 chocolate 56.66 (61) 43.34 (39) 0.89 yellow 43.33 (39) 56.67 (51) 0.89 pink 75.55 (68) 24.45 (22) 13.06*** fourth instar larvae black 28.89 (26) 71.11 (64) 8.91* green 81.11 (73) 18.89 (17) 19.36*** red 48.89 (44) 51.11 (46) 0.02 blue 33.33 (30) 66.67 (60) 5.56 chocolate 43.33 (39) 56.67 (51) 0.89 yellow 50.00 (45) 50.00 (45) 0 pink 67.78 (61) 32.22 (29) 6.32* adults black 28.89 (26) 71.11 (64) 8.91* *p<0.05; ***p<0.001 86 statistical analysis the distribution of larvae and adults on each colored surface was tested using χ2-test. anova was done to examine the effect of the exposure period and stages of t. castaneum on the choice of different colors. results and discussion there is difference for color preference among the larvae and adults of t. castaneum. the second instar larvae were mostly attracted to yellow and pink colors (p<0.001) at both 24and 48-h exposure (tables 1, 2). choice test for the fourth instar larvae showed no preference for any one of the tested colors at 24-h exposure (table 1). at 48-h exposure, the fourth instar larvae preferred the white color (control) more than red and pink (p<0.001) (table 2), and showed a little attraction towards black (p<0.05) (table 2). the adults showed no preference for colored or white surfaces except black (p<0.05) at both exposure periods (tables 1, 2). total avoidance to green surface was observed. during 48 h exposure the adults avoided pink whereas at 24-h exposure the distributions in white and pink colors were similar (tables 1, 2). the present results revealed that advance larvae and adults showed no preferences for colored surface. moreover, they avoided pink, red and green. white (control) was more preferred by the fourth instar larvae and the adults than the second instar larvae. table 3 preference or avoidance to colored surfaces by larvae and adults of t. castaneum preference/avoidance of beetles at two exposures 2nd instar larva 4th instar larva adults colors 24-h 48-h 24-h 48-h 24-h 48-h green mp sp nc nc ma sa red nc nc nc sa nc nc blue nc nc nc nc nc nc chocolate nc nc nc nc nc nc yellow sp sp nc nc nc nc pink sp sp nc sa nc mp black nc nc nc mp mp mp sp = strongly preferred (p<0.001); mp = moderately preferred (p<0.05); sa = strongly avoided; ma = moderately avoided; nc = no choice anova showed that there is no significant difference of color choice at different exposure periods (p>0.05, f= 5.58) by the larvae and the adults. the second instar larvae showed preference for green, yellow and pink colors, but had no significant choice for black. however, black was marginally preferred over white by the fourth instar larvae and adults (table 3). generally, a wide range of insects exhibit attraction towards yellow color (borror et al., 1989). the coleopteran families like chrysomelidae, coccinellidae, curculionidae and scarabaeidae showed preference for yellow traps (p<0.05) as stated by flemming et al. (1940), cross et al. (1976), dominick (1976) and dowell and cherry (1981). these beetles also showed choice for green and white traps. however, in the present investigation except young larvae, t. castaneum showed no choice for yellow color. an experiment on the farmland sawflies showed that all the collected species showed strong responses to colored traps, especially to yellow (barker et al., 1997). moreover, the hymenopteran egg parasitoids of the genus trichogramma showed preference for yellow and white, representing an adaptive preference for the egg color of the primary hosts (pak and de jong, 1987; lobdell et al., 2005). furthermore, romies et al. (1998) observed that naturally occurring trichogramma in sorghum fields in india were poorly attracted to yellow stick-traps as compared to white and green traps. khalil (1991) found that adult t. confusum attracted by red in a narrow space (45 ×1 cm) with a color area of 7×1 cm; while in a wide space (100×4 cm) having a color area of 16×4 cm, they were attracted by blue color. the author observed that sitophilous oryzae adults were attracted by green and black, and these responses were greatly affected by the exposure period. however, in the present experiment the adult t. castaneum no definite choice for either blue or red color was observed. normally, it is expected that there is no possibility of differential preference to colors between different species of an insect in the same habitat. however, different sawfly species showed significance difference between color choices (barker et al., 1997). in the present study no food was given in the choice chambers, which might be the reasons that the advanced larvae and the adults might be distributed evenly on white (control) and colored surfaces for the search of food at short exposure. moreover, they preferred dark colors, as a moderate choice for the black surface, for a site of refuge. the fourth instar larvae showed a strong avoidance to red and pink colors at 48-h post-exposure. the adults totally avoided green, and moderately pink. but the second instar larvae showed strong attraction to green and pink colors along with the yellow. hence, it can be said that the younger larvae are attracted to green, yellow and pink colors, and equally distributed on dark colors. longer exposure period provided to fourth instar larvae and adults gave them sufficient time for a good selection of a suitable color. color preference 87 may change during the insect’s life time. for example, the pre-reproductive adult anthomyiid flies were attracted to yellow colored traps, but reproductive flies were attracted to purple color (jenkins and roques, 1993); similarly tephritid flies are attracted to yellow color prior to maturation and red color during oviposition (kring, 1970). it can be concluded that for tribolium control in the grain stores, green, red and pink grain bags could be used to keep away the older larvae and adults, or the wall and surface of the stores may be painted with these colors. use of colored bags or colored surfaces may give better result in integrated pest management programs for the control of tribolium. acknowledgement the authors are thankful to professor dr md ataur rahman khan, department of zoology, rajshahi university, for his painstaking corrections of the manuscript and kind suggestions. references anderbrant o, lofquist j, jonssen j, marking e. effects of pheromone trap type, position and colour on the catch of the pine sawfly neodiprion sertifer (geoff.) (hym. diprionidae). j. appl. ent. 107: 365-369, 1989. barker am, sanbrooke kj, aebischer nj. the water trap colour preferences of farmland sawflies. ent. exp. et appl. 85: 83-86, 1997. borror dj, triplehorn ca, johnson nf. an introduction to the study of insects. saunders college publishing, philadelphia, pa, 1989. capinera jl, walmsley mr. visual responses of some sugarbeet insects to stick traps and water pan traps of various colours. j. econ. ent. 71: 926-927, 1978. cross wh, mitchell hc, hardee dd. boll weevils: response to light source and colours on traps. environ. ent. 5: 565571, 1976. dominick cb. collection of the tobacco flea beetle on coloured panels. j. econ. ent. 64: 1575, 1976. dowell rv, cherry rh. survey traps for parasitoids and coccinellid predators of the citrus black fly, aleurocanthus woglumi. ent. exp. et appl. 29: 356-362, 1981. flemming we, burgess de, maines ww. relation of colour to the effectiveness of japanese beetle traps. j. econ. ent. 33: 320-327, 1940. hoback ww, svatos tm, spomer sm, higley lg. trap colour and placement affects estimates of insects familylevel abundance and diversity in a nebraska salt marsh. ent. exp. et appl. 91: 393-402, 1999. jenkins mj, roques a. attractiveness of colour traps to strobilomyia spp. (diptera: anthomyiidae). env. ent. 22: 297-304, 1993. khalil me. the orientation responses of some coleopteraous adult, tribolium confusum (tenebrionidae) and sitophilus oryzae (curculionidae) to colours. j. egypt. ger. soc. zool. 005: 303-313, 1991. kring jb. red spheres and yellow panels combined to attract apple maggot flies. j. econ. ent. 63: 466-469, 1970 lobdell ce, yong tze-hei, hoffmann mp. host colour preferences and short-range searching behaviour of the egg parasitoid trichogramma ostriniae. ent. exp. et appl. 116: 127-134, 2005. mondal kamsh. effects of methyloquinine, aggregation pheromone and pirimophos-methyl on tribolium castaneum herbst larvae. ph.d. thesis, university of newcaste upon tyne, uk, 1984. pak ga, de jong ej. behavioural variations among strains of trichogramma spp. host recognition. netherlands j. zool. 37: 137-166, 1987. park t. studies on population physiological effects of conditioned flour upon tribolium confusum duv. and its population. physiol. zool. 8: 91-115, 1935. park t, frank mb. the fecundity and development of the flour beetles, tribolium confusum and tribolium castaneum at three constant temperatures. ecology 29: 368-375, 1948. peitsch d, fietz a, hertel h, desouza j, ventura, df, mendzel r. the spectral input systems of hymenopteran insects and their receptor-based colour vision. j. comp. physiol. 170a: 23-40, 1992. prokopy rj, owens ed. visual detection of plants by herbivorous insects. ann. review ent. 28: 337-364, 1983. romies j, shanowar tg, zebitz cpw. response of trichogramma egg parasitoids to coloured stick traps. biocontrol 43: 17-27, 1998. 88 ams reza, s parween department of zoology, rajshahi university rajshahi 6205, bangladesh accepted july 28, 2006 abstract materials and methods stages of tribolium castaneum used color choice test experimentation statistical analysis results and discussion adults acknowledgement seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, mytilus edulis isj 7: 141-145, 2010 issn 1824-307x research report seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, mytilus edulis: temperature controls physiological processes kj mantione, p cadet, f casares, w zhu, gb stefano neuroscience research institute, state university of new york at old westbury, ny 11568, usa accepted may 13, 2010 abstract it is anticipated that invertebrate processes will be subject to seasonal variations because of their poikilothermal characteristics. in the present study we determined if the morphine coupled nitric oxide (no) release, which is constitutive in nature, exhibits seasonal characteristics, which has previously been shown for catecholamine processes in the marine mollusc mytilus edulis. in this regard, morphine induced no release measured on a monthly basis for one year revealed a peak release value (39 ± 4 nm) during the late spring and early summer. the lowest no release occurred during the months of january (6.0 ± 0.5 nm) through march (6.5 ± 1.1 nm). the lowest sea surface temperatures (1.3 °c) were also recorded in these same three winter months in new york. relative mu opiate receptor gene expression was assessed by real time pcr during these seasons. the mrna expression reached a relative peak during the month of june and was at its lowest in february and march, further demonstrating the direct coupling of morphine with this receptor. we conclude that the temperature an animal is chronically exposed to serves to control cellular processes, i.e., opiate signaling. key words: nitric oxide; mu opiate receptor; morphine introduction through homeostasis, living organisms maintain their survival in the face of both internally and externally generated stimuli. this balance is constantly challenged and therefore the ability to overcome these normal perturbations is essential to survival and longevity (chrousos and gold, 1992; fricchione and stefano, 1994). in this regard, subjecting invertebrate animals to temperature changes has been shown to alter the ganglionic monoamine levels as well as affecting functionality of gill cilia in mytilus edulis (stefano et al., 1977a, 1977b; stefano and catapane, 1977b). additionally, opiate processes respond to various types of ___________________________________________________________________________ corresponding author: kirk j mantione neuroscience research institute suny college at old westbury p.o. box 210 old westbury, ny 11568-0210, usa e-mail: kjmantione@sunynri.org list of abbreviations: nitric oxide, no; phosphate buffered saline, pbs; s-nitroso-n-acetyl-dl-penicillamine, snap; constitutive nitric oxide synthase, cnos stressors in both vertebrates and invertebrates ( lee and spector, 1991; marrazzi et al., 1997; sonetti et al., 1999; zhu et al., 2001; cadet et al., 2002; guarna et al., 2002). we have previously demonstrated that endogenous morphine represents the terminal component of a successful stress response and that its actions are generally down regulating immune responses and metabolic rates (stefano et al., 2000). this down regulation occurs because of coupling of the mu opiate receptor to nitric oxide (no) release (cadet et al., 2003). our group has also demonstrated that opiate receptors and subsequent morphine induced no release can be profoundly impacted by temperature changes in m. edulis (cadet et al., 2002; mantione et al., 2003). furthermore, we have presented molecular evidence on the effect of rapid temperature changes on mu opiate receptor expression and morphine levels in this invertebrate’s nervous system (cadet et al., 2002). in cold stressed organisms, ganglionic mu opiate receptor decreases and morphine levels increase (cadet et al., 2002). in the present study, we investigated the seasonal variation in ganglionic mu opiate receptor expression in m. edulis. in addition, we performed a morphine induced no release assay to determine the functionality of the morphine signaling system in this model organism. 141 month jan febmarchapril may june july aug sept oct nov dec nm n itr ic o xi de 0 10 20 30 40 50 t em pe ra tu re o c 0 10 20 30 40 50 [no] sea surface temperature fig. 1 monthly measurements of sea surface temperature and morphine (1 µm) stimulated no release from mytilus edulis pedal ganglia (20 ganglia per assay, n = 4). paired t-tests revealed statistically significant differences between january or february or march and may or june or july (p = 0.002). material and methods animal collection and nitric oxide (no) determination mytilus edulis collected from shinnecock bay on long island were immediately transported to the laboratory in seawater for processing. the ambient seawater temperature was maintained using insulated coolers until dissection of animals. samples were collected monthly on the 15 day of the month and sea surface temperature was recorded. for no determination, approximately 20 pedal ganglia were placed in 1 ml phosphate buffered saline (pbs) at room temperature. no release from the tissues was immediately directly measured using a 200 μm flexible no-specific amperometric probe (world precision instruments, sarasota, fl) connected to a 4-channel biostat (esa, chelmsford, ma). the system was calibrated daily with s-nitroso-n-acetyl-dl-penicillamine (snap) in 0.1 m cu+2. the amperometric probe was allowed to equilibrate in pbs for at least 10 min prior to being transferred to the tube containing the tissue. morphine-stimulated no release was evaluated at a final concentration of 10-6 m. each experiment was repeated four times (4 groups of 20 ganglia for each month) along with a control (pbs only). a paired student’s t-test was performed to evaluate differences between selected months. mu opiate receptor expression pedal ganglia (15) were immediately processed after dissection. the ganglia were placed in 1.5 ml tubes and then washed with pbs (invitrogen, carlsbad, ca). total rna was isolated using the rneasy mini kit (qiagen, valencia, ca). ganglia were homogenized in 600 µl lysis buffer. the samples were then processed following the manufacturer’s instructions. in the final step, the rna was eluted with 50 µl of rnase-free water. this rna isolation process was repeated four times for each group of 15 ganglia. first-strand cdna synthesis was performed using random primers (invitrogen, carlsbad, ca). 1 µg of total rna was denatured at 95 °c and reverse transcribed at 40 °c for 1 h using superscript iii rnase h-rt (invitrogen, carlsbad, ca). ten microliters of the rt product was added to the pcr mix containing primers for the mu opiate receptor. primers and probes specific for the mu-opiate receptor gene (mor) were designed by the software primer express (applied biosystems, foster city, ca). the forward primer was 5’atgccagtgctcatcattac-3’ and the reverse primer sequence was 5’gatccttcgaagattcctgtcct-3’. the taqman probe was constructed with the 5’-reporter dye, 6carboxyfluorescein (fam), and a 3’-quencher dye, 6-carboxy-tetramethyl-rhodoamine (tamra). the probe sequence was 5’cgcctcaagagtgtccgcatgct-3’. the endogenous control gene, β-actin, was used to normalize the rt-pcr. the 2x universal master mix (applied biosystems) containing the pcr buffer, mgcl2, dntp’s, and the thermal stable amplitaq gold dna polymerase was used in the pcr reactions. in addition, 200 µm reverse and forward primers, 100 µm taqman probe, 3 µl of rt product and rnase/dnase-free water was added to the master mix to a final volume of 50 µl. the pcr reaction mixture was transferred to a microamp optical 96-well reaction plate and incubated at 95 °c for 10 min to activate the amplitaq gold dna polymerase and then run for 40 cycles at 95 °c for 30 s and 60 °c for 1 min on the applied biosystems geneamp 7500 sequence detection system(sds). each pcr was performed in triplicate. the pcr results were analyzed with the geneamp 7500 sds 142 month jan febmarchapril may june july aug sept oct nov dec r el at iv e g en e e xp re ss io n 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 fig. 2 relative mu opiate receptor gene expression in mytilus edulis pedal ganglia determined by real time pcr for each monthly sample (n = 4). paired t-tests revealed statistically significant differences between february or march and may or june or july (p = 0.004). software (applied biosystems). relative gene expression was calculated using the method of yoshikawa et al. (2001). standard curves were generated by serial dilution of the june cdna sample. r values were calculated and used to directly compare the monthly measurements. a paired student’s t-test was performed to evaluate differences between selected months. results morphine induced no release measured on a monthly basis for one year revealed a average peak value of 39 ± 4 nm, during the late spring and early summer (fig. 1). the lowest no release occurred during the months of january (6.0 ± 0.5nm) through march (6.5 ± 1.1nm) (fig. 1). the lowest sea surface temperatures (1.3 °c) were also recorded in these same three months (fig. 1). student’s t-tests revealed a statistically significant difference (p = 0.002) between the warm season high no values and cold season low no values. relative gene expression was assessed by real time pcr. the mrna expression reached a relative peak during the month of june and was at its lowest in february and march (fig. 2). student’s t-tests revealed a statistically significant difference (p = 0.004) between the warm season high r values (1.2 ± 0.10) and cold season low r values (0.46 ± 0.052). a regression analysis using mu opiate receptor expression as the independent variable and no release as the dependent variable showed a correlation between the measurements. the calculated r value was 0.729. discussion m. edulis neural tissues contain the typical biogenic amines, which includes dopamine (stefano et al., 1976). biogenic amines display variations in their ganglionic levels, which corresponds to the seasons and temperature, being high in warmer months and low in the winter months (stefano et al., 1977a, 1977b; stefano and catapane, 1977a, 1977b). interestingly, this same relationship occurs with opioid peptide expression along with their receptors (stefano et al., 1980; stefano and leung, 1986). as demonstrated in this report, this same phenomenon involves no release, which is coupled to opiate receptor activation, namely via μ3 ( liu and stefano, 1996; liu et al., 1996; magazine et al., 1996; stefano and scharrer, 1996; stefano et al., 1996). the coupling of catecholamine, no and morphinergic signaling has recently been reviewed (kream et al., 2009; stefano and kream, 2009; stefano et al., 2009; zhu and stefano, 2009). it is important to note that dopamine is a morphine precursor in this animal, which synthesizes endogenous morphine ( zhu et al., 2005; kream and stefano, 2006). the significance of this precursor status of dopamine emanates from previously noted reports in this document showing seasonal and temperature alterations of catecholamine levels, which can now be directly compared to morphinergic phenomena, including the ability of morphine to release constitutively derived no. we surmise that at colder “winter” temperatures all processes appear to be down regulated, including the homeostasis between an opiate receptor and its ligand levels, as currently demonstrated. this probably occurs because very cold temperatures influence all metabolic processes to decrease their activity levels, including those involved in various survival processes. this homeostasis mechanism occurs in all living organisms, including those with a pathogenic ability. thus, in both types of organisms processes providing a survival benefit are not required. this 143 probably extends into the energy metabolic processes found in mitochondrial-like structures where morphine exerts actions (kream and stefano, 2009). in conclusion, the therapeutic value of performing medical operations, maintaining food, etc., at very low temperatures probably arises from the ability of low temperatures to disassociate adaptive cellular processes, allowing for a rather universal down regulation, which depending on the organism has tremendous survival advantage. in the case of mytilus, if bacteria, viruses can’t survive or have a decreased infectious characteristic, why have a full functioning immune process with the activation of cytokines and opiate components at low temperature ( stefano and scharrer, 1994; stefano and salzet, 1999; stefano et al., 2000, 2008; stefano and kream, 2008). references cadet p, mantione kj, stefano gb. molecular identification and functional expression of mu3, a novel alternatively spliced variant of the human mu opiate receptor gene. j. immunol. 170: 5118-5123, 2003. cadet p, zhu w, mantione k, baggerman g, stefano gb. cold stress alters mytilus edulis pedal ganglia expression of μ opiate receptor transcripts determined by real-time rt-pcr and morphine levels. brain res. mol. brain res. 99: 26-33, 2002. chrousos gp, gold pw. the concepts of stress and stress system disorders: overview of physical and behavioral homeostasis. jama 267: 12441252, 1992. fricchione gl, stefano gb. the stress response and autoimmunoregulation. adv. neuroimmunol. 4: 13-28, 1994. guarna m, bianchi e, bartolini a, ghelardini c, galeotti n, bracci l, et al. endogenous morphine modulates acute thermonociception in mice. j. neurochem. 80: 271-277, 2002. kream rm, mantione kj, sheehan m, and stefano gb. morphine's chemical messenger status in animals. activitas nervosa superior rediviva 51: 2009. kream rm, stefano gb. de novo biosynthesis of morphine in animal cells: an evidence-based model. med. sci. monit. 12: ra207-ra219, 2006. kream rm, stefano gb. endogenous morphine and nitric oxide coupled regulation of mitochondrial processes. med. sci. monit. 15: ra263-ra268, 2009. lee cs, spector s. changes of endogenous morphine and codeine contents in the fasting rat. j. pharmacol. exp. ther. 257: 647-650, 1991. liu y, shenouda d, bilfinger tv, stefano ml, magazine hi, and stefano gb. morphine stimulates nitric oxide release from invertebrate microglia. brain res. 722: 125-131, 1996. liu y, stefano gb. hiv gp120 inhibits human and invertebrate immunocyte phagocytosis. chinese j. immunol. 12: 139-142, 1996. magazine hi, liu y, bilfinger tv, fricchione gl, stefano gb. morphine-induced conformational changes in human monocytes,granulocytes, and endothelial cells and in invertebrate immunocytes and microglia are mediated by nitric oxide. j. immunol. 156: 4845-4850, 1996. mantione k, hong r, im r, nam jh, simon m, cadet p, et al. effects of cold stress on morphine-induced nitric oxide production and mu-opiate receptor gene expression in mytilus edulis pedal ganglia. neuroendocrinol. lett. 24: 68-72, 2003. marrazzi ma, luby ed, kinzie j, munjal id, spector s. endogenous codeine and morphine in anorexia and bulimia nervosa. life sci. 60: 1741-1747, 1997. sonetti d, mola l, casares f, bianchi e, guarna m, stefano gb. endogenous morphine levels increase in molluscan neural and immune tissues after physical trauma. brain res. 835: 137-147, 1999. stefano gb, catapane ej. seasonal monoamine changes in the central nervous system of mytilus edulis. experientia 33: 1341-1342, 1977a. stefano gb, catapane ej. the effects of temperature acclimation on monoamine metabolism. j. pharmacol. exp. ther. 203: 449546, 1977b. stefano gb, catapane ej, aiello e. dopaminergic agents: influence on serotonin in the molluscan nervous system. science 194: 539541, 1976. stefano gb, catapane ej, stefano jm. temperature dependent ciliary rhythmicity in mytilus edulis and the effects of monoaminergic agents on its manifestation. biol. bull. 153: 618629, 1977a. stefano gb, goumon y, casares f, cadet p, fricchione gl, rialas c, et al. endogenous morphine. trends neurosci. 9: 436-442, 2000. stefano gb, hiripi l, catapane ej. the effects of short and long term temperature stress on serotonin, dopamine and norepinephrine concentrations in molluscan ganglia. j. thermal biol. 3: 79-83, 1977b. stefano gb, kream r. endogenous opiates, opioids, and immune function: evolutionary brokerage of defensive behaviors. semin. cancer biol. 18: 190-198, 2008. stefano gb, kream rm. dopamine, morphine, and nitric oxide: an evolutionary signaling triad. cns neurosci. ther. 2009 [epub ahead of print]. stefano gb, kream rm, mantione kj, sheehan m, cadet p, zhu w, et al. endogenous morphine/nitric oxide-coupled regulation of cellular physiology and gene expression: implications for cancer biology. semin. cancer biol. 18: 199-210, 2008. stefano gb, kream rm, zukin rs, catapane ej. seasonal variation of stereospecific enkephalin binding and dopamine responsiveness in mytilus edulis pedal ganglia. in: rozsa ks (ed), neurotransmitters in invertebrates, pergamon press, london, pp 453-459, 1980. stefano gb, leung mk. opioid aging and seasonal variations in invertebrate ganglia: evidence for an opioid compensatory mechanism. in: 144 stefano gb (ed), comparative opioid and related neuropeptide mechanisms, crc press, inc., boca raton, pp 233-242, 1986. stefano gb, salzet m. invertebrate opioid precursors: evolutionary conservation and the significance of enzymatic processing. int. rev. cytol. 187: 261-286, 1999. stefano gb, salzet m, ottaviani e. neuroimmune chemical messengers and their conservation during evolution. in: rinkevich b, matranga v. (eds), stem cells in marine organisms, springer, netherlands, pp 139-164, 2009. stefano gb, scharrer b. endogenous morphine and related opiates, a new class of chemical messengers. adv. neuroimmunol. 4: 57-68, 1994. stefano gb, scharrer b. the presence of the μ3 opiate receptor in invertebrate neural tissues. comp. biochem. physiol. 113c: 369-373, 1996. stefano gb, scharrer b, smith em, hughes tk, magazine hi, bilfinger tv, et al. opioid and opiate immunoregulatory processes. crit. rev. immunol. 16: 109-144, 1996. yoshikawa m, nakayama h, ueno s, nishimine n, furuya h. novel quantitative reversetranscribed polymerase chain reaction of mu opioid receptor mrna level. brain res. brain res. protoc. 7: 147-153, 2001. zhu w, baggerman g, goumon y, casares f, brownawell b, and stefano gb. presence of morphine and morphine-6-glucuronide in the marine mollusk mytilus edulis ganglia determined by gc/ms and q-tof-ms. starvation increases opiate alkaloid levels. brain res. mol. brain res. 88: 155-160, 2001. zhu w, mantione kj, shen l, cadet p, esch t, goumon y, et al. tyrosine and tyramine increase endogenous ganglionic morphine and dopamine levels in vitro and in vivo: cyp2d6 and tyrosine hydroxylase modulation demonstrates a dopamine coupling. med. sci. monit. 11: br397-br404, 2005. zhu w, stefano gb. comparative aspects of endogenous morphine synthesis and signaling in animals. ann. ny acad. sci. 1163: 330-339, 2009. 145 research report seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, mytilus edulis: temperature controls physiological processes isj 3: 89-96, 2006 isj 3: 111-117, 2006 issn 1824-307x research report the effects of okadaic acid on enchytraeus crypticus (annelida: oligochaeta) a franchini, m marchetti department of animal biology, university of modena and reggio emilia, modena, italy accepted november 29, 2006 abstract we describe the morpho-functional effects of different concentrations of okadaic acid (oa) on specimens of enchytraeus crypticus. the results demonstrate that this experimental model is very sensitive to the treatment and presents timeand dose-related effects mainly involving an immune response associated with a reaction in the chloragogenous tissue. at the lower dose (100 nm), the main organs do not appear particularly affected except for a swelling of the coelomatic cavity and an increased number of circulating coelomocytes. at the higher dose (200 nm), the chloragogenous tissue extends in volume to occupy the body cavity almost completely, while the circulating amoebocytes and chloragocytic cells undergo conformational changes. at the highest oa dose (400 nm), there is a general cell suffering in the main animal organs. in control animals, the immunocytochemical reaction with anti-il-6 antibody is positive in neuron cell bodies and fibres from the ventral nerve cord and in circulating amoebocytes. following oa treatment, fewer immunoreactive cells are seen in the damaged nervous tissue, and the high number of recruited amoebocytes is also positive. key words: annelid; enchytraeus crypticus; okadaic acid; il-6-like molecules introduction okadaic acid (oa) is produced by toxigenic dinoflagellates from the dinophysis and prorocentrum genera and is involved in fish death, diarrhetic shellfish poisoning and, consequently, human intoxication (yasumoto et al., 1978, 1985). the molecular mechanisms responsible of the various biological actions attributed to this toxin involve the specific inhibition of serine/threonine protein phosphatases 1 and 2a (bialojan and takai, 1988) which are critical components in signalling cascades regulating a variety of cellular processes in eukariotic cells. in vivo studies using mice as experimental models have described the distribution and excretion of oa following oral administration, as well as the morpho-functional modifications of toxin target organs (edebo et al., 1988; ito and terao, 1994; ito et al., 2002; franchini et al., 2005). the small intestine was particularly affected, with an edema in the lamina propria of villi and desquamation ___________________________________________________________________________ corresponding author: antonella franchini department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: franchini.antonella@unimore.it of degenerated epithelium, while the liver accumulated a significant amount of toxin, but was not damaged (ito and terao, 1994; ito et al., 2002). the mouse thymus was also particularly affected, with a severe alteration in structural architecture and a significant depletion in lymphoid elements. the ability of oa to provoke both immunostimulation and systemic immunotoxicity has also been highlighted (franchini et al., 2005). oa is a well known inducer of proinflammatory responses (stanley et al., 2001) and a stimulator of inflammatory cytokine gene transcription in murine macrophages (tebo and hamilton, 1994). in the present investigation, the effects of oa on the structural organization of a species of enchytraeids are evaluated. the enchytraeids have been widely used for many years in ecotoxicological laboratory tests, in view of their keystone ecological status and the fact that they can serve as indicator organisms for chemical stress (didden and rombke, 2001). we have also examined the induced modifications for the presence and distribution of il6-like molecules. il-6 is a multifunctional cytokine able to modulate a variety of physiological events that play a main role in the immune system and inflammation. 111 mailto:franchini.antonella@unimore.it materials and methods adult specimens of the worm enchytraeus crypticus (annelida, oligochaeta, enchytraeidae) were grown on agar medium for 1 month before the experimental procedure. the animals were cultured in 90 mm petri dishes containing 1.1 % agar powder dissolved in distilled water and maintained at a temperature of 23 °c with a photoperiod of 8 h light and 16 h dark. the worms were fed with sterilized powdered rolled oats and water twice a week, checked daily and subcultured by transfer onto a new agar plate once every 10-14 days. fig. 1 longitudinal sections from e. crypticus controls (a, b, d) and specimens treated with 100 nm oa for 48 h (c) and 200 nm oa for 12 h (e, f) stained with mallory-azan stain (b, c, e, f) and gallocyanin-chrome alum (a, d). the chloragogenous tissue (a, b) from controls formed one or two layers of round vacuolated and basophilic cells (d) surrounding the intestine. after oa treatment, the tissue showed a higher number of cell layers and expanded into the coelomatic cavity (e). the toxin also induced an increase in the number of circulating coelomocytes (c, e). note the enlarged and rounded circulating chloragocytic cells (f). brain, b; ventral nervous cord, v; chloragogenous tissue, c; coelomocytes, arrowheads; intestine, i. bar = 10 μm 112 thirty groups of 5 worms each were placed in 30 mm petri dishes and treated as follows: 3 groups of control animals were fed normally at the beginning of the experiment, while 27 groups received powdered rolled oats mixed with a water solution of oa (calbiochem, usa) at different final concentrations (100, 200 and 400 nm) for 12, 24 or 48 h. treated and control specimens were then collected, immediately fixed in bouin’s mixture and embedded in agar/paraffin, as previously described (franchini et al., 2003). the following histological and histochemical stains were performed on 7 µm transversal or longitudinal paraffin serial sections: hematoxylin-eosin and mallory-azan stains for general morphology, and gallocyanin-chrome alum reaction for nucleic acids (bancroft and gamble, 2002). the immunocytochemical procedure was carried out on controls and the variously treated e. crypticus using goat anti-il-6 polyclonal antibody (r & d systems, usa) diluted 1:500. sections were incubated overnight at 4 °c in the primary antibody, fig. 2 longitudinal sections from e. crypticus treated with 200 nm oa for 48 h (a-c) and 400 nm oa for 48 h (df) stained with mallory-azan stain (d-f) and gallocyanin-chrome alum (a-c). brain, b; ventral nervous cord, v; chloragogenous tissue, c; clamped coelomocytes, arrows; intestine, i. bar = 10 μm. 113 and immunoreactivity was visualised by an immunoperoxidase technique using avidin-biotin peroxidase complex (hsu et al., 1981). controls of the immunocytochemical reactions were performed by substituting the primary antibodies with nonimmune sera and/or absorbing the antibody with homologous antigen. results examination of the histological sections revealed that e. crypticus is sensitive to oa treatment in a timeand dose-related manner. at the lower dose (100 nm), the main organs did not appear particularly affected, except for a swelling of the coelomatic cavity, where, in comparison to controls, an increased number of circulating coelomocytes was observed after 48 h (figs 1a-c). in e. crypticus, two main cell types were seen floating freely in the body cavity, a more numerous population of round or mostly elongated coelomocytes presenting a cytoplasm filled with large inclusions (oligochaete designed chloragocytic cells), and smaller cells devoid of inclusions with an irregular cytoplasm (oligochaete designed amoebocytes). in control worms the chloragogenous tissue presented one or two layers of large, vacuolated and basophilic cells surrounding the intestine (fig. 1d). at the higher oa dose (200 nm) and after 12 h of treatment, this tissue differed in morphology, extended in volume, and an increased number of cell layers was observed (fig. 1e). these cells were irregularly arranged and without cytoplasmic basophilia, while some were detached from the tissue and surrounded by amoebocytes that had undergone conformational changes into an active form. the circulating chloragocytic cells also changed morphology and became enlarged and rounded in shape (fig. 1f). after 48 h, the cells in the chloragogenous tissue appeared highly vacuolated and tended to break. they occupied the body cavity almost completely, so that the coelomocytes were concentrated in the prostomium, where the tissue was not present (figs 2a-c). the nervous system was also affected by a reduction in the nervous area, in particular in the ventral nerve cord extending through the entire animal length. at the highest oa dose (400 nm), a general cell suffering was found, i.e. the extended, irregularly shaped chloragogenous tissue cells embedded clumped coelomocytes (figs 2d, e), the epithelial cell layer of the medio-posterior intestine increased in surface area (fig. 2f), nephridia appeared disorganized in structure, and the ventral nerve cord comprising wrinkled nerve cells presented fewer neuronal cell bodies as a result of the invasion of expanding chloragogenous tissue (figs 3a, d). in control animals, the immunocytochemical reaction with anti-il-6 antibody was positive in the nervous system. in particular, the antibody labelled neuron cell bodies and fibres located in the ventral nerve cord (fig. 3a) and in the anterior ganglia near the mouth, but not brain nerve cells. one population of the circulating coelomocyte, i.e amoebocytes, was also positive (figs 3b, c). after oa treatment, fewer immunoreactive cells were seen in nervous tissue (fig. 3d) and the large number of recruited amoebocytes was positive, especially when seen in an active form with large and heterogeneous vacuola inside the cytoplasm (figs 3e-g). discussion the results from the present study demonstrate that the experimental model used is very sensitive to treatment with oa. the toxin induced timeand dose-related effects that mainly involved an immune response associated with a reaction in the chloragogenous tissue. the multi-functional cells in oligochaete chloragogenous tissue are known to be able to store glycogen, lipids and exogenous materials, such as pigments or metals, and have also been associated to immune defenses (needham, 1966; roots and johnston, 1966; prentø, 1979; cooper 1981; morgan 1981). in these cells, the chloragocytes, two cellular compartments have been implicated in metal trafficking: the cytoplasm organelles, called chloragosomes, undergo changes in autophagic derivatives, the debris vesicles. the subcellular modifications were found to be closely correlated with accumulated metal burdens in the tissue (morgan and turner, 2005). following oa treatment, the tissue increases its volume considerably and shows morphological changes in the cells. the earthworm’s chloragogenous tissue has some liver-like properties (laverack, 1963; prentø, 1987), and in e. crypticus, it may be involved in toxin accumulation and detoxification in a dose-related manner. the induction of a protein in response to toxin exposure, possibly related to detoxification reactions, has been demonstrated in the digestive gland of mytilus galloprovincialis (auriemma and battistella, 2004). oa also induces an inflammatory cell response, and the number of circulating coelomocytes, known to be responsible for the animal’s immune defence responses (valembois et al., 1982; vĕtvička and šima, 1998), increases both in the chloragocytic cell population, which originates in the chloragogenous tissue surrounding the intestine (fischer, 1993), and in the amoebocytes that change to an active form and show greater immunoreactivity to anti-il-6 antibody. oa is a well-known specific inhibitor of serine/threonine protein phosphatase 1 and 2a. these molecules have been found to act as endogenous regulators of inflammatory cell signalling (shanley et al., 2001), and to play a role in the control and stimulation of proinflammatory cytokine gene expression by murine mononuclear phagocytes and other cell types (falk et al., 1994; tebo and hamilton, 1994; wakiya and shibuya, 1999; feng et al., 2006). the enhanced inflammatory reaction is associated, through a protein phosphatase 2a-mediated mechanism, with the regulation of the c-jun nh2-terminal kinase, one of the major mitogen-activated protein kinase signalling pathways (shanley et al., 2001; avdi et al., 2002). it has also been suggested that the dynamic interplay between kinases and phosphatases modulates the activity of several proteins that are crucial in neuronal functions. oa provokes effects in the structure of the nervous system, while the protein hyperphoshorylation due 114 fig. 3 sections from e. crypticus controls (a-c) and specimens treated with 100 nm oa for 24 h (e), 200 nm oa for 24 h (f-h) and 400 nm oa for 48 h (d) immunostained with anti-il-6 polyclonal antibody and haematoxylin nuclear counterstaining. negative controls of the immunocytochemical reaction (h). ventral nervous cord, v; chloragogenous tissue, c; negative chloragocytic cells, arrows; positive amoebocytes, arrowheads. bar = 10 μm. 115 to the inhibition of phosphatases 1, 2a and possibly also calcineurin is known to induce neuronal stress and subsequent neurodegeneration (tapia et al., 1999; arias et al., 2002; ramirez-munguia et al., 2003). acknowledgement we thank prof. g pürschke (osnabruck university, germany) who kindly supplied the enchytraeus crypticus population and prof. e ottaviani (university of modena and reggio emilia, modena, italy) for critical reading of the manuscript. references arias c, montiel t, pena f, ferrera p, tapia r. okadaic acid induces epileptic seizures and hyperphosphorylation of the nr2b subunit of the nmda receptor in rat hippocampus in vivo. exp. neurol. 177: 284 -291, 2002. auriemma r, battistella s. biochemical and histological alterations of mytilus galloprovincialis digestive gland after exposure to okadaic acid and derivatives. inv. surv. j. 1: 66 -71, 2004. avdi nj, malcolm kc, nick ja, worthen gs. a role for protein phosphatase-2a in p38 mitogenactivated protein kinase-mediated regulation of the c-jun nh2-terminal kinase pathway in human neutrophils. j. biol. chem. 277: 4068740696, 2002. bancroft j, gamble m. theory and practice of histological techniques. 5th ed, churchill livingstone, edinburgh, uk, 2002. bialojan c, takai a. inhibitory effect of a marinesponge toxin, okadaic acid, on protein phosphatases. biochem. j. 256: 283 -290, 1988. cooper el, stein ea. oligochaetes. in: ratcliffe na, rowley af (eds), invertebrate blood cells, vol 1, academic press, london, uk, pp 75-140, 1981. didden w, rombke j. enchytraeids as indicator organisms for chemical stress in terrestrial ecosystems. ecotoxicol. environ. safety 50: 25 -43, 2001. ebebo l, lange s, li xp, allenmark s. toxic mussels and okadaic acid induce rapid hypersecretion in the rat small intestine. apmis 96: 1029 -1035, 1988. falk w, stricker k, praast g, tsukitani y, krammer ph, stoeck m. activation of the mouse il-2 by okadaic acid: synergy with interleukin-1. lymphokine cytokine res. 13: 167-174, 1994. feng g, ohmori y, chang pl. production of chemokine cxcl1/kc by okadaic acid through the nuclear factor-κb pathway. carcinogenesis 27: 43-52, 2006. fischer e. the myelo-erythroid nature of the chloragogenous-like tissues of the annelids. comp. biochem. physiol. 106a: 449-453, 1993. franchini a, marchesini e, poletti r, ottaviani e. swiss mice cd1 fed on mussels contaminated by okadaic acid and yessotoxins: effects on thymus and spleen. eur. j. histochem. 49: 179188, 2005. franchini a, peruzzi e, ottaviani e. morphochemical age-related changes in the nematode caenorhabditis elegans: immunoperoxidase localization of cytokine and growth factor-like molecules. eur. j. histochem. 47: 75-80, 2003. hsu sm, raine l, fanger h. use of avidin-biotinperoxidase complex (abc) immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures. j. histochem. cytochem. 29: 577-80, 1981. ito e, terao k. injury and recovery process of intestine caused by okadaic acid and related compounds. nat. toxins 2: 371-377, 1994. ito e, yasumoto t, takai a, imanishi s, harada k. investigation of the distribution and excretion of okadaic acid in mice using immunostaining method. toxicon 40: 159-165, 2002. laverack ms. the physiology of earthworms. pergamon press, oxford, uk, 1963. morgan aj. a morphological and electronmicroprobe study of the inorganic composition of the mineralized secretory products of the calciferous gland and chloragogenous tissue of the earthworms, lumbricus terrestris l. cell tissue res. 220: 829-844, 1981. morgan aj, turner mp. quantitative ultrastructure of metal-sequestering cells reflects intersite and interspecies differences in earthworm metal burdens. arch. environ. contam. toxicol. 49: 45-52, 2005. needham ae. the cloragogen-pigment of earthworms. life sci. 5: 33-93, 1966. prentø p. metals and phosphate in the chloragosomes of lumbricus terrestris and their possible physiological significance. cell tissue res. 196: 123-134, 1979. prentø p. distribution of 20 enzymes in the midgut region of the earthworm, lumbricus terrestris l., with particular emphasis on the physiological role of the chloragog tessue. comp. biochem. physiol. 87a: 135-142, 1987. ramirez-munguia n, vera g, tapia r. epilepsy, neurodegeneration, and extracellular glutamate in the hippocampus of awake and anesthetized rats treated with okadaic acid. neurochem. res. 28: 1517-1524, 2003. roots bi and johnston pv. the lipids and pigments of the chloragosomes of the earthworm lumbricus terrestris l. comp. biochem. physiol. 17: 285-288, 1966. shanley tp, vasi n, denenberg a, wong hr. the serine/threonine phosphatase, pp2a: endogenous regulator of inflammatory cell signaling. j. immunol. 166: 966-972, 2001. tapia r, peña f, arias c. neurotoxic and synaptic effects of okadaic acid, an inhibitor of protein phosphatases. neurochem. res. 24: 14231430, 1999. tebo jm, hamilton ta. okadaic acid stimulates inflammatory cytokine gene transcription in murine peritoneal macrophages. cell. immunol. 153: 479-491, 1994. valembois p, roch p, lassegues m, davant n. bacteriostatic activity of a chloragogen cell 116 secretion. pedobiologia 24: 191-195, 1982. vĕtvička v, šima p. evolutionary mechanisms of defence reactions. birkhäuser verlag, basel, switzerland, 1998. wakiya k, shibuya m. okadaic acid stimulates the expression of vascular endothelial growth factor gene. biochem. biophys. res. commun. 265: 584-588, 1999. yasumoto t, murata m, oshima y, sano m. diarrhetic shellfish toxins. tetrahedron 41: 1019-1025, 1985. yasumoto t, oshima y, yamaguchi m. occurrence of a new type of shellfish poisoning in the tohoku district. bull. jap. soc. sci. fish. 44: 1249-1255, 1978. 117 abstract introduction results discussion acknowledgement because they compete for some of the same limited metabolic resources, the outcome of the combative interaction between host a isj 7: 198-210, 2010 issn 1824-307x review cellular immunity and pathogen strategies in combative interactions involving drosophila hosts and their endoparasitic wasps aj nappi department of biology, loyola university chicago, chicago, illinois, usa accepted september 17, 2010 abstract various cellular innate immune responses protect invertebrates from attack by eukaryotic pathogens. in insects, assessments of the factor(s) causing, or contributing to, pathogen mortality have long considered as toxic components certain molecules associated with enzyme-mediated melanogenesis. in drosophila hosts, observations that have prompted additional or alternative considerations are those that document either the survival of certain endoparasitic wasps despite melanotic encapsulation, or the destruction of the parasite with no evidence of this type of host response. investigations of the production of some reactive intermediates of oxygen and nitrogen during infection provide a basis for proposing that these molecules constitute important elements of the immune arsenal of drosophila. studies of the target specificity of virulence factors injected by female wasps during infection that suppress the host immune response will likely facilitate identification of the toxic host molecules, and contribute to a more detailed understanding of the cellsignaling pathways that regulate their synthesis. key words: cellular innate immunity; reactive intermediates of oxygen; nitric oxide; melanization; encapsulation; drosophila introduction because of competition for some of the same limited metabolic resources, the outcome of the combative interaction between host and pathogen depends in large part on the effectiveness of apposing physiological, biochemical, and behavioral responses. in destroying pathogens, vertebrate hosts benefit from the collaborative interactions of two distinct, but not entirely separate, immune systems; adaptive and innate. the adaptive immune system produces an almost limitless repertoire of pathogen-specific responses, enabled in large part by considerable genetic plasticity that produces specific cell surface receptors, immunoglobulins, and cells possessing immune memory that rapidly initiate and enhance subsequent responses to the same antigen. insects and other invertebrates rely exclusively on innate immune responses, which many authors regard as the first line of defense. these responses are considered to be dependent on constitutive (i.e., germ-line encoded) and dedicated cell membrane-bound pattern recognition ___________________________________________________________________________ corresponding author: anthony j nappi department of biology, loyola university chicago, chicago, illinois, usa e-mail: anappi@luc.edu receptors with limited responsiveness to invariant molecular motifs of certain pathogens (pal and wu, 2009). some of the invertebrate innate immune effector responses elicited by prokaryotic infections include phagocytosis, hemolymph coagulation, the synthesis of pro-inflammatory cytokines, antimicrobial peptides, reactive intermediates of oxygen (roi) and nitrogen (rni), and stress-related proteins (nappi and vass, 2001; beutler, 2004; malagoli and ottaviani, 2007; malagoli et al., 2007, 2010; becker et al., 2010). eukaryotic pathogens succumb to an encapsulation response that is mediated in large part by macrophage-like blood cells (hemocytes). in insects and other arthropods, hemocyte-mediated encapsulations characteristically are accompanied by the synthesis of melanin, with intermediates such as quinones and semiquinones considered potentially toxic to invading pathogens (nappi and ottavani, 2000a; nappi and christensen, 2005; poirie et al., 2009) (fig. 1). in both adaptive and innate systems, the binding of foreign elements to cell-surface receptors leads to the activation of signal transduction pathways, the transcription of immune genes, and the generation of reactive cells and various toxic molecules. as counter strategies, some pathogens produce virulence factors that actively suppress immune responses, while others 198 fig. 1 overview of some toxic molecules manifested in the innate immune responses of various invertebrates. non-self recognition may involve plasma membrane receptors independently functioning, or cooperatively engaging non-self binding molecules in the host’s hemolymph. melanotic encapsulation, which is a common manifestation of the defense reaction made by arthropods infected with eukaryotic pathogens, involves activation of one or more of the following enzymes; dopa decarboxylase (ddc), dopachrome conversion enzyme (dce), phenylalanine hydroxylase (pah), and phenoloxidase (po). enzymes capable of generating reactive intermediates of oxygen (roi) and nitrogen (rni) include myeloperoxidase (myelo-px), nadph oxidase (nadph ox), nitric oxide synthase (nos), and superoxide dismutase (sod). melanogenic intermediates such as quinones and semiquinones can react with roi, rni and the active centers of certain metaloenzymes to contribute additional toxic molecules. passively avoid detection, either by molecular mimicry or by finding sanctuary within host tissues. the focus of this review concerns some unresolved aspects of the cellular innate immune responses of drosophila against certain endoparasitic wasps, including the nature of the toxic molecules generated during infection, as well as the mechanisms employed by pathogens to circumvent these potentially damaging molecules. drosophila-parasitic wasp interactions the availability of well-defined resistant and susceptible species and strains of drosophila, together with both virulent and avirulent lines of endoparasitic wasps (parasitoids), provide exceptionally good models for investigating not only the genetic and biochemical components of insect cellular innate immunity, but also the varied processes by which parasitoids deal with such reactions (fig. 2). leptopilina boulardi and l. heterotoma are two closely related wasp species that parasitize larvae of drosophila with varying degrees of success (vass and nappi, 2000; dubuffet et al., 2007, 2008, 2009). eggs of avirulent wasps characteristically provoke a rapid hemocytemediated melanotic encapsulation response when introduced into the hemocoel of resistant drosophila, 199 fig. 2 leptopilina boulardi and l. heterotoma parasitize larvae of drosophila. genetically resistant hosts exhibit a typical hemocyte-mediated melanotic encapsulation response. susceptible host have a faulty non-self recognition mechanism or fail to produce effective toxic responses. immune suppressive factors (isf) in the venom of virulent wasps block host cellular immune response. an atypical host response exhibited by d. paramelanica readily destroys l. heterotoma, but the response does not involve melanotic encapsulation. fig. 3 comparative hemocyte profiles illustrating involvement of these cells in the host response, and the immune suppressive effects manifested by virulent wasps. 200 fig 4 genetic complexity exhibited by the varying degrees of immune reactivity and parasite survival in different species and stains of drosophila and leptopilina (courtesy s dupas, m poirié, y carton, laboratoire evolution, génomes et spéciation, cnrs, gif-sur-yvette cedex, france). whereas the eggs of virulent wasps survive host defenses. melanin typically appears at the site of infection, generally just before or at about the same time hemocytes are observed adhering to the surface of the dead parasitoid. the drosophila encapsulation response involves the collaborative activities of three types of hemocyte: plasmatocytes, lamellocytes and crystal cells. comparative examinations of hemocyte profiles show significantly elevated numbers of hemocytes in resistant or immune competent hosts (fig 3). during infection, plasmatocytes and lamellocytes show precocious increases in numbers as they participate in the formation of the cellular 201 components of the capsule, while crystal cells, which represent an important source of melanin precursors, decline in number (nappi and streams, 1969). some of the immune-activated hemocytes participating in the encapsulation response appear to be recruited from those already in circulation at the time of infection, while others are mobilized from hematopoietic glands (i.e., lymph glands) (lanot et al., 2001; sorrentino et al., 2002; meister and lagueux, 2003; meister, 2004; carton et al., 2005, 2008; crozatier and meister, 2007; honti et al., 2010) and/or a subepidermal population of normally sessile cells ( krzemien et al., 2007; markus et al., 2009). the genetic complexity of the drosophila-wasp associations is illustrated by the varied outcomes of the combative interactions made by different species and strains of both host are parasite (fig 4). the gene for host resistance, which is associated with the second chromosome, is specific for each species of wasp. reciprocal chromosome exchange between resistant and susceptible host strains virtually completely reverses immune competence in each recipient. immune competence is later restored following reciprocal return of the chromosome (fig. 5). during oviposition, wasp venom also is introduced into the host hemocel. virus-like particles (i.e., immune suppressive factors, isf) in the venom protect the wasp egg from encapsulation, either by lysing host hemocytes, or interfering with essential transcriptional responses so as to abrogate or diminish host responses. unlike isf of virulent wasps, those present in the venom of avirulent species and strains exhibit little or no immune suppressive effect (labrosse et al., 2003; kohler et al., 2007). in experiments involving double infections, first by avirulent wasps followed by a virulent strain of l. boulard, isf from virulent parasitoids were found capable of also protecting avirulent wasps, provided the interval between infections is 12 hrs or less (fig. 6). also, the ability of leptopilina spp. to immune suppress depends on her egg-laying experience. during the latter part of the ovipositional period of l. boulardi, eggs introduced into d. melanogaster larvae are more susceptible to melanotic encapsulation than are eggs laid earlier (fig. 7). the decrease in immune suppression presumably correlates with a corresponding depletion of isf (vass and nappi, 1998). wasps with prior ovipositional experience not only lack or have a diminished capacity to immune suppress, but they also infect far fewer hosts than females with no prior ovipositional experience. if such ovipositional restraint retains eggs that would otherwise be encapsulated, selection pressure in host populations for evolving specific immune reactivity would be reduced. melanization and associated toxic molecules in immune reactive insects, melanin appears in many cases to occur concurrently with early capsule formation, an observation that has long been viewed as evidence that the proteinase cascade leading to activation of one or more enzymes involved in fig. 5 reciprocal exchange of resistant and non-resistant gene reverses the immune capacity of the recipient, which can be restored in subsequent exchanges (carton and nappi, 1997). 202 fig. 6 in superparasitized hosts, immune suppressive factors from virulent parasitoids also protect avirulent wasps provided the interval between infections is 12 hrs or less. fig. 7 the effects of prolonged oviposition on the diminishing capacity of isf from l. boulardi (vass and nappi, 1998) and l. heterotoma (streams, 1968) to suppress the immune response of d. melanogaster. wasp eggs introduced into hosts by females during the latter part of their ovipositional period are more susceptible to destruction than eggs laid earlier. the increase in parasitoid mortality is believed to result from a decline in isf. 203 catechol metabolism and pigment synthesis forms toxic molecules that target and destroy foreign organisms (nappi and vass, 2001; sugumaran, 2002; nappi and christensen, 2005; sideri et al., 2008; an et al., 2009; bidla et al., 2009; nappi et al., 2009). support for this proposal derives in large part from studies showing diminished immune responsiveness when components of the enzymeregulated melanin pathway are experimentally inhibited. reservations about such an interpretation concern the questionable specificity and excessive levels of agents injected into the host to inhibit and thereby demonstrate involvement of melanin intermediates in immune reactions. frequently overlooked in studies assessing the role of melanin in insect immunity is the initial enzyme-mediated reaction involving the hydroxylation of l-phenylalanine to l-tyrosine, a reaction catalyzed by phenylalanine hydroxylase (pah). ensuing oxidations of l-tyrosine and/or ldopa, which can be catalyzed either by phenoloxidase (po; terland et al., 2006), or peroxidase (per; kasraee, 2002; okun, 1996), generate dopaquinoine, a reactive intermediate essential for the formation of eumelanin, a brownish-black pigment, and, in the presence of sufficient levels of thio compounds, pheomelanin, a reddish-brown pigment. precursors of both pigments possess cytoprotective and cytotoxic properties, given their capacity to scavenge potentially toxic organic and inorganic cations and free-radical species, engage in metal-binding and sequestering responses, initiate redox reactions, cross-link proteins and mediate detoxification processes. to date, only eumelanin has been identified as the pigment type formed in the encapsulation response of d. melanogaster (nappi et al., 1992). following the formation of dopaquinone, a series of enzyme-regulated and/or spontaneous oxidoreductions occur yielding dopachrome and fig. 8 overview of the principal pathways involved in the formation of eumelanin and pheomelanin and some their reactive intermediates, including quinones and semiquinones. redox cycling and univalent transfers, which represent important mechanisms for generating cytotoxic molecules, also occur with dhi-derived indolequinone (iq) and indolesemiquine (not illustrated). insects apparently are incapable of forming dhica. 204 fig. 9 effects of injection of spn27a on the percentage of d. melanogaster hosts exhibiting a successful melanotic encapsulation of l. boulardi. enzymes involved in melanin synthesis include dopa decarboxylase (ddc), dopachrome conversion enzyme (dce), phenylalanine hydroxylase (pah), peroxidase (per), and phenoloxidase (po). additionally potentially cytotoxic eumelanin intermediates, including 5,6dihydroxyindole (dhi), 5,6-dihydroxyindole-2-carboxylic acid (dhica), and their respective indole quinones (iq, and iqca) (fig. 8). the dopa decarboxylase (ddc)-mediated pathway to dhi may be a principal route for production of pigment precursors in infected drosophila, as the melanotic encapsulation response against eggs of l. boulardi is severely compromised in temperature-sensitive ddcdeficient mutants (nappi et al., 1992) accordingly, it was recently shown that silencing the genes for ddc and dopachrome conversion enzyme (dce) significantly reduced melanization of foreign objects implanted in the mosquito anopheles gambiae (paskewitz and andreev, 2008). in the medfly ceratitis capitata, ddc-dependent pathways have been shown to regulate such immune functions as phagocytosis, nodulation and melanization by hemocytes (sideri et al., 2008). 205 fig. 10 l. boulardi isf diminishes the in vitro oxidations of two diphenol eumelanin precursors, dopamine and dhi. tyrosine and the diphenols dopa and dhica are not affected by isf. parasite suppression of host melanization melanization in insects is controlled by a cascade of serine proteases that ultimately activates prophenoloxidase (ppo) and leads to activated phenoloxidase (po) and pigment formation (tang et al., 2006, 2008; scherfer et al., 2008; tang, 2009). in drosophila, melanization induced by activated po is a tightly regulated reaction sequence (aggarwal and silverman, 2008; kan et al., 2008) involving at least three ppo isoforms, as well as serine protease inhibitors. two isoforms are expressed in crystal cells, the third is associated with lamellocytes (kan et al., 2008). an important regulating element in the cascade of proteolytic cleavages that converts ppo to po is the serine protease inhibitor serpin 27a (spn27a), which inhibits the terminal protease prophenoloxidase-activating enzyme (ppae) (de gregorio et al., 2002; nappi et al., 2005) (fig. 9). because of the critical role played by spn27a as a negative regulator of melanogenesis, the molecule and the signaling elements mediating its activity likely represent critically important factors in determining immune reactivity against leptopilina. this was established by experiments involving the introduction of spn27a into immune competent d. melanogaster larvae just before infection by l. boulardi. in these hosts, the ability to form melanotic capsules was significantly reduced. the specificity of action of spn27a establishes some of the components of the po-mediated pathway in the insect's defense response against l. boulardi (fig. 9). more recent comparative investigations using isf from virulent and avirulent wasps provide additional evidence that isf inhibits melanization in d. yakuba by affecting one or more steps in the cascade leading to po activation, but not po activity by itself (dubuffet et al., 2009). other experiments designed to determine if venom factors from l. boulardi targeted the principal oxidation pathways leading to synthesis of eumelanin, sensitive electrochemical detection methods showed that venom factors diminished the oxidations of the two diphenol eumelanin precursors, dopamine and dhi, while oxidations of the monophenol tyrosine, and two other related 206 fig. 11 comparative analyses of hemocytes and nitric oxide levels in infected and non-infected larvae of d. paramelanica, and the effects of introducing a nos inhibitor (ng-monomethyl-l-arginine on the fate of l. heterotoma (nappi et al., 2009). diphenols, dopa and dhica, were not significantly inhibited (kohler et al., 2007) (fig. 10). collectively, these related studies suggest that, in addition to targeting specific hemocytes, isf from leptopilina spp. specifically suppresses the oxidation pathways synthesizing certain pigment precursors, especially the decarboxylated pigment precursors derived from dhi. reactions lacking evidence of the involvement of melanization it is generally believed that at least some of the melanogenic enzymes and intermediate pigment products play a role in the defense reactions of insects (cerenius and soderhall, 2004; christensen et al., 2005; nappi et al., 2009), although this issue 207 still remains to be clarified (schnitger et al., 2007). reports that would appear to discredit or at least down-play the role of melanogenesis in insect immunity and prompt additional or alternative proposals are those that document parasite mortality prior to, or in the absence of, melanotic encapsulation (tardieu and rabasse, 1988; henter and via, 1995; vernick et al., 1995), and those that clearly show successful parasite development despite extensive melanotic responses (shin et al., 2003). in the d. paramelanica–l. heterotoma association, eggs of the endoparasite succumb with no evidence of blood cell-mediated encapsulation and no pigment reaction (fig. 2) (nappi and streams, 1970; carton et al., 2008). if melanization is not a universal feature of insect cellular immunity, the destruction of some pathogen must involve host molecules other than those associated with melanogenesis, and one would expect successful parasites to have evolved specific inhibition strategies that suppress or detoxify such potentially biochemically hostile reactions. although identity of the cytotoxic molecules remains unknown, attention has focused on roi and rni, given that elevated levels of some of these molecules have been found in immune responsive hosts (luckhart and li, 2001; foley and o'farrell, 2003; novas et al., 2004; whitten et al., 2007; molina-cruz et al., 2008), including those in which hemocyte-mediated melanotic encapsulation reactions are typically formed (nappi and vass, 1998, 2001a, b; nappi et al., 1995, 2000b), and in wasp-infected d. paramelanica where parasites are destroyed but no melanotic capsules are produced (carton et al., 1992). potentially damaging roi and rni can form during normal metabolism as a result of successive univalent reductions of molecular oxygen. initially, superoxide anion is produced, with subsequent electron transfers ultimately generating highly reactive and potentially cytotoxic molecules, including the hydroxyl radical (.oh-), peroxynitrite (onoo–) and hypochlorous acid (hocl-). interestingly, melanogenic intermediates may serve to promote or augment cytotoxic activity by reacting with certain transition metal ions, roi and rni (fig. 1). the univalent oxidations of redox active o-diphenols (qh2) such as l-dopa and dopamine by po and/or per can form semiquinones (.qh-) and quinones (q), which then can interact with roi and rni. reactions involving per and tyrosine can lead to the production of potentially injurious molecules, such as the tyrosyl radical, dityrosine, and tyrosine peroxide (fig. 1), without producing melanin. an important issue to consider is that the production of toxic molecules in response to infection must by a tightly regulated and localized reaction in order to avoid damage to nonspecific sites within the host's open circulatory system. the binding of the copper-containing po or the heme-containing per to pathogens would expose the metal active sites of these enzymes, a response that would facilitate their interaction with roi and rni and form .ohand other reactive molecules, and also serve to localize metal ionmediated cytotoxicity. because of its intrinsic coordination properties, copper can induce a more site-specific .ohcytotoxicity to bound ligands than can iron (berthon, 1993). recent studies (carton et al., 2009) support earlier reports that document the involvement of .no in mediating various toxic responses in drosophila and other invertebrates. nitric oxide is a well known signaling molecule associated with certain innate immune pathways. the radical serves an equally important role as a toxic effector molecule in eliminating pathogens (nappi and ottavani, 2000a; nappi et al., 2000b; luckhart and li, 2001; dimopoulos, 2003; han et al., 2009). in d. paramelanica where elevated levels of .no are produced almost immediately following infection by l. heterotoma, immune capacity is diminished when a specific nitric oxide synthase (nos) inhibitor is introduced in larvae prior to infection (fig. 11). these observations suggest .no is involved in the host immune response, either a critical signaling molecule in recruiting hemocytes to sites of infection, or as a component of the insect's arsenal of defense, given the capacity of the radical to readily react with various roi and rni. conclusions the associations between host and pathogen represent coevolved adaptations of great complexity. insects typically manifest a unique defense response against metazoan parasites that involves hemocyte-mediated melanotic encapsulation. the use of melanin for protection from foreign insult involves a multifaceted biochemistry and an equally complex genetic regulation. an equally fascinating component of insect host-parasitoid combative relationships is the ability of some wasp species and strains to develop unmolested within otherwise immune competent hosts. either such parasitoids evolve with passive immune evasion strategies that effectively preclude host detection, or with the capacity to actively combat and render ineffective host defenses. despite numerous descriptive accounts of non-self responses and associated cell-signaling molecules that summon blood cells to sites of infection, much remains to be learned about the identity of the killing molecules employed by insect hosts, knowledge of which would enhance current efforts to better define immune cell-signaling pathways, and most likely contribute to a more comprehensive understanding than presently exists of receptor-mediated processes involved in detection of non-self. the mechanism employed by pathogens to suppress cellular innate immunity in insects and other invertebrate hosts may likewise contribute to our understanding of immune signaling and non-self discrimination. studies that merely correlate host immune competence or parasite virulence with the presence or absence of melanin fail to provide substantive information about the actual cytotoxic mechanism(s) involved and how the pathogen circumvents such hostile chemistry. future proteomic and transcriptomic studies of parasitoid isf will likely facilitate identification of the cytotoxic molecules, the cell-signaling pathways that regulate their synthesis, and their mode of target-specific engagement with foreign organisms. 208 references aggarwal k, silverman n. positive and negative regulation of the drosophila immune response. bmb rep. 41: 267-277, 2008. an c, ishibashi j, ragan ej, jiang h, kanost mr. functions of manduca sexta hemolymph proteinases hp6 and hp8 in two innate immune pathways. j. biol. chem. 284: 1971619726, 2009. becker t, loch g, beyer m, zinke i, aschenbrenner ac, carrera p, et al. foxo-dependent regulation of innate immune homeostasis. nature 463: 369-373, 2010. berthon g. is copper proor anti-inflammatory? a reconciling view and a novel approach for the use of copper in the control of inflammation. agents actions 39: 210-217, 1993. beutler b. innate immunity: an overview. mol. immunol. 40: 845-859, 2004. bidla g, hauling t, dushay ms, theopold u. activation of insect phenoloxidase after injury: endogenous versus foreign elicitors. j. innate immunol. 1: 301-308, 2009. carton y, frey f, nappi aj. inheritance of cellular immune resistance in drosophila melanogaster. heredity 69: 393-399, 1992. carton y, frey f, nappi aj. parasite-induced changes in nitric oxide levels in drosophila paramelanica. j. parasitol. 95: 1134-1141, 2009. carton y, nappi aj. drosophila cellular immunity against parasitoids. parasitol. today 13: 218227, 1997. carton y, nappi aj, poirie m. genetics of antiparasite resistance in invertebrates. dev. comp. immunol. 29: 9-32, 2005. carton y, poirie m, nappi aj. insect resistance to parasitoids. insect sci. 15: 67-87, 2008. cerenius l, soderhall k. the prophenoloxidaseactivating system in invertebrates. immunol. rev. 198: 116-126, 2004. christensen bm, li j, chen c-c, nappi aj. melanization immune responses in mosquito vectors. trends parasitol. 21: 192-199, 2005. crozatier m, meister m. drosophila haematopoiesis. cell. microbiol. 9: 1117-1126, 2007. de gregorio e, han sj, lee wj, baek mj, osaki t, kawabata si, et al. an immune-responsive serpin regulates the melanization cascade in drosophila. dev. cell 3: 581-592, 2002. dimopoulos g. insect immunity and its implication in mosquito-malaria interactions. cell. microbiol. 5: 3-14, 2003. dubuffet a, colinet d, anselme c, dupas s, carton y, poirie m. variation of leptopilina boulardi success in drosophila hosts: what is inside the black box? adv. parasitol. 70: 147188, 2009. dubuffet a, doury g, labrousse c, drezen jm, carton y, poirie m. variation of success of leptopilina boulardi in drosophila yakuba: the mechanisms explored. dev. comp. immunol. 32: 597-602, 2008. dubuffet a, dupas s, frey f, drezen jm, poirie m, carton y. genetic interactions between the parasitoid wasp leptopilina boulardi and its drosophila hosts. heredity 98: 21-27, 2007. foley e, o'farrell ph. nitric oxide contributes to induction of innate immune responses to gramnegative bacteria in drosophila. genes dev. 17: 115-125, 2003. han y, niu m, an l, li w. upregulation of proinflammatory cytokines and no production in bv-activated avian macrophage-like cell line (hd11) requires mapk and nf-kappab pathways. int. immunopharmacol. 9: 817-823, 2009. henter h, via s. the potential for coevolution in a host-parsitoid system. i. genetic variation within an aphid population in susceptibility to a parasitic wasp. evolution 49: 427-438, 1995. honti v, csordás g, márkus r, kurucz e, jankovics f, andó i. cell lineage tracing reveals the plasticity of the hemocyte lineages and of the hematopoietic compartments in drosophila melanogaster. mol. immunol. 47: 1997-2004, 2010. kan h, kim ch, kwon hm, park jw, roh kb, lee h, et al. molecular control of phenoloxidaseinduced melanin synthesis in an insect. j. biol. chem. 283: 25316-25323, 2008. kohler lj, carton y, mastore m, nappi aj. parasite suppression of the oxidations of eumelanin precursors in drosophila melanogaster. archiv. insect biochem. physiol. 66: 64-75, 2007. krzemien j, dubois l, makki r, meister m, vincent a, crozatier m. control of blood cell homeostasis in drosophila larvae by the posterior signalling centre. nature 446: 325328, 2007. labrosse c, carton y, dubuffet a, drezen jm, poirie m. active suppression of d. melanogaster immune response by long gland products of the parasitic wasp leptopilina boulardi. j. insect physiol. 49: 513-522, 2003. lanot r, zachary d, holder f, meister m. postembryonic hematopoiesis in drosophila. dev. biol. 230: 243-257, 2001. luckhart s, li k. transcriptional complexity of the anopheles stephensi nitric oxide synthase gene. insect biochem. mol. biol. 31: 249-256, 2001. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. malagoli d, ottaviani e. helical cytokines and invertebrate immunity: a new field of research. scand. j. immunol. 66: 484-485, 2007. malagoli d, sacchi s, ottaviani e. lectins and cytokines in celomatic invertebrates: two tales with the same end. inv. surv. j. 7: 1-10, 2010. markus r, laurinyecz b, kurucz e, honti v, bajusz i, sipos b, et al. sessile hemocytes as a hematopoietic compartment in drosophila melanogaster. proc. natl. acad. sci. usa 106: 4805-4809, 2009. meister m. blood cells of drosophila: cell lineages and role in host defence. curr. opin. immunol. 16: 10-15, 2004. meister m, lagueux m. drosophila blood cells. cell. microbiol. 5: 573-580, 2003. 209 molina-cruz a, dejong rj, charles b, gupta l, kumar s, jaramillo-gutierrez g, et al. reactive oxygen species modulate anopheles gambiae immunity against bacteria and plasmodium. j. biol. chem. 283: 3217-3223, 2008. nappi a, poirie m, carton y. the role of melanization and cytotoxic by-products in the cellular immune responses of drosophila against parasitic wasps. adv. parasitol. 70: 99121, 2009. nappi aj, christensen bm. melanogenesis and associated cytotoxic reactions: applications to insect innate immunity. insect biochem. mol. biol. 35: 443-459, 2005. nappi aj, frey f, carton y. drosophila serpin 27a is a likely target for immune suppression of the blood cell-mediated melanotic encapsulation response. j. insect physiol. 51: 197-205, 2005. nappi aj, ottavani e. cytotoxicity and cytotoxic molecules in invertebrates. bioessays 22: 469480, 2000. nappi aj, streams fa. haemocytic reactions of drosophila melanogaster to the parasites pseudocoila mellipes and p. bochei. j. insect. physiol. 15: 1551-1566, 1969. nappi aj, streams fa. abortive development of the cynipid parasite pseudocoila bochei (hymenoptera) in species of the drosophila melanica group. ann. entomol. soc. am. 63: 321-327, 1970. nappi aj, vass e (2001). cytotoxic reactions associated with insect immunity. in: beck g, sugumaran m, cooper el (eds), phylogenetic perspectives on the vertebrate immune system, kluwer academic-plemum, new york, pp 329348, 2001. nappi aj, vass e, carton y, frey f. identification of 3,4-dihydroxyphenylalanine, 5,6dihydroxyindole, and n-acetylarterenone during eumelanin formation in immune reactive larvae of drosophila melanogaster. arch. insect biochem. physiol. 20: 181-191, 1992. nappi aj, vass e, frey f, carton y. nitric oxide involvement in drosophila immunity. nitric oxide 4: 423-430, 2000b. novas a, cao a, barcia r, ramos-martinez ji. nitric oxide release by hemocytes of the mussel mytilus galloprovincialis lmk was provoked by interleukin-2 but not by lipopolysaccharide. int. j. biochem. cell biol. 36: 390-394, 2004. pal s, wu lp. lessons from the fly: pattern recognition in drosophila melanogaster. adv. exp. med. biol. 653: 162-174, 2009. paskewitz sm, andreev o. silencing the genes for dopa decarboxylase or dopachrome conversion enzyme reduces melanization of foreign targets in anopheles gambiae. comp. biochem. physiol. 150b: 403-408, 2008. scherfer c, tang h, kambris z, lhocine n, hashimoto c, lemaitre b. drosophila serpin28d regulates hemolymph phenoloxidase activity and adult pigmentation. dev. biol. 323: 189-196, 2008. schnitger ak, kafatos fc, osta ma. the melanization reaction is not required for survival of anopheles gambiae mosquitoes after bacterial infections. j. biol. chem. 282: 2188421888, 2007. shin sw, kokoza va, raikhel as. transgenesis and reverse genetics of mosquito innate immunity. j. exp. biol. 206: 3835-3843, 2003. sideri m, tsakas s, markoutsa e, lampropoulou m, marmaras vj. innate immunity in insects: surface-associated dopa decarboxylasedependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes. immunology 123: 528-537, 2008. sorrentino rp, carton y, govind s. cellular immune response to parasite infection in the drosophila lymph gland is developmentally regulated. dev. biol. 243: 65-80, 2002. streams fa. factors affecting the susceptibility of pseudeucoila bochei eggs to encapsulation by drosophila melanogaster. j. invertebr. pathol. 12: 379-387, 1968. sugumaran h. comparative biochemistry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects. pigm. cell. res. 15: 2-9, 2002. tang h. regulation and function of the melanization reaction in drosophila. fly (austin) 3: 105-111, 2009. tang h, kambris z, lemaitre b, hashimoto c. two proteases defining a melanization cascade in the immune system of drosophila. j. biol. chem. 281: 28097-28104, 2006. tang h, kambris z, lemaitre b, hashimoto c. a serpin that regulates immune melanization in the respiratory system of drosophila. dev. cell 15: 617-626, 2008. tardieu i, rabasse m. some aspects of host immunity and physiological suitability in aphids attacked by aphidius colemani. in: anderson afg (ed), ecology and effectiveness of aphophaga, the hague, academic publishing, pp 311-315, 1988. vass e, nappi aj. prolonged oviposition decreases the ability of the parasitoid leptopilina boulardi to suppress the cellular immune response of its host drosophila melanogaster. exp. parasitol. 89: 86-91, 1998. vass e, nappi aj. developmental and immunological aspects of drosophilaparasitoid relationships. j. parasitol. 86: 12591270, 2000. vernick kd, fujioka h, seeley dc, tandler b, aikawa m, miller lh. plasmodium gallinaceum: a refractory mechanism of ookinete killing in the mosquito, anopheles gambiae. exp. parasitol. 80: 583-595, 1995. whitten m, sun f, tew i, schaub g, soukou c, nappi a, et al. differential modulation of rhodnius prolixus nitric oxide activities following challenge with trypanosoma rangeli, t. cruzi and bacterial cell wall components. insect biochem. mol. biol. 37: 440-452, 2007. 210 comparative aspect of propo system in invertebrates isj 6: s67-s76, 2009 issn 1824-307x review the ascidian prophenoloxidase activating system m cammarata, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 abstract phenoloxidases/tyrosinases initiate melanin synthesis in almost all organisms, and are involved in different biological activities such as the colour change of human hair and the browning or blackening of fruit skin etc. in many invertebrates, defence reactions are linked to phenoloxidase activity and/or melanization. contacts with foreign molecules are able to trigger the prophenoloxidase (propo) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (po). it is generally accepted that the propo system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect propo activation steps have been achieved. after cells were stimulated by components of pathogen associated molecular pattern (pamp), propo activation takes place via zimogenic serine proteinase in turn activated by pamps followed by cascade, spatial and temporal control. the propo activating system plays a defensive role in arthropods, molluscs, annelids, ascidians and the cephalochordate branchiostoma belcheri. in the present paper, we report on ascidian propo system and related molecules, with particular focus on the biochemical, cellular and molecular aspects of the ciona intestinalis, propo system of circulating hemocytes from naïve ascidians as well as of body wall following lps inflammatory challenge. key words: ciona intestinalis; ascidians; propo; phenoloxidase; immune response; hemocytes phenoloxidases and related enzymes melanin is a pigment ubiquitous throughout the animal kingdom. in invertebrates melanization is related to phenoloxidase (po) and in part to tyrosinase, both are copper-dependent enzymes (monophenol, l-dopa; oxygen oxidoreductase; ec 1.14.18.1), that share similar active sites and catalyse the o-hydroxylation of monophenols (monophenoloxidase or cresolase activity) and the subsequent oxidation of the reaction products (odiphenols) to quinones. thus, substances forming copper ion complexes can be enzyme inhibitors (kahn, 1985; sugumaran et al., 1988). tyrosine is the natural substrate of tyrosinase, which exhibits a lag–phase during the tyrosine conversion ascribed to an autocatalytic mechanism due to the production of l-dopa in the initial phase of the reaction pathway (lerner, 1949). ________________________________________________________________________ corresponding author: matteo cammarata marine immunobiology laboratory department of animal biology university of palermo via archirafi, 90123 palermo, italy e-mail: camat@unipa.it vertebrate tyrosinases form dimers whereas pos, only found in invertebrates, form oligomers, from monomers to pentamers. although, both present two sites containing copper vary in their remaining sequence. in invertebrates, the prophenoloxidase (propo) is converted to po by proteolytic cleavage. the activation depends upon a cascade due to hemolymph proteases which are sensitive to peptidoglycans and lipopolysaccharides (lpss) or other bacterial carbohydrates or fungal β-glucans. in crayfish hemocytes propo is a 76 kda glycoprotein that, after activation by β-1.3-glucans, is cleaved by specific serine proteases to produce the 60 kda active po (aspan and söderhäll, 1991). a similar cascade has been reported in other invertebrates (beschin et al., 1998; parrinello et al., 2001; lunagonzales et al., 2003). molecular analysis of po and related protein pos can be sharply distinguished from tyrosinase and an independent evolution with short sequence traits conservation has been proposed. s67 fig. 1 a. the conserved domain architecture of c. intestinalis phenoloxidases performed by similarity searches of the ncbi entrez protein database (cdart). b. sequence alignments of c. intestinalis prophenoloxidases for comparison with arthropod pos, hemocyanins and various tyrosinases. the sequences shown are segments corresponding to the copper a and b binding sites of the hemocyanins. the homologous aa are shown in boldface, the three histidine residues (h) participating in the cu atom are shown in red. conversely, a close similarity between arthropod phenoloxidases and hemocyanin, an oxygen carrier protein, has been shown (söderhäll et al., 1996). both proteins present sequence similarity and contain two oxygen-binding sites that reversibly bind two copper atoms (decker and tuczek, 2000), moreover hemocyanin-like proteins can act as phenoloxidases (immesberger and burmester, 2004). like propo, hemocyanins can be activated in vitro by sds, trypsin and other effectors with denaturating property (decker et al., 2001; lee et al., 2004), or by chitin-binding antimicrobial peptides (nagai et al., 2001). this activation has been imputed to the cleavage of crayfish hemocyanin subunit 2 at the n-terminal part (lee et al., 2004). decker et al. (2004) described similar results on the tarantula hemocyanin, and hypothesized that after n-terminal cleavage the hemocyanin active site becomes accessible also for phenolic substances. propos from different arthropods have been cloned and sequenced (aspan et al., 1995; fujimoto et al., 1995; hall et al., 1995; kawabata et al., 1995). comparative sequence analysis (söderhäll et al., 1996), including several copper-containing proteins, showed that propos disclose sequence similarity to hemocyanins higher (49-59%) than to tyrosinases (3335 %) (parrinello et al., 2003). s68 table 1 possible genes involved in the c. intestinalis propo activated system cinpo-1 cinpo-2 peroxinectin-like gene cu-zn sod-like gene mrna aj7547813 aj7547814 predicted xp_002126285 predicted xp_002122526 protein complete; 794 aa partial at n-term; start atg absent. 774 aa (768 aa in jgi:279870) complete; 960 aa complete; 154 aa putative size (kda) 92.0 86.9 105.0 15.6 domain/binding sites cu binding sites cu binding sites peroxidase/cell binding site cu-zn reference burmester et al 2003 burmester et al 2003 present paper present paper immesberger and burmester (2004) cloned and sequenced two ciona intestinalis po cdnas (cinpo-1 and cinpo-2). cinpo-1 and cinpo-2, with predicted molecular masses of 92.0 kda and 86.9 kda respectively, showed 43.2 % identity and do not contain signal peptides indicating a non classical release. figure 1 shows the sequence alignment of c. intestinalis po sites compared to arthopod pos, hemocyanins and some tyrosinases, and displays a close similarity between pos and hemocyanins. a third putative cinpo showing high similarity to cinpo-2 had been identified by a search on jgi ciona intestinalis v2 as cinpo-3 (cammarata et al., 2008). however, after a further analysis cinpo-3 appeared to be a product of an uncorrect assembly of the sequences in the v2 genome version. in fact, a long stretch of undetermined nucleotides, at the scaffold end, explained the assumed high similarity of introns and esons as well as of the presumptive protein sequences. propo activating system the first defence line of the innate immunity includes po pathway products that participate in several responses such as melanization and encapsulation, cytotoxicity, phagocytosis, clotting, microbial killing and wound repair (söderhäll, 1982; cammarata et al., 1997; gillespie et al., 1997; huang et al., 2000; nagai and kawabata, 2000; nappi and ottaviani, 2000; cerenius and söderhäll, 2004; jiravanichpaisal et al., 2006; cerenius et al., 2008). it has been proposed that a molecular crosstalk takes place between the propo system and cellular defence responses which share activation by microbial products signals, such as coagulation and blood cell degranulation (lemaitre and hoffmann 2007; cerenius et al., 2008). crustacean granular and semigranular hemocytes contain propo, and, upon exposure to bacteria, they undergo degranulation in vitro leading to exocytosis (johansson and söderhäll, 1989b, 1999). in crayfish, pacifastacus leniusculus, the 76kda peroxinectin, purified from the hemocytes (johansson et al., 1995), mediates the attachment and spreading of hemocytes in vitro (johansson and söderhäll, 1988), and stimulates degranulation events when added to granular cells monolayer (johansson and söderhäll, 1989a, b). peroxinectinlike is a cell adhesion protein also detected in shrimp (poeneus monodon) hemocyte lysate supernatant (sritunyalucksana et al., 2001). sequence comparison shows that the shrimp protein is similar to crayfish peroxinectin (69 %) and to various peroxidases or putative peroxidases from invertebrates and vertebrates. since the biological effect of crayfish hemocyte peroxinectin is mimicked by the peptide gly-arg-glyasp-ser (grgds) (johansson and söderhäll, 1989c), the possibility exists that crayfish hemocyte integrin-like receptors recognize and bind rgd or kdg (rouslahti, 1996; holmblad et al., 1997). vertebrate integrins form a family of integral membrane proteins that act in cell-cell adhesion and as receptors in trans-membrane signalling (hines, 1992). peroxinectin may act as opsonin, and promote the adhesion of bacteria or other particles to the phagocyte surface, facilitating their subsequent ingestion by the cell. peroxonectin also binds cuzn-superoxide dismutase (cuznsod) at the surface of circulating hemocytes, and this interaction, facilitated by the close localization, modulates both the enzyme activities. the hydrogen peroxide, produced by the superoxide dismutases, can be substrate for the peroxinectin and antimicrobical substances (johansson et al., 1999). therefore, the cuznsod is involved in arthropod propo activating system. we carried out a bioinformatic analysis and, in table 1, show for the first time that the c. intestinalis predicted peroxinectin-like gene contains both the active site of the peroxidase and the cell binding site (gly-arg-gly-asp-ser, lkkgdr), moreover the deduced aminoacid complete sequence reveals 35 % identity with p. leniulusculus peroxinectin. in c. intestinalis genome, the presence of eleven alpha and five β chain integrin genes, suggest putative peroxinectin cell surface s69 table 2 properties and modulation of the ascidian phenoloxidases c. intestinalis s. plicata b.schlosseri p. mamillata h. roretzi references (1-7) (5,7,8) (9,10) (5,11) (12) treatment hls ths hls trypsin trypsin + sti -------------------- lps po inhibitors calcium effect no no no yes no no po containing cell types urg granular amebocytes morula morula compartment cell granular hemocytes nd po subunit mw 74 90 nd 80 70 62 method cloned hls isolated hls isolated biological activities cytotoxicity cytotoxicity non fusion reaction nd antimicrobial activity hemocyte lysate supernatant (hls); tunic homogenate supernatant (ths); unilocular refractile granulocytes(urg) (1) söderhäll and smith, 1992; (2) peddie and smith, 1993; (3) parrinello et al., 1995; (4) cammarata et al., 1996; (5) parrinello et al., 2003; (6) cammarata et al., 2008; (7) arizza et al., 1995; (8) cammarata et al., 1997; (9) ballarin et al., 1994; (10) ballarin et al., 2008; (11) cammarata et al., 1999; (12) hata et al., 1998 adhesion receptors (ewan et al., 2005). in table 1, we also report the sequence of a c. intestinalis putative gene cu-zn sod with 46 % identity to the predicted aminoacid sequence of the h. roretzi enzyme, obtained by using the aminoacid sequence of h. roretzi cu-znsod (abe et al., 1999) as a query in an aminoacid-based blast search (tblastn) versus the ncbi/genbank database. ascidian propo activating system and innate immunity ascidians occupy a key phylogenetic position in the evolutionary line leading to vertebrates (hori and osawa 1987; field et al., 1988; zeng and swalla 2005; delsuc et al., 2006), therefore both solitary and colonial ascidians are of interest in studying the evolution of defence mechanisms. phagocytosis, cytotoxicity, encapsulation and tissue damage (wright and cooper, 1983; parrinello and patricolo, 1984; parrinello et al., 1984, 2001, 2007; ballarin et al., 2008) in inflammatory responses, as well as in inflammatory events linked to allorecognition responses, have been shown (sabbadin, 1982; rinkevich, 1992; raftos et al., 1988). both share hemocytes degranulation in tissues of solitary ascidians (parrinello et al., 1984) and in the contact area of incompatible botryllid colonies (sabbadin, 1982; ballarin, 2008). differently than arthropod pos, which are monophenoloxidases, ascidian hemocyte pos are orthodiphenoloxidases. o-diphenol oxidase activity and phenolic compounds were at first identified by histochemical reaction (barrington and thorpe, 1968) in the tunic hemocytes suggesting a quinonetanning system involved in the production of tunic scleroprotein (chaga, 1980). although ascidian pos show the highest activity at 6-9 ph range (jackson et al., 1993; arizza et al., 1995; ballarin et al., 1994), they can differ in several biochemical properties. in botryllus schlosseri the ldopa oxydizing activity is enhanced by divalent cations (ballarin el al., 1994), whereas the po activity of other ascidian species does not appear to be ca2+ or mg2+-dependent (jackson et al., 1993; arizza et al., 1995; cammarata et al., 1999). in crustaceans, although calcium ions are requested, a high cation concentration exerts a suppressive effect. a further difference concerns the activating mechanism: β 1,3-glucans and oligosaccharides induce propo-activation in arthopods (söderhäll and smith, 1986; söderhäll, 1992) and in solitary ascidian c. intestinalis (jackson et al., 1993), whereas do not activate styela plicata (arizza et al., 1995) and b. schlosseri (ballarin et al., 1994) pos. ascidian orthodiphenoloxidases are copperdependent enzymes inhibitable by copper chelating substances (kahn, 1985; sugumaran et al., 1988). like in arthropods, the ascidian propo requires proteolytic cleavage for its activation. the level of po activity is significantly higher after incubation with serin-proteases, decreases as an effect of protease inhibitors, and it is activated by lps (table 2). smith and peddie (1992) and jackson et al. (1993) reported that the lps-sensitive protease activity, contained in the c. intestinalis hemocyte lysate supernatants, may be associated with in vivo prophenoloxidase activaction. benzamidine, soybean trypsin inhibitor (sti), phenylmethylsulphonyl fluoride, tosyl phenylalanyl s70 chloromethyl ketone, tosyl-l-lysin-chloromethyl ketone inhibited both protease and propo activation, this inhibitory activity is short lived and precedes hemocyte po activity (jackson and smith, 1993). at least three proteases are contained in solitary ascidians c. intestinalis, s. plicata (unpublished data) and phallusia mamillata hemocytes (guerrieri et al., 2000), and could regulate the otherwise uncontrolled protease activity. po containing hemocytes phenoloxidase participates in tunic formation (chaga, 1980), melanin production and non fusion reaction (ballarin et al., 1996), and an increased po activity can be found in c. intestinalis tissues inflamed by lps inoculation (parrinello et al., 2001). in c. intestinalis (smith and söderhäll, 1991) and b. schlosseri (ballarin et al., 1993) po oxidative activity of circulating hemocytes from naïve ascidians as well as hemocyte lysate supernatants has been revealed by l-dopa reaction. a similar activity has also been reported in ascidia mentula, ascidia virginea, ascidiella scabra, ascidiella aspersa, polycarpa pomaria, dendrodoa grossularia and morchellium argus (jackson et al., 1993). the specificity of the po reaction of s. plicata, p. mamillata and c. intestinalis hemocytes (arizza et al., 1995; parrinello et al., 2003) has been supported by tropolone, phenylthiourea and diethyltiocarbamate usually used as inhibitors (sugumaran et al., 1988; cadenas, 1989; kahn, 1995). the enzymatic assay of the hemocyte lysate and the cytochemical reaction of the hemocytes revealed a limited heterogeneity in the ascidian pocontaining cell types. po-activity as well as the possible substrates tunichrome and halocyaminereducing polyphenol, have mainly been identified in the hemocytes called "morula cells" (azumi et al., 1990; he et al., 1992; ballarin et al., 1996; parrinello et al., 2003). evidence of po positive “morula cells” have also been drawn by assaying hemocyte populations enriched through a density gradient separation of the hemolymph from c. intestinalis (jackson et al., 1993; parrinello et al., 2003), b. schosseri (ballarin et al., 1994) and s. plicata (arizza et al., 2005). this hemocyte-type is a round globular granulocyte (berry-like under the light microscope; 5.5-11.0 µm diameter) with large granules containing material of various electrondensity (de leo, 1992). light microscopy observations show large globular granules varying in number, shape and content feature. although smith and peddie (1992) suggested that c. intestinalis morula cells contain po, we found a weak reaction after the treatment with l-dopa-mbth. on the contrary, a strong po activity was found in the unilocular refractile granulocytes (urgs) identified in the hemolymph and characterized by a single popositive large granule that occupies the largest part of the cytoplasm (parrinello et al., 2003). in addition po-positive large granules were also found in granulocytes. although a defined differentiation line was not established, the possibility exists that granulocytes with large granules, urgs and morula cells may be components of a lineage characterized by a different state of the granule content including po activity level as well as hemocyte functions. accordingly, urgs exert po-dependent cytotoxic activity whereas morula cells do not show any cytotoxic activity against erythrocytes (parrinello et al., 1996) and tumour cell lines (peddie and smith, 1993). in p. mamillata, “hemocytes with large granules” show a low po activity whereas the morula cells do not show any positive reaction with l-dopa-mbth. the activity of the hemocyte lysate supernatant of enriched populations, obtained through a percoll density gradient, can be enhanced by trypsin that presumably activates the propo remaining after cell lysis. usually, hemocytes preparation and further treatments may cause the activation of a part of propo content by endogeneous proteases. in this ascidian, another hemocyte type named compartment cells characterized by few large vacuoles show spontaneous propo activation, and vacuoles appear to be po-positive. however, the lysate supernatant from enriched populations are less sensitive to trypsin proteolysis activation indicating that these cells may be more reactive to handling which can activate propo. proteolytic activation of propo is sustained by electrophoresis and l-dopa-mbth stain of trypsin-treated and untreated hemocytes with large granule lysate supernatants; the electropherograms show an increased mobility of the enzyme after proteolysis and, accordingly, reveal an additional protein fragment (parrinello et al., 2003). tunic pos and inflammatory response recently, the po activity of tunic homogenate supernatant (ths) from naïve c. intestinalis has been assayed (cammarata et al., 2008). as already reported upon the hemocyte lysate supernatant (hls), the po activity of ths is ca2+-independent, but, unlike hls (ph 6-9) requires a lower ph (7-8) and is more thermo-stable. the ths activity is lost after two days at 0 °c and after about one year at 80 °c, whereas the hls activity disappears after 2-3 h at 0 °c and 3-4 week at -80 °c. likewise the hlspropo, the treatment with exogenous trypsin enhances the activity and sti inhibits it, whereas a further difference resulted from a more effective activation due to lps acting at a lower concentration than that needed for activating hls-propo. in addition, since lps inoculation enhances the thspo activity of samples assayed in the absence of trypsin the possibility exists that in the tunic several serine proteases or different proteases could be involved in the activation phase (cammarata et al., 2008). to check for tunic matrix and tunic cells po activity in the inflammatory response, an in vitro enzyme assay of the inflamed tunic tissue excised at 24 or 48 h after lps inoculation has been performed. the po activity appears to be distributed throughout the tunic matrix, as well as inside the large granule of urgs and hemocytes with large granules. these observations are in accordance with the report on circulating hemocytes from naïve ascidians, whereas it is of interest that morula cells in the inflamed tunic disclose an evident po activity suggesting a distinct step of the cell lineage not found in circulating hemolymph from naïve ascidians s71 and presumably due to the lps challenge. microscope observations of wet inflamed tunic fragments after l-dopa-mbth treatment, showed both a strong po reaction and a high density of popositive hemocyte populations (fig. 2). since the deduced aminoacid sequence of a c. intestinalis cinpo-2 isoform has been reported (genbank accession n. aj547814) by immersberg and burmester (2004), a peptide (11-aa, efhndrrnrgf) has been selected through an antigen-prediction program, and anti-cinpo-2peptide specific antibodies has been raised in rabbits (cammarata et al., 2008). the immunohistochemistry reaction shows an intense dye of the inflammatory cells supporting that propo2 synthesis could be enhanced by lps inoculation (cammarata et al., 2008). the same assay revealed that, after lps inoculation, the enzyme is distributed in the tunic matrix, mainly in the outer layer, in addition anti-cinpo-2 antibodies also marked the pharynx vessel epithelia. immesberger and burmester (2004) reported that 86.9 kda is the predicted molecular size of cinpo-2, and this value fits with the 90kda revealed by the l-dopa-mbth reaction and immunoblotting analysis of ths from naïve ascidians (cammarata et al., 2008). after the lps inoculation, an additional 120 kda band reacts with l-dopa-mbth and anticinpo2 antibodies, whereas a 170 kda l-dopambth positive band does not react with the antibodies. presumably, this band could be considered as a dimeric form of cinpo-1 (92.0 kda) which cannot be identified by antibodies directed to a cinpo-2 peptide. anyway, an oligomerisation process may be responsible of cinpos higher in size (120 and 170 kda). a similar process may be taken in account for the differences observed in the thsand hls-po (parrinello et al. 2003) molecular sizes, and suggests that further analyses are needed. biological activity of po and related molecules in invertebrates, substances with cytotoxic activity include molecular derivates of oxygen and nitrogen, antimicrobial peptides, lectins and quinoid intermediates of melanin (parrinello 1996; nappi and ottaviani 2000). the oxygen reactive forms, such as superoxide anions, hydroxyl radicals, and hydrogen peroxide anions have been implicated as components of the cytotoxic mechanisms of vertebrates (cadenas, 1989) and invertebrates (bell and smith, 1993; anderson, 1994; valembois and lassegues, 1995; nappi and ottaviani 2000). the propensity of quinones for redox-cycling makes these eumelanin precursors potential sources of reactive forms of oxygen (riley, 1988; o'brien, 1991). indeed, it has been demonstrated (smit et al, 1996) that phenolic compounds, converted into toxic products by tyrosinase, exhibit cytotoxic activity towards human melanoma cells. we reported that the morula cells form s. plicata (cammarata et al., 1997) and urgs from c. intestinalis (parrinello et al., 1995) display podependent cytotoxic activity against tumour cell lines (unpublished data) and rabbit erythrocytes due to quinones which are known to be cytotoxic (pawelek and lerner, 1978; cotelle et al., 1991; fu et al., 1994; parrinello et al., 2003).the presence of quinones, possibly originated by po-driven tunichrome oxidation, has been reported in ascidians. differently, roi derived from the po pathway are cytotoxic factors in b. schosseri nonfusion alloreaction (ballarin et al., 2002, 2008). indeed, in vivo, quinones could be activated by enzymatic reduction as, under aerobic conditions, the semiquinone radical autoxidizes and forms superoxide anion radicals (nappi and vass, 1993). the superoxide anion, hydrogen peroxide and trace amounts of transitional-metal ions form a hydroxyl radical (fenton reaction), which is the most toxic of all oxygen products. there is only indirect evidence that, in c. intestinalis, propo-activation products have a stimulatory influence on hemocyte behaviour in vitro. in this ascidian, the crude hemocyte lysate supernatant, which contains active po and lpssensitive proteases, has a marked opsonic influence on the uptake of bacteria by phagocytic amoebocytes (smith and peddie, 1992). such opsonic potential match to the degree of the enzyme activity; in addition, po and proteases inhibition with benzamidine or sti precludes the opsonic effect, while pre-treatment of the lysate supernatants with lps produces a greatly elevated phagocytic response (smith and peddie, 1992). figure 3 shows a tentative model of the c. intestinalis propo activating system, based on present knowledge. finally, morula cells can release propo-activation products for tunic formation and regeneration in goniocarpa fig. 2 tunic fragment of c. intestinalis 24 h treated with l-dopa-mbth after the inoculation of lps. arrowhead indicates the po reaction at the injection site. bar (a) = 1 cm; (b) = 200 µm. s72 fig. 3 a model for the activation and modulation of the pro-po system in c. intestinalis, based on the following reference results: söderhäll and smith, 1992; peddie and smith, 1993; parrinello et al., 1995; cammarata et al., 1996, 1997; parrinello et al., 2003; cammarata et al., 2008. rustica, halocynthia aurantium (chaga, 1980) and b. schlosseri (zaniolo, 1981), and can be involved in the formation of clotting (wright, 1981), and in the encapsulation of foreign particles (anderson, 1971; parrinello, 1996). conclusions the melanogenetic pathway can be dependent on tyrosinase or phenoloxidase activity, the propo activating system appears to be a sophisticated system which represents an evolutionary independent defence mechanism characteristic of invertebrates. apparently protostomes and deuterostomes share propo and po-related factors and activities. phenoloxidase activity and propo-like components have been reported in the hemolymph of arthropods, annelids, molluscs, echinoderms and cephalocordata (roch et al., 1992; nappi and vass, 1993; beschin et al., 1998; luna-gonzales et al., 2003; pang et al., 2005). preliminary sequence analysis suggest that hemocyanin could be the evolutionary ancestor of po and a parallel molecular evolution cannot be excluded. taking in account the c. intestinalis inflammatory response challenged by lps, the propo system appears to be a component (cammarata et al., 2008) of a very complex reaction that presumably involves several interacting cell types and factors, namely inducible cytokine-like molecules (parrinello et al., 2007), cifacitcollagen (vizzini et al., 2007), and citnfα (parrinello et al., 2008). the lps or glucans activating effect could be associated with one or more serine proteases modulated by trypsin inhibitors, cu-znsod and peroxinectin-like molecules. therefore, lpsand glucan-binding proteins appear to be suitable candidates as recognition molecules central to several defence reactions. although some insight has been gained into the invertebrate propo system, host recognition proteins, biological activities, molecular interaction, and signal transduction pathways, little is known on the ascidian cascade. acknowledgements this work was supported by a miur (cofin2006 n. 2006059857) research grant. references abe y, ishikawa g, satoh h, azumi k, yokosawa h. primary structure and function of superoxide s73 dismutase from the ascidian halocynthia roretzi comp. biochem. physiol. 122b: 321-326, 1999. anderson rs. cellular responses to foreign bodies in the tunicate molgula manhattensis. biol. bull. 141: 91-98, 1971 anderson rs. hemocyte-derived reactive oxygen intermediate production in four bivalve mollusks. dev. comp. immunol. 18: 89-96, 1994. arizza v, cammarata m, tomasino mc, parrinello n. phenoloxidase characterization in vacuolar hemocytes from the solitary ascidians styela plicata. j. invertebr. pathol. 66: 297-302, 1995. aspán a, huang ts, cerenius l, söderhäll k. cdna cloning of prophenoloxidase from the freshwater crayfish pacifastacus leniusculus and its activation. proc. natl. acad. sci. usa 92: 939-943,1995. aspán a, söderhäll k. purification of prophenoloxidase from crayfish blood cells and its activation by an endogenous serine protease. insect biochem. 21: 363-373, 1991. azumi k, yokosawa h, s ishii. presence of 3-4 dihydroxyphenylalanine-containing peptides in hemocytes of the ascidian, halocynthia roretzi. experientia. 46: 1020-1023, 1990. ballarin l, cima f, floreani m, sabbadin a. oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian botryllus schlosseri. comp. biochem. physiol. 133c: 411-4118, 2002. ballarin l, cima f, sabbadin a, morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1996. ballarin l, cima f, sabbadin a. phenoloxidase in the colonial ascidian botryllus schlosseri (urochordata, ascidiacea). anim. biol. 3: 4148, 1994. ballarin l, cima f, sabbadin a. histochemical staining and characterization of the colonial ascidian botryllus schlosseri hemocytes. boll. zool. 60: 19-24, 1993. ballarin l. immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art. inv. surv. j. 5: 54-74, 2008 barrington ejw, thorpe a. histochemical and biochemical aspects of iodine binding in the tunic of ascidian dendrodoa grossularia. proc. roy. soc. b171: 91-109, 1968. bell k, smith vj. in vitro superoxide production by hyaline cells of the shore crab carcinus maenas (l.) dev. comp. immunol. 17: 211219,1993. beschin a, bilej m, hanssens f, raymakers j, van dyck e, revets h, et al. identification and cloning of a glucanand lipopolysaccharidebinding protein from eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. j. biol. chem. 273: 24948-54, 1998. cadenas e. biochemistry of oxygen toxicity. ann. res. biochem. 58: 79-110, 1989. cammarata m, arizza v, cianciolo c, parrinello d, vazzana m, vizzini a, et al., the prophenoloxidase system is activated during the tunic inflammatory reaction of ciona intestinalis.cell tissue res. 333: 481-492, 2008. cammarata m, arizza v, parrinello n, candore g, caruso c. phenoloxidase-dependent cytotoxic mechanism in ascidian (styela plicata) hemocytes active against erythrocytes and k562 tumor cells. eur. j. cell biol. 74: 302-307, 1997. cammarata m, arizza v, savona b, vazzana m, parrinello d. prophenoloxidase in the hemocyte of phallusia mamillata anim. biol. 8: 15-17, 1999. cammarata m, arizza v, vazzana m, parrinello n, prophenoloxidase activating system in tunicate hemolymph. it. j. zool. 63: 345-351, 1996. cerenius l, söderhäll k. the prophenoloxidaseactivating system in invertebrates immunol. rev. 198: 116-126, 2004. cerenius l, lee bl, söderhäll k. the propo-system: pros and cons for its role in invertebrate immunity. trends immunol. 29: 263-271, 2008. chaga oy. ortho-diphenoloxidase system of ascidians. tsitologia 22: 619-625, 1980. cotelle n, moreau s, cotelle p, catteau jp, bernier jl, henicard. generation of free radicals by simple prenylated hydroquinone derivativs, natural antitumor agents from the marine urochordate aplidium californicum. chem. res. toxicol. 4: 300-305, 1991. de leo g. ascidian hemocytes and their involvement in defence reactions. boll. zool. 59: 195-213, 1992. decker h, jaenicke e. recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins. dev. comp. immunol. 28: 673687, 2004. decker h, ryan m, jaenicke e, terwilliger n. sdsinduced phenoloxidase activity of hemocyanins from limulus polyphemus, eurypelma californicum, and cancer magister. j. biol. chem. 276: 17796-17799, 2001. decker h, tuczek f.tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism. trends biochem. sci. 25: 392-397, 2000. delsuc f, brinkmann h, chourrout d, philippe h. tunicates and not cephalochordates are the closest living relatives of vertebrates. nature 439: 965-968, 2006. ewan r, huxley-jones j, mould ap, humphries mj, robertson dl, boot-handford rp. the integrins of the urochordate ciona intestinalis provide novel insights into the molecular evolution of the vertebrate integrin family. bmc evol. biol. 5:31 2005. field kg, olsen gj, lane dj, giovannoni sj, ghiselin mt, et al., molecular phylogeny of the animal kingdom science 239: 748-753, 1988. fu x, hossain mb, van der helm d, schmitz fj, longithorone a. unprecedented dimeric prenylated quinone from the tunicate aplydium longithorax. j. amer. chem. soc. 116: 1212512126, 1994. fujimoto k, okino n, kawabata s, iwanaga s, ohnishi e. nucleotide sequence of the cdna encoding the proenzyme of phenol oxidase a1 of drosophila melanogaster. proc. natl. acd. sci. usa 92: 7769-7773, 1995. guerrieri n, scippa s, maietti m, de vincentiis m, cerletti. protease activity in fractionated blood s74 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22arizza%20v%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/pubmed/16495997?ordinalpos=9&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/16495997?ordinalpos=9&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22ohnishi%20e%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus cells of the vanadium accumulating ascidian phallusia mammillata. comp. biochem. physiol. 125a : 445-450, 2000. hall m, scott t, sugumaran m, söderhäll k, law j. prophenoloxidase of the hawkmoth, manduca sexta. purification, activation, substrate specificity of the active enzyme, and molecular cloning. proc. natl. acad. sci. usa 92: 77647768, 1995. hata s, azumi k, yokosawa h. ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles. comp. biochem. physiol. 119b: 769776, 1998. he x, kustin k, parry dl, robinson we, ruberto g, nakanishi k. in vivo incorporation of 14cphenylalanine into ascidian tunichrome. experientia 48: 367-371, 1992. holmblad t, thörnqvist po, söderhäll k, johansson mw. identification and cloning of an integrin beta subunit from hemocytes of the freshwater crayfish pacifastacus leniusculus. j. exp. zool. 277: 255-261, 1997. huang ts, wang h, lee sy johansson mw, söderhäll k, cerenius l. a cell adhesion protein from the crayfish pacifastacus leniusculus, a serine proteinase homologue similar to drosophila masquerade. j. biol. chem. 275: 9996-10001, 2000. immesberger a, burmester t. putative phenoloxidases in the tunicate ciona intestinalis and the origin of the arthropod hemocyanin superfamily. j. comp. physiol. [b] 174: 169-180, 2004. jackson ad, smith vj, peddie cm. in vitro phenoloxidase activity in the blood of ciona intestinalis and other ascidians. dev. comp. immunol. 17: 97-108, 1993. jackson ad, smith vj. lps-sensitive protease activity in the blood cells of the solitary ascidian, ciona intestinalis (l) comp. biochem. physiol. 106b: 505-512, 1993. jiravanichpaisal p, lee bl, söderhäll k. cellmediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. immunobiology 211: 213-236, 2006 johansson mw, holmblad t, thörnqvist po, cammarata m, parrinello n, söderhäll k. a cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish. j. cell sci. 112: 917925, 1999. johansson mw, lind mi, homblad t, thörnqvist po, söderhäll k. peroxinectin, a novel cell adhesion protein from crayfish blood. biochem. biophys. res. comm. 216: 1079-1087, 1995. johansson mw, söderhäll k. a cell adhesion factor from crayfish hemocytes has degranulating activity towards crayfish granular cells. insect biochem.19: 183-190, 1989a. johansson mw, söderhäll k. cellular immunity in crustaceans and propo system. parasitol. today 5: 171-176, 1989b. johansson mw, söderhäll k. a peptide containing the cell adhesion sequence rgd can mediate degranulation and cell adhesion of crayfish hemocytes in vitro. insect biochem.19: 573579, 1989c. johansson mw, söderhäll k. isolation and purification of a cell adhesion factor from crayfish blood cells. j. cell biol. 106: 17951803, 1988. kahn v. tropolone a compound that can aid in differentiating between tyrosinase and peroxidase. biochemistry 24: 915-920, 1985. kawabata t, yasuhara y, ochiai m, matsuura s, ashida m. molecular cloning of insect prophenol oxidase: a copper-containing protein homologous to arthropod hemocyanin. proc. natl. acad. sci. usa 92: 7774-7778, 1995. lee sy, lee bl, söderhäll k. processing of crayfish hemocyanin subunits into phenoloxidase. biochem. biophys. res. com. 322: 490-496, 2004. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. lerner ab. on the metabolism of phenylalanine and tyrosine. j. bio. chem.181: 281-294, 1949. luna-gonzalez a, maeda-martınez an, vargasalbores f, ascencio-valle f, robles-mungaray m. phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs fish shellfish immunol. 15: 275-282, 2003. nagai t, kawabata s. a link between blood coagulation and prophenoloxidase activation in arthropod host defense. j. biol. chem. 275: 29264-29267, 2000. nagai t, osaki t, kawabata s. functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. j. biol. chem. 276: 27166-27170, 2001. nappi aj, ottaviani e. cytotoxicity and cytotoxic molecules in invertebrates. bioessays 22: 469480, 2000. nappi aj, vass e. melanogenesis and the generation of cytotoxic molecules during insect cellular immune reactions. pigment cell res. 6: 117-126, 1993. o'brien pj. molecular mechanisms of quinone cytotoxicity. chem. biol. interact. 80: 1-41, 1991. pang q, zhang s, wang c, shi x, sun y. presence of prophenoloxidase in the humoral fluid of amphioxus branchiostoma belcheri tsingtauense fish shellfish immunol. 17: 477-487, 2004. parrinello n, arizza v, cammarata m, giaramita ft, pergolizzi m, vazzana m, et al., inducible lectins with galectin properties and human il1α epitopes opsonize yeasts in the ascidian ciona intestinalis inflammatory response. cell tissue res. 329: 79-90, 2007. parrinello n, arizza v, chinnici c, parrinello d, cammarata m. phenoloxidases in ascidian hemocytes: characterization of the prophenoloxidase activating system. comp. biochem. physiol. 135b: 583-591, 2003. parrinello n, cammarata m, arizza v. univacuolar refractile hemocytes from the tunicate ciona intestinalis are cytotoxic for mammalian erythrocytes in vitro. biol. bull. 190: 418-425, 1996. s75 javascript:al_get(this,%20'jour',%20'j%20exp%20zool.'); javascript:al_get(this,%20'jour',%20'fish%20shellfish%20immunol.'); parrinello n, cammarata m, lipari l, arizza v. sphingomyelin inhibition of ciona intestinalis (tunicata) cytotoxic hemocytes assayed against sheep erythrocytes. dev. comp. immunol. 19: 31-41, 1995. parrinello n, cammarata m, vazzana m, arizza v, vizzini a, cooper el in: yokosawa h, lambert cc (eds), immunological activity of ascidian hemocytes. the biology of ascidians, springer-verlag, tokyo, pp 395-401, 2001. parrinello n, patricolo e, canicattì c, inflammatory like reaction in the tunic of ciona intestinalis (tunicata) i. encapsulation and tissue injury. biol. bull. 167: 229-273, 1984. parrinello n, patricolo e. inflammatory-like reaction in the tunic of ciona intestinalis (tunicata). ii. capsule components biol. bull. 167: 238-250, 1984. parrinello n, vizzini a, arizza v, salerno g, parrinello d, cammarata m, et al. enhanced expression of a cloned and sequenced ciona intestinalis tnfalpha-like (citnf alpha) gene during the lps-induced inflammatory response. cell tissue res. 334: 305-317, 2008. parrinello n. cytotoxic activity of tunicate hemocytes. in: rinkevich b, müller weg (eds), invertebrate immunology, springer, berlin, pp 190-217, 1996. pawelek jm, lerner ab. 5,6-dihydroxyindol is a melanin precursor showing potent cytotoxicity. nature 276: 627-628, 1978. peddie cm, smith vj. in vitro spontaneous cytotoxic activity against mammalian target cells by the hemocytes of the solitary ascidian, ciona intestinalis. j. exp. zool. 267: 616-623, 1993. raftos da, briscoe da, tait nn. the mode of recognition of allogeneic tissue in the solitary urochordate styela plicata. transplantation 45: 1123-1126, 1988. riley pa. radicals in melanin biochemistry. ann. ny acad. .sci. 551: 111-120, 1988. rinkevich b. aspects of incompatibility nature in botryllid ascidians. anim. biol. 1: 17-28, 1992. roch p, canicattì c, sammarco s. tetrameric structure of the active phenoloxidase evidenced in the coelomocytes of the echinoderm holothuria tubulosa. comp. biochem. physiol. 102b: 349-355, 1992. ruoslahti e. integrin signalling and matrix assembly. tumour biol. 17: 117-124, 1996. sabbadin a. formal genetics of ascidians. amer. zool. 22: 765-777, 1982. smit npm, peters k, menko w, westerhof w, pavel s, riley pa. cytotoxicity of a selected series of substituted phenols towards cultured melanoma cells. melanoma res. 2: 295-304, 1996. smith vj, peddie cm. cell cooperation during host defence in the solitary tunicate ciona intestinalis (l.). biol. bull. 116: 211-219, 1992. smith vj, söderhäll k. a comparison of phenoloxidase activity in the blood of marine invertebrates. dev .comp. immunol. 15: 251-261, 1991. söderhäll k, cerenius l. crustacean immunity. annu. rev. fish dis. 3-23, 1992. söderhäll k, smith vj. the prophenoloxidase activating system as a recognition and defence system in arthropods. in: gupta ap (ed), hemocytic and humoral immunity in arthropods, wiley, new york, pp 251-286, 1986. söderhäll k. biochemical and molecular aspects of cellular communication in arthropods. boll. zool. 59: 141-151, 1992. söderhäll k. molecular analysis of the prophenoloxidase activating system in: söderhäll k, iwanaga s, vasta g (eds), new directions in invertebrate immunology, sos publications , nj, pp 229-253, 1996. söderhäll k. prophenoloxidase activating system and melanizationa recognition mechanism of arthopods? a review. dev. comp. immunol. 6: 601-611, 1982. sritunyalucksana k, wongsuebsantati k, johansson mw, söderhäll k. peroxinectin, a cell adhesive protein associated with the propo system from the black tiger shrimp, penaeus monodon. dev. comp. immunol. 25: 353-363, 2001. sugumaran m, hennigan b, semensi v, mitchell m, rivera t. differential mechanism of oxidation of n-acetyldopamine and n-acetylnorepinefrine by cuticular phenoloxidase from sarcophaga bullata. arch. insect biochem. physiol. 8: 229-241, 1988. valembois p, lassegues m, in vitro generation of reactive oxygen species by free coelomic cells of the annelid eisenia fetida andrei: an analysis by chemioluminescence and nitro blue tetrazolium reduction. dev. comp. immunol. 19: 195-204, 1995. vizzini a, pergolizzi m, vazzana m, salerno g, di sano c, macaluso p, et al. facit collagen (1alpha-chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian ciona intestinalis. dev. comp. immunol. 32: 682-692, 2008. wright rk, cooper el. inflammatory reactions of the protochordata. amer. zool. 23: 205-211, 1983. wright rk. urochordates. in: invertebrate blood cells. ratcliffe na, rowley af (eds) vol. 2, academic press, london, 1981. zaniolo g. histology of the ascidian botryllus schlosseri tunic: in particular, the test cells. boll. zool. 48: 169-178, 1981. zeng l, swalla bj. molecular phylogeny of the protochordates: chordate evolution can. j. zool. 83: 24-33, 2005. s76 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22raftos%20da%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22briscoe%20da%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22tait%20nn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus report of meeting isj 4: 24-36, 2007 issn 1824-307x report of meeting viiith scientific meeting of the italian association for developmental and comparative immunology (iadci), 1 and 2 march 2007, area della ricerca, cnr, naples, italy organizers: u oreste, mr coscia institute of protein biochemistry, cnr, naples, italy session 1. chairman: e ottaviani, university of modena and reggio emilia, italy variomics of novel immunoglobulin-like transcripts in teleost fish rjm stet, e boumans, a oestergaard scottish fish immunology research centre, aberdeen university, aberdeen, uk the immune system uses a variety of rearranging and non-rearranging receptors to distinguish between self and non-self antigens. the rearranging receptors of the adaptive immune system such as the immunoglobulin proteins and t cell receptors have been studied in great detail in fish. it is hypothesized that the rearranging receptors, which form a subset of the immunoglobulin super family, arose from innate immune precursors that are not associated with somatic reorganization. non-rearranging receptors of the innate immune system, which recognise largely unknown ligands, collectively denoted as pathogen-associated molecular patterns, have only recently been described in fish. examples of these receptors are the toll-like receptors and novel immune-type receptors (nitrs). recently, highly polymorphic novel immunoglobulin-like transcripts (nilts) have been described in common carp, which encode leukocyte receptors composed of a single extra-cellular immunoglobulin domain, a stalk, transmembrane and cytoplasmic region. two receptors were isolated with opposing signal ability as indicated by the presence of either an immunoreceptor tyrosine-based activating (itam) or inhibitory (itim) motif. recently, we have extended these studies to salmonid fish. in atlantic salmon an est was retrieved with similarity to carp nilts. the complete sasa-nilt sequence was obtained by 3’ and 5’ race and was shown to encode the activating nilt receptor. we have analysed the presence of sasa-nilt receptors in two unrelated atlantic salmon individuals. this showed the presence of three framework sasa-nilt sequences that were present in both individuals. in addition to these framework sequences, each individual has its own set of polymorphic sasa-nilt receptors. the mechanisms that generate this receptor diversity are at present unknown. however, the haplotypic and allelic variation is reminiscent of the massive expansion and diversification of immunoglobulin-like loci encoded in the leukocyte receptor complex in chicken. self/nonself discrimination in ciliated protozoa: the molecular basis a vallesi, c alimenti, p luporini department of biology, university of camerino, camerino as also a recent review in cell on the "quality control in self/nonself discrimination" points out (boehm t. cell 125: 845-858, 2006), comparative studies of the mechanisms that avoid self-mating in more ancient eukaryotes are thought to be of key relevance for shedding light on the control of specificity in self/nonself discrimination, as well as on the evolutionary emergence of the antigen receptors in the adaptive immune system. these studies, however, traditionally drive most attention on the self-incompatibility of plants, self-sterility and allo-recognition of tunicates, mating types of fungi. scarce or no reference at all is made to ciliates. nevertheless, the ciliate highly multiple mating-type systems are providing insightful information not only on the molecular basis of self/nonself recognition in more ancient organisms, but also on the central question of how new receptor/ligand pairs are generated in complex recognition systems. this information essentially derives from: (i) nmr and crystallographic analyses (mostly carried 24 http://www.isj.unimo.it/articoli/isj093.pdf http://www.isj.unimo.it/articoli/isj093.pdf out in collaboration with the kurt wuthrich’s laboratory at the eth in zurich) of the threedimensional structures of a set of water-born protein signals (pheromones) produced by euplotes species (luporini et al. curr. pharm. des. 12: 30153024, 2006), and (ii) the determination of the splicing mechanism by which the same cell controls its own specific diffusible signal and the (autocrine) binding receptor of this signal (vallesi et al. eukaryot. cell 4: 1221-1227, 2005). a novel approach for studying hematopoietic cells in hirudo medicinalis a grimaldi, g tettamanti, c bianchi, g greco, r valvassori, m de eguileor department of structural and functional biology, university of insubria, varese, italy leeches have evolved a complex immune system that can recognize foreign antigens and can respond with a wide repertoire of reactions in relation to the non-self. surgical wounds or cytokine injection induce the formation of an extensive network of new vessels and the proliferation of hematopoietic precursors. these cells exert their functional role migrating through the extracellular matrix towards the stimulated areas. the different types of cells involved in leeches immune response have been characterized only in vivo, since its very difficult to isolate and culture these cells entrapped in the thick connective tissue. we establish an innovative system to isolate and culture specific population of leeches immune cells utilizing the injection of matrigel matrix gel added with different cytokines known to play a pivotal role in regulating proliferation, migration and differentiation of leech immune cells. after 48h from injection the matrigel sponge is infiltrated by cells. the infiltrated matrigel sponge dissected from leech is cultured in an appropriate medium. two types of cultured cells have been obtained depending on the added cytokines. after one week cells cultured in matrigel containing the vascular endothelial growth factor (vegf) differentiated in endothelial cells cd34+ while those cultured in matrigel containing the monocyte chemoattractant protein-1 (mcp-1) differentiated in macrophages cd14+. our method provides a controllable protocol for repetitive isolation and culture of precursors cells from a “parenchimatous” animal and it is an excellent tool to select different types of cell population. aging and il-6 immunoreactivity changes in the polychaete ophryotrocha labronica a franchini, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy the aging process is associated with dysregulation of the immune and inflammatory responses including modifications in the regulation and production of cytokines. il-6 is a pleiotropic proinflammatory cytokine thought to play a role in age physiology, even if its possible modulation by aging mechanisms has not been fully defined. the morpho-functional modifications and il-6 immunoreactivity during the aging process in a simple invertebrate model, the polychaete ophryotrocha labronica, are reported. the comparison between newly-hatched, juveniles (at 611 setigerous segments, max 2 weeks), young adult females (at 14 setigerous segments, about 3 weeks) and 3 month old females showed significant structural differences in the nervous and genital systems. a reduction in the nerve area with a substantial depletion in neurons of the central system was found. a decline in oocyte growth and maturation was observed at the gonad level, even if sexually mature o. labronica continued to produce egg mass until week 16 of their lives. the age induced morphological modifications were associated to a different distribution of il6-like molecules, that were detected in the central nervous system. a decreased number of reactive nerve cells and in particular in the anterior region of the brain of aged o. labronica was observed. session 2. chairman: l ballarin, university of padova, italy evolution of helical cytokines: a structural approach d malagoli, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy cytokines are small soluble factors retrieved in mammals and involved in several processes such as immunity and development. they are typically characterized by pleiotropicity, functional and receptor redundancy. in consideration of functional parallels between mammalian and invertebrate immunity, a lot of experiments have been dedicated to the unravelling of cytokine network in both protostomian and deuterostomian invertebrates. the presence of cytokine-like molecules has been evidenced by several morphological and functional investigations in different taxa of invertebrates, leading to the hypothesis that cytokines are molecules of ancient origin, present in metazoans before the division of protostomian and deuterostomian phyletic lines. however, all the recent molecular biology advances indicate that no sequence similarity can be retrieved between the known vertebrate cytokines and the whole genome of invertebrate species. on these basis, functional convergence has been proposed between 25 vertebrate and invertebrate cytokines. the functional convergence would be due to the lectinlike activity of vertebrate cytokines that can be retrieved also in some invertebrate molecules. in order to unravel this unsolved matter, we have adopted a new bioinformatics approach able to isolate proteins whose structure is comparable to that of mammalian helical cytokines from est and protein databases. through this method we have isolated a molecule from drosophila melanogaster databases that presents the structural characteristics of a helical cytokine (drosophila helical factor, dhf). functional experiments performed on third instars larvae and sl2 embryonic hemocyte cell line of d. melanogaster demonstrated that dhf expression was increased after different immune challenges. from the present findings, it emerges that the contradiction between the amount of morphological and functional evidences and the absence of any homology between mammalian and invertebrate cytokines, could be explained by evolution of cytokine genes thereby conserving specific protein structures rather than amino acid or nucleotide sequences. from the present findings, it emerges that the contradiction between the amount of morphological and functional evidences and the absence of any homology between mammalian and invertebrate cytokines, could be explained by evolution of cytokine genes thereby conserving specific protein structures rather than amino acid or nucleotide sequences. haemocytes of the cockle cerastoderma glaucum: cell types and involvement in immune responses v matozzo, mg marin department of biology, university of padua, padua, italy for the first time, morpho-functional characterisation of haemocytes from the cockle cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. haemocyte mean number was 5.5 (x105) cells/ml haemolymph (n=10). two main haemocyte types were found in haemolymph: granulocytes (85 %), about 10 µm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 µm in diameter, with a few or no granules. most of the cytoplasmic granules stained in vivo with neutral red, indicating that they were lysosomes. on the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. acidophil hyalinocytes were the largest haemocyte type (about 14 µm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2 %). it increased significantly (up to 26 %) after preincubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. haemocytes also produced superoxide anion. moreover, both granulocytes and hyalinocytes (except acidophils) were positive to some important hydrolytic and oxidative enzymes, such as acid phosphatase, non-specific esterase, acid esterase, and peroxidase. lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. results indicate that haemocytes from c. glaucum are effective cells in immune responses. evidence for stem cell factor-induced proliferation/differentiation in bivalve hemocytes m betti*, b canonico°, c ciacci*, lc lorusso*, s papa°, l canesi^ *institute of physiological sciences, °cytometry and cytomorphology, university of urbino “carlo bo”, urbino, italy ^department of biology, university of genoa, genoa, italy bivalve hemocytes comprise both granular and agranular circulating cells that are capable of nonself recognition through lectins and chemotaxis, and, most of all, phagocytosis. as with vertebrate phagocytes, bivalve phagocytes are equipped with both oxidative and non-oxidative killing systems related to activities of lysosomal enzymes. in bivalves, the process of hematopoiesis is still unknown. the most generally accepted belief is that hemocytes may originate from connective tissue cells, although hemocyte proliferation at sites of inflammation has been demonstrated. stem cell factor (scf) is a member of hematopoietic cytokines, a group of glycoproteins that regulate the growth and differentiation of hematopoietic progenitor cells and functionally activate mature neutrophils or macrophages. in this work the possible effects of recombinant human scf on the hemocytes of the marine bivalve mytilus sp. were investigated. the in vitro effects of scf (50 ng/ml) on hemocyte functional parameters (lysosomal membrane stability-lms and lysozyme release-lr) were first evaluated. scf induced significant increase in lms and decrease in lr, this indicating a reduction in lysosomal membrane fusion processes. moreover, flow cytometry analysis showed that scf significantly affected both hemocyte number and cell cycle; in particular, increases in the number of both granular and agranular hemocytes were observed. the results obtained with heterologous sfc support the hypothesis that common pathways involved in modulating activity, differentiation and proliferation 26 of immune cells are shared by invertebrates and vertebrates. characterization of hemocytes from polistes dominulus (insecta, hymenoptera), target of the strepsipteran endoparasite xenos vesparum f manfredini*, e ottaviani°, r dallai* *department of evolutionary biology, university of siena, siena, italy °department of animal biology, university of modena and reggio emilia, modena, italy xenos vesparum (insecta, strepsiptera) is a macro-parasite of polistes dominulus, a primitively eusocial paper wasp. sem and tem observations after artificial infections allowed us to follow step by step the parasite development inside the hemocoel of the wasp. the host-seeking stage is the triungulin (free-living 1st instar larva) which is able to “softly” overcome the structural barriers of the larval wasp (cuticle and epidermis) without any traumatic reaction at the entry site. the parasite molts 48h later to a 2nd instar larva, which moves away from the 1st instar exuvium, molts twice more without ecdysis and pupates, if male, or develops into a neotenic female. some features result unusual: the encapsulation reaction involves the 1st instar exuvium (not the living parasite) and it starts only 48 h after host invasion; in addiction, no signs of melanization are visible. we suspect that x. vesparum inhibits host defense reactions during the early events of the infection and then the parasite seems to operate an elusion of p. dominulus immunity starting from the 2nd larval instar. we characterized the hemocytes present in the hemolymph of p. dominulus 3rd and 4th instar larvae (the main targets of triungulins) through morphological observations at tem, sem, light and phase-contrast microscopy; moreover we performed adhesion and phagocytosis functional tests and immunocytochemistry essays to check the presence of pomc-derived peptides and nep-like molecules. apart from the prohemocyte, the stem cell from which other hemocytes originate, two “types” or functional states are discernable, both of them adhering on glass slides and phagocytizing fluorescent beads, one type provided with structured and/or amorphous granules, the other one devoid of them. effect of cadmium exposure on phagocytosis and plaque lysis activity of paracentrotus lividus coelomocyte v arizza, f giaramita, g salerno, m vazzana, s basiricò, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy phagocytosis and plaque lysis activity (pla) of coelomocyte from paracentrotus lividus were examined after exposure to cadmium chloride (cdcl2·h2o), a potentially toxic metal salt, widely used in industry. p. lividus specimens were exposed at different cd concentration (100, 200, and 400 µg l-1) for 24 hours (sampled at 0, 6, 12 and 24 hrs) at 15°c, in tanks containing artificial sea water (asw) or injected with asw containing the metal at 50, 100 and 200 µg l-1 in to the coelomic cavity. the treatment without cd did not affect phagocytosis and pla, whereas treatment with cd, significantly lowered. this effect was dose and time dependent, presumably dependent on the cytotoxic effect of cadmium on coelomocyte as indicated by neutral uptake assay. session 3. chairman: l abelli, university of ferrara, italy invertebrate lectins present cytokine properties n parrinello, v arizza, m cammarata, m vazzana, a vizzini, d parrinello, ml di bella, m pergolizzi, f giaramita, m celi marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy the origin and evolution of the innate immunity, including cell-cell, cell-cytokines, cell-lectins, cellmatrix interactions, appear to be product of cells and genetic markers for self recognition that grow with molecules and mechanisms to identify and destroy non-self. lectins are components of a wellconserved protein-carbohydrate recognition system, the activity of most of them resides in a carbohydrate-recognition domain (crd). they present an ample repertoire and have been proposed to mediate cell-cell or cell-extracellular matrix interactions in developmental processes, cell adhesion, inflammation and metastasis. several immunomodulatory functions have been reported, among them mitogenic properties, opsonic properties, complement pathway activation and several immune responses. cytokines are the major regulators of the host defence processes and are involved in responses to exogenous and endogenous insults, tissue repair and recovery of homeostasis. many cytokines are bifunctional molecules having, beside a receptorbinding domain, a crd. the expression of the biological activity relies on the association between both domains. several reports have also shown that cytokine-crd can interact with various pathogens and presents the recognition site that contributes to pathogen elimination via opsonization and/or leukocyte activation. lectin-like activities of several cytokines, including il-1, have been described. our understanding of invertebrate cytokine-lectin 27 biological functions and evolution are lacking. in some studies, similarities at the physicochemical level of vertebrate cytokines and functional invertebrate analogues have also been described. among experimental approaches to identify cytokine-like molecules, antibodies neutralizing the activity of mammalian cytokines, have been used to screen for cross-reactivity with invertebrate factors in hemolymph. tunicates are a key group in chordate phylogenesis. in ascidian species, lectins are responsible for the in vitro opsonization, modulate cell proliferation activity, phagocytosis and complement activation, stimulate proliferation of mouse thymocytes and l-929 fibroblasts. recently we have shown that, in the serum from the lpschallenged ciona intestinalis, il1-like inducible components cross-reacted with anti-humanril1a antibodies. ciil1 with hemagglutinating properties, appears to be involved in sugar specific opsonization of yeast in an in vitro phagocytosis assay. therefore a putative structural model of the opsonin include both crd and il1 epitopes. we propose an evolutionary model in which multifunctional costituive/inducible lectins can express cytokine activity with crd responsible for pleiotropy and redundance, evolutionary conserved to guaranty a basic recogniton mechanism. several inflammatory factors have been hypothesized as responsible for this process including components known in mammal innate immunity (cytokines, complement components, collagens) and phenoloxidase activity. a putative evolutionary model in which lectins are represented as multifunctional inducible molecules (cytokine-like function) involved in adult defence mechanisms, could be hypothesized. the lectin crd, conserved in the evolution to guaranty a basic recognition mechanism, could explain lectin pleiotropy and redundancy as already suggested for mammalian cytokines. a novel rhamnose-binding lectin from the compound ascidian botryllus schlosseri l ballarin°, n franchi°, b spolaore*, f gasparini° °department of biology, university of padua, padua, italy *cribi, university of padua, padua, italy animal lectins play a fundamental role in invertebrate immunity, as they are involved in the recognition of microbial molecular patterns which, in turn, triggers various effector responses, such as opsonisation, encapsulation, activation of the propo activating system, phagocytosis. in a previous study, we purified by affinity chromatography and partially characterised a soluble ca2+-independent lectin, with specificity for b-galactosides, from the blood of the colonial ascidian botryllus schlosseri. the molecule can agglutinate rabbit erythrocytes, is secreted by haemocytes upon the recognition of foreign particles and behaves as an opsonins (ballarin et al., 1999, 2000). recently, we purified further this protein by rphplc, obtaining 4 lightly different peaks, likely isoforms of the same molecule. the mws estimated using mass spectrometry ranged between 10.7 and 11.1 kda. the lectin was digested with trypsin and tryptic fragments were sequenced by mass spectrometry. blast analysis of the main sequences obtained indicated a high degree of homology with rhamnose-binding proteins, a family of s-type lectins described in sea urchin and teleosts. the specificity for rhamnose (and the similar melibiose) was successively demonstrated in haemoagglutinating inhibition assays. we prepared a full length cdna library from botryllus colonies from which we obtained three full sequences of transcripts which, after blast analysis, resulted highly homologous to known genes for rhamnose-binding lectins. their putative aminoacid sequences contained our tryptic peptide sequences. serum lectins in fish innate immunity: molecular and functional aspects m cammarata*, g benenati*, g parisi*, d parrinello*, g salerno*, m vazzana*, a vizzini*, gr vasta°, n parrinello* *marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy °center of marine biotechnology, university of maryland, biotechnology institute, baltimore md, usa fucose-binding lectins (fbl) are present in tissues and fluids from invertebrates and vertebrates. the lectin repertoires in teleost fish are highly diversified and recently has been described the structure of the fucose-binding agglutinin that revealed a novel lectin fold (the “f-type” eel (anguilla anguilla) fold), which shared a unique fucose-binding sequence motif contained both in carbohydrate-binding proteins and unrelated proteins. in this report, we describe serum fbl from sea bass dicentrarchus labrax and sea bream sparus aurata. these lectins were purified, characterized, cloned and sequenced. studies on structural aspects, biological activity, tissue distribution as well as ontogenetic aspects were carried out. in addition, results on inflammatory response and opsonic activity against bacteria suggested that d. labrax fbl is involved in innate immunity. finally a new galactose binding lectin with agglutinating activity against bacteria purified from dicentrarchus labrax serum was also described and compared with fbl. 28 indifferentiating cells in the blood of the colonial ascidian botryllus schlosseri: a morphofunctional charaterisation f cima, l ballarin department of biology, university of padua, padua, italy colonies of the ascidian botryllus schlosseri undergo a periodic tissue renewal in the take-over stage of the colonial blastogenetic cycle, during which an extensive apoptosis occurs in the adult zooid tissues and the senescent cells are progressively removed by circulating phagocytes. the haemocytes which circulate in the common vascular system also die partly by apoptosis during this stage. these cells are replaced by new haemocytes, likely differentiating from stem cells. up to now, haemopoiesis was observed only in solitary ascidians in which haematopoietic noduli were described in the branchial wall. nothing is known on haemopoiesis in colonial species, in the blood circulation of which two cell types with the morphology of undifferentiated cells are recognizable: haemoblast and lymphocyte. we have studied the cytochemical and immunocytochemical properties of these haemocytes: results indicate the haemoblast as a pluripotent stem cell since it shows a basophilic nucleus labeled either with hoechst 33342 for euchromatin or anti-ki-67 and anti-pcna antibodies specific markers of nuclear proteins involved in cell proliferation and its plasma membrane is labeled by anti-cd34 and anti-cd100 antibodies, specific for haemopoietic cells in vertebrates. commercial antibodies for cytokine receptors, like interleukin 1 receptor i (il-1ri) and stem cell factor receptor (scf-r) label haemoblast plasma membrane, suggesting the presence of growth factor receptors. both lymphocytes and haemoblasts during the colonial cycle show a significant increase in concentration during the blastogenetic replacement. however, mitosis figures were rarely observed in circulating haemocytes. in vitro assays of haemocyte exposure to colchicine showed the presence of mitosis figures, which significantly increase after exposure to bacteria indicating a proliferating capability in blood circulation mainly as an immune response as observed in other invertebrates like molluscs. effects of different carbon dioxide concentrations on the adaptive immune system of cultured sea bass (dicentrarchus labrax) n romano^, e caccia^, t petochi°, s meloni^, l mastrolia^, g scapigliati^, l abelli*, g marino° ^department of environmental sciences, university of tuscia, viterbo, italy *department of biology and evolution, section of comparative anatomy, university of ferrara, ferrara, italy °icram, central institute for marine applied research, rome, italy low oxygen and high carbon dioxide concentrations could affect health and welfare of farmed fish. this study evaluated acute and chronic effects of different dissolved carbon dioxide concentration (2-45 co2 mg/l) on specific immune response of sea bass. fish were vaccinated against vibrio anguillarum before exposure to co2 and after 45 days were analysed for: a) the percentage of t and b lymphocytes in the leukocyte fraction of blood and head kidney (by flow cytometry using specific mabs dlt15 and dlig3 for t and b cells, respectively), b) proliferation capability of lymphocytes exposed to vibrio; c) the serum content of anti-vibrio ig by captured-elisa method and d) the agglutinating capacity of serum against vibrio bacteria. t and b lymphocytes significantly decreased (p<0.001) in fish maintained for 45 days at the highest co2 concentration respect to controls. the proliferation capability of head kidney lymphocytes was also significantly reduced in co2 treated fish. also anti-vibrio ig content decreased (50 %; p>0.001) in co2 exposed fish. non-immunised showed a lower vibrio-agglutination capability. these findings evidenced the strong effect of co2 on circulating lymphocytes and in their specific immune function and the sensitivity of farmed sea bass to carbon dioxide concentration higher than 40 mg/l. thymic morpho-functional changes after hypophysectomy and bursectomy in chicken embryos m aita*, n romano° *department of human physiology and pharmacology, faculty of medicine, university “la sapienza”, rome, italy °department of environmental sciences, university of tuscia, viterbo, italy experiments of hypophysectomy or bursectomy were performed in chicken embryos in order to give more information on the role of hypophysis and fabricius’ bursa in the thymus development. hypophysectomy was performed on chick embryos at 36-40 hr of incubation. the thymuses were collected on day 18 and tested for: 1) antithymostimulin (ts) immune reaction; 2) histoenzymatic activities (ldh, sdh, nadh, nadph, alfa-gpdh, ca2+-atp-ase). the total thymic size was reduced and anti-ts, sdh, atpase yielded negative reactions in the medullary epithelial cells. when hypophysectomized embryos received on day 12 a hypophyseal allograft from 18 day-old donor embryos, the thymic compartments improved and anti-ts immune reaction and enzymatic activities were partially recovered. bursectomy was performed at 68-72 hr of incubation. the thymuses were collected on day 17 and were tested for the pcna (proliferating cell 29 nuclear antigen) and cd3, cd8 and cd4 markers. a significant reduction of pcna-immunoreactive lymphocytes was observed in cortex (p<0,001) and a significant decrease of anti-cd3,-cd4,-cd8 lymphocytes was evidenced in medulla (p<0,01). these findings confirm, at one hand, the key role of the hypophysis in thymic ontogenic development and, on the other hand, that bursectomy interferes with a correct differentiation of thymocytes and that there is an interrelationship between thymus and bursa at least during embryonic life. session 4. chairman: n parrinello, university of palermo, italy evolution of the complement system: invertebrate animal models mr pinto stazione zoologica “anton dohrn”, naples, italy in the past decade, in the context of the renewed interest in innate immunity, the complement system has been investigated in increasing depth. one successful approach to analyzing the complement system has involved the study of its evolutionary origin. the search for complement components has been carried out in very divergent species, aided by the powerful tools provided by computational biology and the genome projects that are ongoing in many invertebrate species. a surprising result of these endeavours has been the finding that both c3, the key molecule of the complement system, and factor b have a very ancient origin. in cnidaria, these molecules seem to form a basic complement assembly that is able to opsonize bacteria through a primordial alternative pathway. carbohydrate-recognizing molecules, structural homologues of vertebrate ficolins, have been recruited by the complement system more recently in the protostomian lineage. high levels of complement complexity, comparable to those of mammals, have been reached in ascidians (urochordata), which experienced many gene duplication events specific to the urochordate lineage. as a result of this gene expansion, ascidians exhibit a complex complement system that operates through alternative and lectin pathways and exhibits proinflammatory and opsonic effector activities. expression pattern of c3 during ciona intestinalis embryo development d melillo*, r de santis*, s giacomelli*, jd lambris^, mr pinto* *stazione zoologica “anton dohrn”, naples, italy ^protein chemistry laboratory, university of pennsylvania, philadelphia, usa the identification of many complement components in ascidians indicates the presence of a complex complement system, comparable to that of mammals, activated via an alternative and an mbl-mediated pathways. c3 activation-dependent pro-inflammatory and opsonic effector activities have been demonstrated in this subphylum. in particular, c3, the central molecule of the system, is expressed in ciona blood granular amoebocytes and compartment cells, and the gene product is present in blood serum. while the immune defense mechanisms and molecules of adult ascidians have received some attention, no information is available on the immune surveillance, if any, during embryo development. to approach this topic, we have analyzed, in the present study, the spatial and temporal expression of c3 in ciona intestinalis embryo. following rt-pcr indications, we have carried out in situ hybridization experiments on developmental stages, from the unfertilized egg to the swimming larva. c3 shows an expression pattern restricted to mesenchyme cells and neural tissue cells. to extend these results western blot and immunochemical experiments have been also carried out. our preliminary data provide a first hint in defining the role of the c3 molecule, crucial in the innate immune response in the adults, during embryogenesis. cikll, the ascidian multipurpose c-type lectinlike receptor i zucchetti*, r marino*, mr pinto*, l du pasquier°, r de santis* *stazione zoologica “anton dohrn”, naples; italy °institute of zoology, university of basel, basel, switzerland c-type lectins, a family of diverse animal lectins characterized by a c-type lectin domain that serve for a broad range of biological processes, such as adhesion, endocytosis and pathogen recognition and neutralization, have been originally identified as carbohydrate-recognition molecules found in both invertebrates and vertebrates. the carbohydrate binding c-type lectin domains are part of a larger family of domains called c-type lectin-like domains (ctlds) that seem to have originated by a process of divergent evolution from a common ancestor. vertebrate cd94, one of the ctld-containing molecules characterized by a lack of ca2+-binding sites, and therefore, a putative lack of sugar-binding activity, is one of several vertebrate natural killer lymphocyte receptors. cd94, forming heterodimers with nkg2 family molecules, regulates cytotoxic activity of nk cells towards target cells by interacting specifically with the mhc class i molecules, thus representing a trait d’union between innate and adaptive immunity. 30 in order to provide further information on the evolution of c-type lectins, we have carried out an in depth study on cikll, a homolog of the bscd94/nkr-p1, a cd94-like gene identified in the colonial tunicate botryllus schlosseri, present in ciona intestinalis genome. cikll, showing intermediate structural features between a carbohydrate-binding protein and an nk cell receptor, is expressed in a blood cell type that is involved in the phagocytic activity during the immune response. furthermore, cikll is expressed in the larva and during early metamorphosis in structures related to the nervous system. these observations are in line with the current speculations on gene cooption in the course of evolution particularly between genes involved in immunity and those related to developmental processes of the nervous system. preliminary characterization of a c1q-like transcript from the ascidian ciona intestinalis s giacomelli, d melillo, r de santis, mr pinto stazione zoologica “anton dohrn”, naples, italy c1q is a subcomponent of the c1 enzyme complex that triggers the activation of the classical pathway of the complement system in the adaptive immune system of the mammalian species. c1q is known for its ability to bind antibodies as well as other ligands, including bacteria, viruses, parasites etc. c1q shows an hexameric assembly of three polypeptide chains, a, b and c chain. these chains possess the same topology, consisting in a collagen-like gly/pro-rich region, and a conserved c-terminal globular domain (c1q domain). the analysis of the urochordate ciona intestinalis genome has confirmed the absence of the pivotal genes for adaptive immunity in this species, which is phylogenetically at the basis of the vertebrate lineage. at the same time, this analysis has confirmed the presence of many complement genes, including a c1q domain-containing gene, cic1q-like, encoding a protein with the same modular organization of the mammalian c1q chains. to trace back to the invertebrates the origin of the classical activation pathway of the complement system, we have undertaken the molecular and functional characterization of the cic1q-like protein. we have verified the presence of cic1q-like mrna in ciona blood cells, by pcr analysis: pcr products have been cloned and sequenced. the 3’utr sequence has been determined on clones obtained from the 3’-race procedure. the c1q domain of the cic1q-like sequence has the 24-28 % of identity with the human orthologs. a phylogenetic tree generated by neighbour-joining method shows the relationship between the ciona c1q-domain and the other c1q-domain orthologs. the ascidian c1q could either act as a lectin, like the lamprey c1q, or interact with other unknown membrane bound receptor/s, as in the case of murine sign-r1. further investigations on cic1q-like expression and biological function may help to shed light on the origin and evolution of the complement system classical pathway and the c1q domain family. toll-like receptors in haemocytes of the colonial ascidian botryllus schlosseri: preliminary results a menin, l ballarin department of biology, university of padua, padua, italy toll-like receptors (tlrs) represent a wellknown family of pattern recognition receptors, expressed by immunocytes, the importance of which in non-self recognition was demonstrated in both vertebrates and invertebrates. in the colonial ascidian botryllus schlosseri, we used commercial anti-tlr2 and anti-tlr4 antibodies to inquire into the presence of toll-like receptors (tlr) in haemocytes lysates. after sdspage, the immunoblot analysis revealed single protein bands recognised by the two antibodies, of 34 kda and 32 kda for tlr2 and tlr4, respectively. immunocytochemical investigation on monolayers of fixed haemocytes, previously exposed to e. coli lps and yeast cells, revealed the expression of molecules recognised by tlr2 on activated phagocytes, whereas no labelling was observed with tlr4. we also studied the role of nf-kb in the signal transduction pathway related to phagocytosis. immunocytochemical analysis with anti-nf-kbp65 antibody revealed the labelling of the cytoplasm of untreated cells, whereas haemocytes exposed to yeast cells or bacillus clausii spores showed a marked staining of the phagocyte nucleus. the nfkb inhibitors na-pyrrolidinedithiocarbamate and parthenolide, at sublethal concentrations, significantly inhibits both the ingestion of yeast cells by botryllus phagocytes and the nuclear translocation of the activated factor. the same molecules have no effects on the morphology of haemocytes. on the whole, our data suggest that, in our species, tlr are involved in phagocytosis and act through the activation of nf-kb. signal transduction in phagocytosis of the colonial ascidian botryllus schlosseri: a preliminary approach a menin, e chemello, l ballarin department of biology, university of padua, padua, italy 31 in the course of our study on the role of immunocytes of the colonial ascidian botryllus schlosseri in immune responses, we began to investigate the signal transduction pathways involved in yeast cell phagocytosis. both calphostin c, a specific inhibitor of protein kinase c (pkc), and h-89, a specific inhibitor of protein kinase a (pka) significantly inhibit the increase in the phagocytic index. this indicates that both cyclic amp, which activates pka, and phospholipase c, which results in the production of ip3 and dag (the former mobilising ca2+ from intracellular stores, the latter activating pkc), are routinely required for phagocytosis. in addition, manumycin a, inhibiting ras activation, pd98059, inhibitor of erk activation, sp600125, preventing jnk activation, sb202190, inhibiting p38 kinase, significantly inhibit yeast phagocytosis by botryllus phagocytes. this suggest that the main map kinase pathways are involved in the ingestion of foreign cells. the frequency of phagocytes expressing molecules recognised by anti-pan ras antibody increase significantly when haemocytes were preincubated in the presence of foreign cells. activated haemocytes also express molecules recognised by anti-p-erk and anti-p38. therefore, a complex network of intersecting pathways is emerging and future research will aim to a better clarification of the main steps of signal transduction in ascidian phagocytosis. session 5. chairman: mr coscia, institute of protein biochemistry, cnr, naples, italy teleost immunoglobulins: genes and proteins u oreste institute of protein biochemistry, cnr, naples, italy the ability of teleosts to mount an antibodymediated immune response has been reported for the first time in the last century, at the beginning of the forties. serological methods, such as bacterial agglutination or hemolysis, provided the indirect evidence of the presence of antibodies in the teleost serum. the introduction of protein chemistry and immunochemistry methods in the sixties allowed the purification and characterization of the immunoglobulin (ig) molecules. the comparison with mammalian ig showed evidence of many peculiar features of teleost ig, such as isotype composition, polymeric assembly, lack of either secondary response or isotype switch. molecular biology tools have been introduced in fish immunology in 1989, when an ictalurus punctatus ig heavy chain (igh) cdna has been sequenced. afterwards, nucleotide sequences encoding ig genes have been obtained from more than 30 different teleost species. at present, data on ig gene structure, regulation, and expression, are also available. the antibody repertoire, the vh gene segment diversity, the occurrence of different igh and igl isotypes, the alternative splicing of primary igh transcripts have been deeply investigated in some model species. the ongoing genome and est sequencing of several teleost species, aided by computational biology, has enormously increased the knowledge of the immune gene organisation and functions: new igh isotypes, new igl gene segment organisation have been disclosed, allowing to draw a scheme of the vertebrate ig evolution. the knowledge on the most important molecule of vertebrate immunity has increased in the last decades at a very high rate thus prospecting fascinating results in the next future. molecular cloning, structural analysis and antigen-induced “in vivo” expression of interleukin-10 in sea bass (dicentrarchus labrax l.) f buonocore*, e randelli*, s bird°, cj secombes°, s costantini^, a facchiano^, s benedetti*, g scapigliati* *department of environmental sciences, university of tuscia, viterbo, italy °scottish fish immunology research centre, university of aberdeen, aberdeen, uk ^ institute of food science, cnr, avellino, italy interleukin-10 (il-10) is a regulatory cytokine mainly involved in the suppression or deactivation of immune responses and is produced by macrophages and by the t-helper cells (subset th2). recently il-10 has been discovered in the fugu genome and cloned in different fish species. here we describe the homology cloning of this cytokine in the mediterranean sea bass (dicentrarchus labrax l.) and investigate its structure and in vitro and in vivo expression upon various stimulants. the full-length il-10 cdna consists of 1015 bp and is translated in one reading frame to give the entire il-10 molecule containing 187 amino acids. a multiple alignment of the predicted translation of il-10 sea bass molecule with other known il-10 sequences showed the conservation of the fundamental features corresponding to il-10 molecules. a comparative 3d modelling using human il-10 as template showed that sea bass molecule is a symmetric homodimer, topologically similar to the structure of interferon-γ and about 70 % of the residues in each monomer assumes an α-helical conformation. expression analysis by real-time pcr was studied at a basal level in the main lymphatic tissues and after in vitro stimulation with lps and pha in the head kidney. moreover, an in vivo stimulation with the t-dependent antigen dnp human gamma 32 globulins (dnp-hgg), alone or in combination with an aluminium hydroxide emulsion, was performed. structural study of complex of mhc class i and co-receptor cd8 in sea bream by computational methods s costantini*, f buonocore°, e randelli°, g scapigliati°, am facchiano* *laboratory of bioinformatics and computational biology, institute of food science, cnr, avellino, italy °department of environmental sciences, university of tuscia, viterbo, italy comparative modelling represents the best predictive method for modelling the 3d structure of proteins. this method is applicable when the protein to be modelled is homologous to a protein whose 3d structure is known. the basis of this strategy is the observation that homologous proteins from different organisms can have low or high level of sequence identity, depending on the evolutive distance among them, but the 3d structure should be similar, being strongly related to the function played by that protein. on this basis, the 3d model of a protein can be created by similarity to the experimental model of known homologous protein. in this work we have applied the comparative modelling strategy to model mhc class i and homodimer cd8aa sequences in sea bream and the related complex. the three-dimensional models of the sea bream molecules and complexes were created by using human and mouse template models. as the sequence identities between the sea bream proteins and the homologous template models were about to 30 %, we used an accurate procedure to search for the best alignment of sequences, in order to improve the quality of the modelling results. the obtained models present a global structure similar to the reference proteins. comparing the complexes obtained for sea bream and the mammals ones, we have evidenced some differences both in structural and in energetic terms. cloning, expression and structural analysis of the mhc class ii β from sea bass dicentrarchus labrax e randelli*, f buonocore*, d casani*, rjm stet°, a facchiano^, s costantini^, g scapigliati* *department of environmental sciences, university of tuscia, viterbo, italy °scottish fish immunology research centre aberdeen university, aberdeen, uk ^institute of food science, cnr, avellino, italy major histocompatibility complex class ii (mhcii) molecules present foreign peptides to t cells of the cd4 subset, and are thus fundamental components of the adaptive immune system. the mhcii proteins belong to the immunoglobulin gene family, bind to lysosomally generated peptides and are expressed only by b cells and antigenpresenting cells. they consist in a heterodimer (α and β chain) having two extracellular domains, a short hydrophobic transmembrane section and a hydrophilic cytoplasmic domain. mhcii genes exhibit an extraordinary degree of allelic polymorphism and are likely candidates as gene markers associated with disease resistance. using degenerate primers corresponding to mhcii conserved regions of vertebrate sequences, we obtained an initial 190 bp product that, once sequenced and analysed by blastx search, corresponded to a mhcii-β gene fragment. from this fragment we designed specific primers that were used in 3’ and 5’ race pcr to complete the cdna sequence. an alignment was performed using available mhcii-β aminoacid sequences and a phylogenetic tree was generated with the putative aminoacid sequences lacking the signal peptide. moreover, two 3d mhcii sea bass models were obtained based on crystallographic mouse mhcii structures complexed with d10 t-cell antigen receptors and human cd4. finally, specific primers were used to analyse by real time-pcr the mhciiβ expression in kidney macrophages stimulated with different concentration of sea bass ril-1β and with lps for 4h and 24 h, which are known to modulate mhcii expression. molecular models of chionodraco hamatus igm transmembrane region s varriale*^, a merlino^, mr coscia*, l mazzarella^, u oreste* *institute of protein biochemistry, cnr, naples, italy ^department of chemistry, university of naples “federico ii”, naples, italy membrane-bound immunoglobulin m (igm) participates to the assembly of the b cell receptor (bcr). igm consists of two μ heavy chains crossing the cell membrane and two light chains. the μ chain region traversing the lipid bilayer (tm region) is highly conserved among species and contains a universal motif for antigen receptors that is important for bcr assembly and function. we analysed the tm region of igμ from the antarctic teleost chionodraco hamatus, belonging to the channichthyidae family. its membrane μ chain is particularly interesting because generated by an unusual mrna splicing mechanism. we determined the complete nucleotide sequence of c. hamatus membrane μ chain and analyzed the deduced amino acid sequence encoded by the tm exons. using different computational methods, we predicted the length and 33 the polarity of the α-helical region crossing the cell membrane, and build a molecular model of the c. hamatus μ chain tm region, using the h helix of the photosynthetic reaction center of rhodobacter sphaeroides as template. the stability of the model was investigated by molecular dynamics (md) simulations. models of a tm homodimer were also obtained by performing md simulations using two copies of the helix, at a 14-16 å distance between the centers of mass and in different orientations, as starting model. the obtained structures were related to the available experimental data collected on igm tm region of different species. identification of a new trematomus bernacchii immunoglobulin light chain isotype c de santi*, v morea^ mr coscia*, s giacomelli*, a tramontano°, u oreste* *institute of protein biochemistry, cnr, naples, italy ^ institute of molecular biology and pathology, cnr, rome, italy °department of biochemical sciences “a. rossi fanelli”, university of rome “la sapienza”, rome, italy immunoglobulin light chains (igl) have been sequenced in 20 different teleost species. in each species one, two or three different isotypes have been described. we have previously identified in the antarctic teleost trematomus bernacchii, two immunoglobulin light chain (igl) isotypes, referred to as trbel1 (distinguishable into two subgroups, trbel1a and trbel1b), and trbel3, based on comparison with the isotypes defined in other teleosts. in order to verify the presence of igl isotype 2 in t. bernacchii, a pcr approach was chosen. based on multiple alignment of igl2 sequences from different teleost species, two oligonucleotide primers, complementary to the most conserved part of the fr2 region (sense) and to the 3’ end of cl (antisense), were designed. the resulting pcr products were cloned into pgem-t easy vector and recombinant clones were isolated, sequenced, and found to belong to the trbel2 isotype. additional igl cdna clones were obtained by rt-pcr and 5’ race using isotype-specific primers. the percentages of identity of the total 30 clones confirmed their distribution in three isotypes, one of them distinguishable into two subisotypes. by multiple alignment of the cl domains, conserved positions and isotype-specific residues were identified. to compare the molecular structure of each isotype specific cl domain, molecular models were built. multiple alignment and phylogenetic tree of the cl sequences from t. bernacchii and other teleosts indicated that each t. bernacchii isotype fell into one of the three teleost isotype groups. session 6. chairman: l mastrolia, university of tuscia, viterbo, italy allelic polymorphism of the igµ exons in the antarctic teleost trematomus bernacchii mr coscia, r de feo, s giacomelli, u oreste institute of protein biochemistry, cnr, naples, italy in mammals, the immunoglobulin constant domains are relatively invariant, despite small amino acid differences are known to exist between products of the genes encoding iga, igm, and igg among the population. comparative studies in nonmammalian organisms have occasionally shown more than one igμ constant domain sequence, but they were usually attributed to gene duplication. to search for polymorphism in the antarctic teleost trematomus bernacchii igμ gene, total rna was extracted from the spleen of eight specimens caught in the same area. two specific pcr primers were designed to amplify the entire constant region. multiple alignment of the eight igμ sequences, revealed that at least 51 positions were polymorphic. the individuals analyzed were found to be heterozygous or homozygous for each polymorphic position as expressing one or two variants. the highest number of polymorphic positions was observed in a particular region. in fact 30 out of 51 nucleotide substitutions were found to fall within the “hinge” region which connects the ch2 and ch3 domains. this region not only displayed extensive nucleotide variation, but also length diversity; in fact several sequences were one amino acid shorter as resulting from the usage of a different splice acceptor site as demonstrated by the analysis of the genomic dna. polymorphism was observed also at some potential n-glycosylation sites. the ka/ks ratios of the polymorphic positions showed typical values higher than one, indicative of positive selection acting to polymorphic residues to favor amino acid replacements and maintain allelic polymorphism. comparison with other non-antarctic teleosts revealed that the high level of polymorphism in the “hinge” region is a peculiar feature of t. bernacchii. these results suggest that this property may have some biological significance, possibly related to modulating susceptibility/resistance to cleavage by bacterial or parasitic protease. secretory immunoglobulins in the skin of the antarctic teleost trematomus bernacchii c motta*, mr coscia^, a de santis*^, s tammaro*, u oreste^ *department of biological sciences, university of naples “federico ii”, naples, italy ^institute of protein biochemistry, cnr, naples, italy 34 the presence and localization of secretory immunoglobulins (ig) in the skin of the antarctic teleost trematomus bernacchii has been investigated by using specific antisera in situ and in western blots. analyses have indicated that l and h chains are present, that their molecular weights are similar to that of mucus ig and that they are localised in filamentous, but not in mucous cells. immuno-gold investigations have demonstrated that the ig are dispersed in the cytoplasm and concentrated at the level of the endoplasmic reticulum thus suggesting a local production. in situ hybridizations confirm the hypothesis since demonstrate that filamentous cells synthesize mrna for the ig h chain. hybridization also reveals that a significant synthesis of h chain mrna occurs in mucous cells indicating a selective activation of post-transcriptional control mechanisms in the different cell types forming the skin in trematomus. b cells in lymphoid tissues of the antarctic teleost trematomus bernacchii l abelli*, mr coscia°, f bertoni*, c caprera^, n romano^, u oreste° *department of biology and evolution, university of ferrara, ferrara, italy °institute of protein biochemistry, cnr, naples, italy ^department of environmental sciences, university of tuscia, viterbo, italy original data are presented on cytology and distribution of b cells in lymphoid tissues of the emerald rockcod (trematomus bernacchii, boulenger 1902), a bony fish living at sub-zero temperatures. b cells were identified by immunohistochemistry using antisera against homologous ig h and l chains. expression of membrane and secretory h chain transcripts and proteins was analised by rt-pcr and immunoblotting. according to general features of the teleost immune system, b cells were numerous in the head kidney and spleen, while were fewer in the gills and intestine. on the other hand, a striking finding was the concentration of ig+ cells in the thymus, clearly exceeding that found in all fish species studied so far. the differential distribution of membraneand cytoplasmic-ig+ cells (likely mature b cells, resting or stimulated) in the different vascular districts suggested a key role of head kidney and thymus in early b development, and of the spleen as the site of definitive maturation. in the gills, ig+ cells were scattered around the filament artery, in the pluristratified filament epithelium and around the outer marginal channel near the lamellar tip. in the intestine, ig+ cells constituted a minor leucocyte population, localised almost exclusively in the lamina propria and submucosa. an increasing gradient (towards the stomach) in the number of ig+ cells and eosinophilic granulocytes along the intestine suggested a higher inflammatory and immune responsiveness in the anterior segment, also mediated by hepatobiliary transport of immunoglobulins. localization of cd8α expressing t-cells in developing sea bass thymus: an hypothesis of positive selection s picchietti°, l guerra°, f buonocore°, am fausto°, m mazzini°, l abelli * °department of environmental sciences, university of tuscia, viterbo, italy *department of biology and evolution, section of comparative anatomy, university of ferrara, ferrara, italy these studies on thymus of the teleost dicentrarchus labrax (l.) focused on differentiation and positive selection of t cells, crucial steps in the development of a functional immune system in all jawed vertebrates. sea bass eggs, larvae (from day 2 until day 92 ph) and juveniles were analysed for developmental appearance of transcripts of two important genes in t cell function, cd8α and tcrβ. rt-pcrs detected tcrβ transcripts in larvae from 25 days ph. otherwise, cd8α expression was detected at day 51 ph when the thymus was well developed. in situ hybridization of cd8α  mrna identified thymocytes in the outer and lateral zones of the thymic paired glands. from day 75 ph on, the signal was mainly detected in the cortex and the corticomedullary junction. in one-year-old specimens cd8α and tcrβ expression patterns nearly overlapped, drawing a cortex-medulla demarcation in each thymic lobe. large cords of cd8α + tcrβ + cells lay in the medulla. a cd8α tcrβ+ subcapsular zone was evident near the septa coming from the inner connective capsule that delimited the thymus. no signal was found in subcapsular and cortical epithelial cells. the tcrβ and cd8α expression patterns demonstrated a compartmentalization of the thymus due to distinct localization of thymocytes at different developmental stages and suggested an hypothesis about positive selection in teleost thymus as described in mammals. localization of glucocorticoid receptor dlgr1 in tissues of teleost dicentrarchus labrax m vazzana, a vizzini, g salerno, ml di bella, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy cortisol is a glucocorticoid that affects a wide variety of biological responses, including immune 35 functions. virtually all tissues in the body are target organs and they can respond in different ways. the physiological response to cortisol is mediated through binding at two classes of intracellular receptors like mineral corticoids (mr) and glucocorticoids (gr) that act as ligand-dependant transcription factors. a cortisol receptor (dlgr1), (vizzini et al., 2007), from leukocytes of peritoneal cavity, previously cloned and sequenced, were employed for in situ hybridization and immunohistochemical assays. the experiments were performed on brain, head kidney, spleen, gills, intestine and hearth. the mrna and protein were expressed in the examined tissues. the wide expression and distribution of dlgr1 confirm the importance of the cortisol role in the maintenance of the homeostasis in the organisms. innate immune response of reared european sea bass dicentrarchus labrax to different environmental and husbandry conditions t petochi°, p di marco*, a priori*, mg finoia*, g marino* °icram, central institute for marine applied research, rome, italy *department of environmental sciences, university of tuscia, viterbo, italy innate immune parameters were evaluated as indicators of health and welfare in reared sea bass dicentrarchus labrax in relation to seasonal temperature changes, experimental carbon dioxide exposure (0-5, 15-20, 30-35 and 50-55 mg/l) for 45 days and different stocking densities (15, 30 and 45 kg/m3) for 35 days. complement activity (ach50) showed a seasonal trend, increasing during the summer and reaching maximum levels in september, with a positive correlation to high water temperature. significant increase in serum lysozyme was measured in late summer and autumn, but any evident correlation with water temperature was found. hypercapnia induced a transient decrease in ach50 activity within the first 24 hours of co2 exposure and in lysozyme levels in the first week of exposure. at the end of the experiment (45 days), there were no significant differences in lysozyme and ach50 among hypercapnic groups and controls and compared to initial levels. the respiratory burst activity in hypercapnic groups was significantly reduced compared to controls after 45 days of co2 exposure. high stocking density affected the ach50 activity in sea bass with a significant decrease in fish maintained at 30 and 45 kg/m3 for 35 days. lysozyme activity did not change in fish reared up to 45 kg/m3. results of present investigations indicate an influence of low water temperature, hypercapnia and high stocking density on innate immune response of sea bass that could be detrimental for health and welfare status. transferrins, nitric oxide and cytokines: complex responses to marek’s disease virus reinfection in a chicken lymphoblastoid cell line mf giardi, f giansanti, d botti department of basic and applied biology, university of l’aquila, l’aquila, italy in previous works, we have shown that a relevant antiviral activity against marek’s disease virus (mdv) is exterted by ovotransferrin (otrf), lactoferrin (lf) (giansanti et al. biochem. cell. biol. 80: 125-130, 2002) and two ovotransferrin derived peptides (giansanti et al. biochem. biophys. res. commun. 331: 69-73, 2005). in addition, we have also evidenced the production of otrf by a chicken lymphoblastoid cell line (mdcc-msb1) induced by mdv, following reinfection with mdv (giansanti et al. biochem. cell biol. in press). the aim of the present work is to verify if mechanisms other than tranferrins are involved in the defense against the above mentioned reinfection. for this reason, the possible role of no, il-8 and ifn-γ has been investigated. the data obtained indicate that no production by mdccmsb1 is strongly enhanced in the presence of transferrins and after the reinfection. the no production is completely inhibited by aminoguanidine (ag), an inhibitor of inos. it has also been observed that both cytokines stimulate the production of no, and that the maximum stimulatory effect was obtained in the presence of ifn-γ plus otrf or in the presence of il-8 plus lf. also in this case, ag was able to completely inhibit the production of no. 36 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj 4: xxx-yyy, 2007 isj 4: 112-118, 2007 issn 1824-307x review longevity genes across species: conservation versus evolvability s salvioli1,2,3, p tieri1,2, g castellani2,4, m capri1, c barbi1, a santoro1,2, serena altilia1,2, l invidia1,2, m pierini1,2, e bellavista1,2, d monti5, c franceschi1,2,3,6 1 department of experimental pathology, university of bologna, 40126 bologna, italy 2 interdepartmental center “l. galvani” (c.i.g.), university of bologna, 40126 bologna, italy 3 er-gentech laboratory, 44100 ferrara, italy 4 dimorfipa, university of bologna,, 40064 ozzano dell'emilia, italy 5 department of experimental pathology and oncology, university of florence, 50134 florence, italy 6 department of gerontological science, italian national research centre on aging (inrca), 60131 ancona, italy accepted november 11, 2007 abstract the search for longevity genes has greatly developed in recent years basing on the idea that a consistent part of longevity is determined by genetics. the ultimate goal of this research is to identify possible genetic determinants of human aging and longevity, but studies on humans are limited by a series of critical restrictions. for this reason, most of the studies in this field have been, and still are, performed on animal models, basing on the assumption that fundamental biological mechanisms are highly conserved throughout evolution and that, accordingly, extrapolation from model systems to humans is quite reasonable. indeed, many comparative data obtained on single genes or gene families fit with this assumption. however, it is also clear that, despite such a basic conservative scenario, major changes also occurred in evolution, particularly regarding biological regulatory processes and integration between and among pathways. this consideration raises the fundamental question of the transferability of the results obtained from model systems to humans. in this review, we discuss the differences between animal models and men regarding the genetics of aging and longevity, and the possible reasons that can explain such discrepancies, with a particular emphasis on the phenomena of conservation and evolvability of biological systems. finally we will suggest a possible strategy to identify putative longevity genes basing on their position inside conserved metabolic structures. key words: genetics of longevity; animal models; conservation; evolvability introduction in biological studies, the reductionist approach has been very successful, and the use of simple experimental models allowed the researcher to obtain an exceptional number of results. the most popular example of this approach is represented by the bacteriovorous nematode caenorhabditis elegans, a small worm composed of 959 cells in the adult hermaphrodite form. a defined subset of 131 cells undergoing programmed cell death has allowed to the identification of specific apoptosisrelated genes, the so called ced genes (hengartner ___________________________________________________________________________ corresponding author: claudio franceschi department of experimental pathology university of bologna via s. giacomo 12, 40126 bologna, italy e-mail: claudio.franceschi@unibo.it and horvitz, 1994). cloning of these c. elegans genes has revealed that nematodes and mammals share a common pathway for programmed cell death. the same reductionist approach has led to the identification of longevity genes in c. elegans since 1988 (friedman and johnson, 1988). in 1992, johnson and lithgow wrote: “if we are fortunate and aging processes exhibit evolutionary conservation, many exciting possibilities await”. at least in some cases, human homologs of invertebrate longevity genes have been found (in particular genes belonging to the igf-1 signalling pathway, see barbieri et al., 2003). such similarities are striking and suggest that the insulin/igf-i regulatory system arose early in evolution and that the fundamental mechanisms that control aging and longevity may be evolutionarily conserved from invertebrates (and even yeast) to mammals, not excluding humans. 112 table 1 comparison between animals and humans as experimental models for genetic studies on aging and longevity. *shorter life span and genetic inbreeding can be both advantageous and disadvantageous. for example, it is questioned whether the effect of the accumulation of mtdna mutations on aging can be seen in mice that live only two years at maximum (santoro et al., 2006). genetic inbreeding is generally advantageous, but it has hampered to discover the effects of mtdna haplogroups on longevity, that can only be seen in outbred populations as humans are (de benedictis et al., 1999). animal models humans advantages shorter life span* genetically inbred* possibility to use very high number of subjects controlled environment simple organisms (especially if invertebrates) subjects can be easily genetically manipulated many genes involved in longevity has been discovered only in human studies disadvantages shorter life span* genetically inbred* genetic and epigenetic differences with respect to humans different level of complexity with respect to humans long life span complex organisms no possibility of genetic manipulation large populationspecific genetic differences profound influence of the environmental background presence of cultural factors other than the relative simplicity of the organism to be studied, c. elegans has many other evident advantages over different experimental models: for example the short life-span, that allows the execution of experiments in short times (experiments that otherwise will be intolerably longer in animal species with a life span of many years, like primates). another advantage is the great number of animals that can be studied with a relatively small effort, and most importantly, the possibility to easily manipulate the genome of such animals, in order to obtain data on the role of particular genes in the aging process or in life span control. these and other pros and cons of invertebrates experimental models are listed in table 1. the main problem with these experimental models is that, for a series of reasons, we were not so fortunate as hoped by johnson and lithgow. indeed, many data obtained on animal models were not confirmed on humans or, on the contrary, some contributions to the genetics of longevity were discovered in humans and not always have been replicated in animals (franceschi et al., 2007). in this review we will briefly summarise some paradigmatic cases in which results found in animals have not been reproduced in men and vice versa, and we will discuss the possible reasons that can explain these differences, with a particular emphasis on two striking features of the conserved biological structures, i.e. conservation and evolvability. genetics of aging and longevity 1: the case of p66shc gene possible reason for the discrepancies between data obtained from animal models and humans can be the profound differences in the environment where experimental animals and humans live in. only recently it has been considered by researchers that the animals used for experiments live all their entire life in safe and pathogen-free environment. they do not have to cope with predators or pathogens, they do not suffer famine, they even do not have to struggle for food, nor to compete for reproduction. this setting assures basic requirement of scientific research, i.e. the possibility to replicate the results in different laboratories, but of course it is a strongly unnatural situation that may lead to confounding results, especially when studying the genetics of aging and longevity. an example of such situation is given by the discovery that the ablation of p66shc gene leads to an enhancement of longevity in mice (migliaccio et al., 1999). a fraction of p66shc has a mitochondrial localization where it can oxidise cytochrome c and give rise to the production of radical oxygen species (ros) (giorgio et al., 2005), and the localization to mitochondria seems to be regulated by pkc-β and pin-1 (pinton et al., 2006). thus, it seems that p66shc gene impinges upon aging and longevity by regulating the resistance to oxidative stress-mediated apoptosis (trinei et al., 2002). then, if p66shc is a bona fide 113 pro-aging gene, one should expect that long living animals do express this gene at low levels. to date, this hypothesis has not yet been tested. however, we studied p66shc expression in humans, and, unexpectedly, we found that centenarians have higher levels of p66shc with respect to young people (pandolfi et al., 2005). these findings lead to the conclusion that the effect on longevity of p66shc is either species-specific, or highly dependent on the environmental conditions. supposing that such effects are not species-specific, it has to be considered that all animal species including humans are evolved and still live in an environment that is profoundly different from that of a laboratory (for example it is very dirty from an immunological point of view), thus it can not be excluded that p66shc has important effects for the fitness of the organism in the wild that are not necessary in a cage. to say, it is possible that p66shc-/animals can have some disadvantages in survival with respect to wild type animals, and that such effects only become evident in the environment where genetic selection occurred. finally, it must be also considered that the ablation of p66shc gene can lead to an altered stress response that may be the real responsible for the increased life span observed in p66shc-/animals. genetics of aging and longevity 2: the case of mitochondrial dna the contribution to longevity of the inherited variants of mitochondrial dna (mtdna) has been neglected until studies performed on humans addressed the question. in particular, some haplogroups of the mtdna have been found to be more represented among centenarians and longliving subjects with respect to younger people (tanaka et al., 1998; de benedictis et al., 1999). this discovery would have never come from inbred animals which share the same mtdna molecule (mtdna does not recombine and it is inherited only from the mother). the involvement of mtdna inherited variants in longevity has not yet been confirmed in animals, but other very elegant experiments in mice have indicated that such variants can modulate functions that are likely important for survival, such as memory and learning (roubertoux et al., 2003). a possible positive role in longevity for specific somatic mutations of the mtdna has also been postulated basing on studies on humans (zhang et al., 2003; niemi et al., 2005; rose et al., 2007). the c150t mutation in the dloop of the mtdna molecule in particular has been found to be more represented in long-living people with respect to younger counterparts. such a mutation seems to cause an alternative origin of replication of the mtdna strands and would be thus advantageous for mtdna pool maintenance (zhang et al., 2003). more recently it has been reported that the tendency to accumulate somatic mutations at the mtdna seems to be genetically controlled, since centenarians and their offspring and nephews share the same levels of mtdna heteroplasmy at the level of d-loop, which differ from that of unrelated people (rose et al., 2007). these figures indicate that, despite their usefulness, animal models are unable to identify all the genetic determinants of human longevity, because of intrinsic limitations of the model, or for specific differences between animals and humans. the opposite situation is also possible, that is, results obtained in animal models that can not be reproduced in humans. for example, recent findings indicate that mtdna point mutations can accumulate in organs and tissues without affecting aging in mice (vermulst et al., 2007), but still the levels of mtdna mutations found in such a model (polgmut/+ mice) are much higher than that found in aged human colonic crypts (taylor et al., 2003), thus suggesting that the functional impact of mtdna mutations is likely different in mice and humans (khrapko and vijg, 2007). genetics of aging and longevity 3: population heterogeneity, heterozigosity and homozygosity a peculiar contribution to the study of the genetics of aging and longevity has come from observations on the levels of heterozygosity and homozygosity of genetic loci of interest. it is generally accepted that natural selection awards heterozygosity as a way to attain high levels of fitness during the reproductive period. nevertheless, it was not known whether the levels of heterozygosity were also fitting with longevity. by studying people of different ages including centenarians we observed that, contrary to what it was expected, the level of homozygosity of a locus in chromosome 1p35 was increased in centenarians with respect to young people (bonafè et al., 2001). in a following study, we observed that this locus, rich in alu sequences, contains a gene called ythdf2 that shows an increase in homozygosity in centenarians (cardelli et al., 2006). in this study we genotyped 412 participants of different ages, including 137 centenarians, and we confirmed the increased homozygosity in centenarians at this locus, and observed a concomitantly increased frequency of the most frequent allele and the corresponding homozygous genotype. remarkably, the same genotype was associated with increased ythdf2 messenger rna levels in immortalized lymphocytes. thus, these data suggest a possible role of this locus in human longevity, and more interesting, they suggest the counterintuitive concept that increased homozygosity can contribute to human longevity. these results has been obtained thanks to the study of a genetically heterogeneous population, as humans are. in animal models, different strains are used, but the animals of each strain are rather homogeneous from a genetic point of view, thus making this discovery almost impossible in such models. when these results will undergo replication in animal models, genetically heterogeneous animals must be used. the role of the environment 1: the antigenic load an important environmental factor impinging upon longevity is the antigenic load, i.e. the number and intensity of antigenic stimuli to which everybody is exposed during lifetime (de martinis et al., 2005). besides the life-threatening risks of the exposure to highly pathogenic microorganisms, it is becoming 114 evident that also the mild chronic exposure to antigenic stimuli (for example to viruses such as cytomegalovirus, cmv) for a period of time largely unpredicted by evolution profoundly affects the possibility to attain longevity (pawelec et al., 2006; vescovini et al., 2007). this life-long exposure to antigenic stimuli is one of the main causes of modifications occurring with age of the immune system, leading to the phenomenon known as immunosenescence and to an increase in inflammatory reactions that favours the onset of many age-associated diseases which do share an inflammatory pathogenic background, such as type ii diabetes, cardiovascular diseases, neurodegenerative diseases and many types of cancer. this agerelated increase in inflammatory markers has been termed “inflammaging” by our group (franceschi et al., 2000). the effect of such (acute or chronic) antigenic exposure is forcedly skipped out in most experimental models, where animals live in a pathogen-free environment. in particular, the role of chronic antigenic burden on longevity is extremely difficult to assess in animals living in a clean or even sterile environment. furthermore, the immune responses are clearly very important in order to attain longevity, and not only the immune system of a mammal is much more complicate than that of an invertebrate, but also the characteristics of immunosenescence, i.e. the age-related modifications of the immune responses, can be strikingly different among species (pawelec et al., 2002). such a confounding factor can overcome the effect of the gene of interest, thus leading the researcher to the conclusion that such a gene does not play a role in longevity. the role of the environment 2: the dietary restriction as mentioned at the beginning of this review, a striking difference between men and animal models regards metabolic regulatory processes. to date, the so called directionality theory distinguishes between species according to the ecological constraints they have to face within their ecological niche (braeckman et al., 2006). according to this theory, two groups of species are recognized: equilibrium species and opportunistic species. the first group is composed mainly by large mammals, like humans, that spent the most part of their evolutionary history in a stationary growth phase, with a limited but roughly constant amount of resources. the latter group includes many small mammals like rodents, insects and worms like c. elegans. these species are subject to fluctuations in size because of irregular availability of resources (periods of abundance and scarcity). it is very likely that species belonging to the two groups, despite the fact that they can share genes or even entire metabolic pathways, may have a different behaviour in terms of survival in face of environmental challenges. for example, the best known treatment that extends life span in animal models (mainly opportunistic species, according to the directionality theory) is dietary restriction (dr), i.e. the reduction in food intake. studies on c. elegans have showed that dr increases life span of such animals (klass, 1977; lakowski and hekimi, 1998; houthoofd et al., 2002). the same has been demonstrated for other “opportunistic” species that are classical animal experimental models, such as drosophila and mice, but contrasting results have been reported on long living mammals and humans (kayo et al., 2001; roth et al., 2004; everitt and le couteur, 2007). many data suggest that dr acts as a low intensity stressor and increases metabolic stability (masoro 1998, butov et al., 2001). equilibrium species, like humans, already have a strong metabolic stability, and thus dr would have a relatively small effect on such species (braekman et al., 2006; demetrius, 2006). from survival studies on overweight and obese people, it is estimated that long-term dr could add 3 to 13 years to human life expectancy (holloszy and fontana, 2007), quite far from the dramatic effects observed in worms and rodents, and in any case much lower than the effects of improved life-style conditions. one of the effects of dr is the reduction of the insulin/igf-1 signalling pathway (clancy et al., 2002), and, as mentioned, this pathway appears to be conserved throughout evolution from yeast to mammals. genetic studies on humans have indicated that some polymorphisms of genes involved in insulin/igf-1 pathway (igf-1r, pi3kcb) are associated with longevity (bonafè et al., 2003). these polymorphisms are correlated to low levels of igf-1, and this finding fits with the data obtained in experimental models, confirming that lower activity of the insulin/igf-1 pathway is a major determinant of longevity and that genes involved in such a pathway can be considered longevity genes. it is to note however that these and other genes, such as mtor and p66shc, increase longevity when they are (at least partially) turned off, and their ablation often produces dwarf animals with a series of defects such as obesity and decreased fertility (longo and finch, 2003). moreover, it has been reported that high levels of igf-1 and low levels of proinflammatory cytokines such as il-6 are beneficial for muscle mass maintenance and are associated with decreased risk of mortality in old people (barbieri et al., 2003). thus, there is a clear trade off between early life fitness and longevity, and every species is the result of a million-years evolution that tuned between these two contrasting goals, reaching a peculiar equilibrium for any species. hence, comparison between species in which this trade-off has led to a different equilibrium is risky. however, a series of possible pharmacological treatments have been envisaged to mimic the prolongevity effects of igf-1 deficiency escaping in the same time the detrimental side-effects (obesity, dwarfism, infertility, sarcopenia) of this deficiency (longo and finch, 2003). conservation and evolvability recent conceptualizations stressed the importance of robustness as one of the fundamental properties of living organisms, from cellular (stelling, 2004) to organismal level (kitano and oda, 2006; kitano, 2007), a characteristic acquired during evolution which allow them to be error-tolerant and to easily respond to external perturbations (jeong et 115 al., 2000). metabolic stability appears to be genetically determined and respond to the requisites of robustness. on the basis of available data obtained from different organisms, especially yeast and invertebrates, a “bow-tie” organizational architecture is likely to be a common feature of highly organized, robust systems (csete and doyle, 2002, 2004), which enables them to accommodate perturbations and fluctuations on many temporal and spatial scales. a bow-tie model is present when many inputs converge on, and are integrated by, few elements, and many different outputs come out as the product of the integration. the elements composing the core (“knot”) of the bow-tie in a biological system are “hub proteins” and the genes that encode them are likely to be in many cases robustness genes. such a structure has also an inner fragility, because few enzymes responsible for robustness can be easily hijacked by pathogenic microorganisms or used to amplify pathological processes (csete and doyle, 2002; kitano and oda, 2006; kitano, 2007). much of the core of a bow-tie is often conserved throughout evolution, but this conservation does not prevent, but rather facilitates the variability of the possible outputs. thus, a bowtie structure allows both robustness and evolvability (gerhart and kirschner, 1997; caporale, 2003). longevity could be intended as a consequence of the robustness of the animal system. indeed, it is conceivable that the outputs of a bow-tie structure include elements that ultimately control the life-span of the animal. if these elements can vary, as a feature of the bow-tie evolution, this variation may explain, at least in part, why knot genes, yet conserved, not always result to be involved in longevity on evolutionarily distant animals according to classical association or functional studies, thus casting some doubts on their role as real “longevity genes”. conclusions homo sapiens is one of the most long-living species among animals, but his longevity has had a dramatic increase in recent times, at least in western countries, due to profound modifications in lifestyle. thus, human longevity has two major components, whose effects are very difficult to distinguish, genetics and culture. this raises an insurmountable obstacle, since no animal species depends on culture as humans do. thereafter, results obtained on animals studies not only must be always confirmed on humans, but they are also not completely satisfactory for a series of reasons: these studies do not consider neither the genetic and epigenetic differences existing between men and animal species, even those much close to humans, nor the influence of culture on the duration of the life after the age of reproduction, i.e. after the period influenced by natural selection. as an example of specific genetic differences between man and animals, it can be mentioned the case of tp53 gene. this gene is crucial for a series of biological processes such as apoptosis, cell senescence, dna repair, and energy metabolism, and is considered a longevity gene in both animals and humans (donehower, 2005). in humans tp53 gene harbours a common functional polymorphism at codon 72 which alters the protein' functions (thomas et al., 1999; dumont et al., 2003; bonafè et al., 2004). this polymorphism has been and still is under investigation for its possible involvement in longevity (bonafè et al., 2002; van heemst et al., 2005), and such effects, if any, can be detected only in humans, because this polymorphism does not exist in chimpanzee, while in mice it seems to have no effects (phang and sabapathy, 2007). moreover, our data indicate that the effects of such a polymorphism become evident as the age of the studied subjects increases (bonafè et al., 2004; salvioli et al., 2005) thus suggesting that the role of such a gene does change with age. this could be a case of antagonistic pleiotropy, that is, a gene that has a positive effect for fitness at young age could turn to be detrimental later on, in a period of time not selected for reproductive fitness, or vice versa. the genetics of human longevity has thus been proposed to be described as a post-reproductive one (de benedictis and franceschi, 2006). it is at present unknown whether also laboratory animals have a post-reproductive genetics, and which similarities it has with the human one. in conclusion, animal models remain an irreplaceable tool to shed light into the genetics of aging and longevity, but the transferability of the results to humans is always an issue. as discussed all along this review, longevity genes are classically identified by their association with an extended life-span, but for a series of reasons this strategy is not always successful, and results can not always be reproduced in different experimental models. here we propose the idea that longevity genes could be identified by an alternative strategy. as mentioned, conservation and evolvability are inherent features of the bow-tie structures, that determine robustness. if robustness is important for longevity, it can be hypothesised that longevity genes should participate with a core position to a bow-tie metabolic structure. we propose that a new way to identify putative genetic determinants of longevity could be the conservation of their products in bow-tie structures all along evolution, rather than for their association with prolonged life-span. in this perspective, studies of comparative biology associated with a systems biology approach could be useful tools to get some insights also into the genetics of human longevity. acknowledgements this work was supported by eu (european union) grant “geha – genetics of healthy aging” fp6-503270; eu grant “proteomage” fp6518230; the prriitt program of the emilia-romagna region (and fondi strutturali obiettivo 2); italian ministry of health grant “progetto finalizzato <<studio delle differenze uomo-donna nei meccanismi patogenetici delle malattie cardiovascolari>>” to cf; italian ministry of university and research (miur) prin 2006 project to cf and dm (# 2006061707), and ss (# 2006063387); university of bologna grant “ricerca fondamentale orientata (rfo ex 60%) 2005”; roberto and cornelia pallotti legacy for cancer research grants to cf and ss. references barbieri m, bonafe m, franceschi c, paolisso g. insulin/igf-i-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans. am. j. physiol. 116 endocrinol. metab. 285: e1064-e1071, 2003. barbieri m, ferrucci l, ragno e, corsi a, bandinelli s, bonafe m, et al. chronic inflammation and the effect of igf-i on muscle strength and power in older persons. am. j. physiol. endocrinol. metab. 284: e481-487, 2003. bonafe m, barbi c, storci g, salvioli s, capri m, olivieri f, et al. what studies on human longevity tell us about the risk for cancer in the oldest old: data and hypotheses on the genetics and immunology of centenarians. exp. gerontol. 37: 1263-1271, 2002. bonafe m, barbieri m, marchegiani f, olivieri f, ragno e, giampieri c, et al. polymorphic variants of insulin-like growth factor i (igf-i) receptor and phosphoinositide 3-kinase genes affect igf-i plasma levels and human longevity: cues for an evolutionarily conserved mechanism of life span control. j. clin. endocrinol. metab. 88: 3299-3304, 2003. bonafe m, cardelli m, marchegiani f, cavallone l, giovagnetti s, olivieri f, et al. increase of homozygosity in centenarians revealed by a new inter-alu pcr technique. exp. gerontol. 36: 1063-1073, 2001. bonafe m, salvioli s, barbi c, trapassi c, tocco f, storci g, et al. the different apoptotic potential of the p53 codon 72 alleles increases with age and modulates in vivo ischaemia-induced cell death. cell death differ. 11: 962-973, 2004. braeckman bp, demetrius l, vanfleteren jr. the dietary restriction effect in c. elegans and humans: is the worm a one-millimeter human? biogerontology 7: 127-133, 2006. butov a, johnson t, cypser j, sannikov i, volkov m, sehl m, et al. hormesis and debilitation effects in stress experiments using the nematode worm caenorhabditis elegans: the model of balance between cell damage and hsp levels. exp. gerontol. 37: 57-66, 2001. caporale lh. natural selection and the emergence of a mutation phenotype: an update of the evolutionary synthesis considering mechanisms that affect genome variation. annu. rev. microbiol. 57: 467-485, 2003. cardelli m, marchegiani f, cavallone l, olivieri f, giovagnetti s, mugianesi e, et al. a polymorphism of the ythdf2 gene (1p35) located in an alu-rich genomic domain is associated with human longevity. j. gerontol. a biol. sci. med. sci. 61: 547-556, 2006. clancy dj, gems d, hafen e, leevers sj, partridge l. dietary restriction in long-lived dwarf flies. science 296: 319, 2002. csete m, doyle j. reverse engineering of biological complexity. science 295: 1664-1669, 2002. csete m, doyle j. bow ties, metabolism and disease. trends biotechnol. 22: 446-450, 2004. de benedictis g, franceschi c. the unusual genetics of human longevity. sci. aging knowledge environ. 2006(10): pe20, 2006. de benedictis g, rose g, carrieri g, de luca m, falcone e, passarino g, et al. mitochondrial dna inherited variants are associated with successful aging and longevity in humans. faseb j. 13: 1532-1536, 1999. de martinis m, franceschi c, monti d, ginaldi l. inflamm-ageing and lifelong antigenic load as major determinants of ageing rate and longevity. febs lett. 579: 2035-2039, 2005. demetrius l. aging in mouse and human systems: a comparative study. ann. n. y. acad. sci. 1067: 66-82, 2006. donehower la. p53: guardian and suppressor of longevity? exp. gerontol. 40: 7-9, 2005. dumont p, leu ji, della pietra ac 3rd, george dl, murphy m. the codon 72 polymorphic variants of p53 have markedly different apoptotic potential. nat. genet. 33: 357-365, 2003. everitt av, le couteur dg. life extension by calorie restriction in humans. ann. n.y. acad. sci. [epub ahead of print], 2007 franceschi c, bonafe m, valensin s, olivieri f, de luca m, ottaviani e, et al. inflamm-aging. an evolutionary perspective on immunosenescence. ann. n.y. acad. sci. 908: 244-254, 2000. franceschi c, capri m, monti d, giunta s, olivieri f, sevini f, et al. inflammaging and antiinflammaging: a systemic perspective on aging and longevity emerged from studies in humans. mech. ageing dev. 128: 92-105, 2007. friedman db, johnson te. a mutation in the age-1 gene in caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. genetics 118: 75-86, 1988. gerhart j, kirschner m. cells, embryos, and evolution. blackwell science, uk, 1997. giorgio m, migliaccio e, orsini f, paolucci d, moroni m, contursi c, et al. electron transfer between cytochrome c and p66shc generates reactive oxygen species that trigger mitochondrial apoptosis. cell 122: 221-233, 2005. hengartner mo, horvitz hr. programmed cell death in caenorhabditis elegans. curr. opin. genet. dev. 4: 581-586, 1994. holloszy jo, fontana l. caloric restriction in humans. exp. gerontol. 42: 709-712, 2007. houthoofd k, braeckman bp, lenaerts i, brys k, de vreese a, van eygen s, et al. axenic growth upregulates mass-specific metabolic rate, stress resistance, and extends life span in caenorhabditis elegans. exp. gerontol. 37: 13711378, 2002. jeong h, tombor b, albert r, oltvai zn, barabasi al. the large-scale organization of metabolic networks. nature 407: 651-654, 2000. johnson te, lithgow gj. the search for the genetic basis of aging: the identification of gerontogenes in the nematode caenorhabditis elegans. j. am. geriatr. soc. 40: 936-945, 1992. kayo t, allison db, weindruch r, prolla ta. influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. proc. natl. acad. sci. usa 98: 5093-5098, 2001. khrapko k, vijg j. mitochondrial dna mutations and aging: a case closed? nat. genet. 39: 445-456, 2007. kitano h, oda k. robustness trade-offs and hostmicrobial symbiosis in the immune system. mol. syst. biol. 2: 2006-2022, 2006. kitano h. a robustness-based approach to systemsoriented drug design. nat. rev. drug discov. 6: 117 202-210, 2007. klass mr. aging in the nematode caenorhabditis elegans: major biological and environmental factors influencing life span. mech. ageing dev. 6: 413-429, 1977. lakowski b, hekimi s. the genetics of caloric restriction in caenorhabditis elegans. proc. natl. acad. sci. usa 95: 13091-13096, 1998. longo vd, finch ce. evolutionary medicine: from dwarf model systems to healthy centenarians? science 299: 1342-1346, 2003. masoro ej. hormesis and the antiaging action of dietary restriction. exp. gerontol. 33: 61-66, 1998. migliaccio e, giorgio m, mele s, pelicci g, reboldi p, pandolfi pp, et al. the p66shc adaptor protein controls oxidative stress response and life span in mammals. nature 402: 309-313, 1999. niemi ak, moilanen js, tanaka m, hervonen a, hurme m, lehtimaki t, et al. a combination of three common inherited mitochondrial dna polymorphisms promotes longevity in finnish and japanese subjects. eur. j. hum. genet. 13: 166170, 2005. pandolfi s, bonafe m, di tella l, tiberi l, salvioli s, monti d, et al. p66(shc) is highly expressed in fibroblasts from centenarians. mech. ageing dev. 126: 839-844, 2005. pawelec g, barnett y, forsey r, frasca d, globerson a, mcleod j, et al. t cells and aging, january 2002 update. front. biosci. 7: d10561d1083, 2002. pawelec g, koch s, franceschi c, wikby a. human immunosenescence: does it have an infectious component? ann. n. y. acad. sci. 1067: 56-65, 2006. phang bh, sabapathy k. the codon 72 polymorphism-specific effects of human p53 are absent in mouse cells: implications on generation of mouse models. oncogene 26: 2964-2974, 2007. pinton p, rimessi a, marchi s, orsini f, migliaccio e, giorgio m, et al. protein kinase c beta and prolyl isomerase 1 regulate mitochondrial effects of the life-span determinant p66shc. science 315: 659-663, 2007. rose g, passarino g, scornaienchi v, romeo g, dato s, bellizzi d, et al. the mitochondrial dna control region shows genetically correlated levels of heteroplasmy in leukocytes of centenarians and their offspring. bmc genomics 8: 293 [epub ahead of print], 2007. roth gs, mattison ja, ottinger ma, chachich m, lane ma, ingram dk. aging in rhesus monkeys: relevance to human health interventions, science 305: 1423-1426, 2004. roubertoux pl, sluyter f, carlier m, marcet b, maarouf-veray f, cherif c, et al. mitochondrial dna modifies cognition in interaction with the nuclear genome and age in mice. nat. genet. 35: 65-69, 2003. salvioli s, bonafe m, barbi c, storci g, trapassi c, tocco f, et al. p53 codon 72 alleles influence the response to anticancer drugs in cells from aged people by regulating the cell cycle inhibitor p21waf1. cell cycle 4: 1264-1271, 2005. santoro a, salvioli s, raule n, capri m, sevini f, valensin s, et al. mitochondrial dna involvement in human longevity biochim. biophys. acta 1757: 1388-1399, 2006. stelling j, sauer u, szallasi z, doyle fj 3rd, doyle j. robustness of cellular functions. cell 118: 675685, 2004. tanaka m, gong js, zhang j, yoneda m, yagi k. mitochondrial genotype associated with longevity. lancet 351: 185-186, 1998. taylor rw, barron mj, borthwick gm, gospel a, chinnery pf, samuels dc, et al. mitochondrial dna mutations in human colonic crypt stem cells. j. clin. invest. 112: 1351-1360, 2003. thomas m, kalita a, labrecque s, pim d, banks l, matlashewski g. two polymorphic variants of wild-type p53 differ biochemically and biologically. mol. cell biol. 19: 1092-1100, 1999. trinei m, giorgio m, cicalese a, barozzi s, ventura a, migliaccio e, et al. a p53-p66shc signalling pathway controls intracellular redox status, levels of oxidation-damaged dna and oxidative stress-induced apoptosis. oncogene 21: 38723878, 2002. van heemst d, mooijaart sp, beekman m, schreuder j, de craen aj, brandt bw, et al. variation in the human tp53 gene affects old age survival and cancer mortality. exp. gerontol. 40: 11-15, 2005. vermulst m, bielas jh, kujoth gc, ladiges wc, rabinovitch ps, prolla ta, et al. mitochondrial point mutations do not limit the natural lifespan of mice. nat. genet. 39: 540-543, 2007. vescovini r, biasini c, fagnoni ff, telera ar, zanlari l, pedrazzoni m, et al. massive load of functional effector cd4+ and cd8+ t cells against cytomegalovirus in very old subjects. j immunol. 179: 4283-4291, 2007. zhang j, asin-cayuela j, fish j, michikawa y, bonafe m, olivieri f, et al. strikingly higher frequency in centenarians and twins of mtdna mutation causing remodeling of replication origin in leukocytes. proc. natl. acad. sci. usa 100: 11161121, 2003. 118 isj 3: 89-96, 2006 isj 4: 10-12, 2007 issn 1824-307x short communication monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2006: regular changes of cytotoxicity during the year d malagoli, l casarini, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted february 01, 2007 abstract by monitoring the course of hemolymph cytolytic activity in mytilus galloprovincialis during 2006, we have observed important fluctuations in the percentage of cytotoxic animals over the year. the changes seem to be correlated with seasonal variations in the temperature, but observations in mussels kept in aquaria indicated that this parameter is not the main cause of the fluctuations. data presented here suggest that normal levels of cytotoxicity can be predicted in a population for a specific period of the year, therefore confirming the value of this parameter in determining the immune efficiency of mussels at a given time. key words: mytilus galloprovincialis; cytotoxicity; immune efficiency __________________________________________________________________________________________ introduction the presence of hemolytic molecules in the hemolymph of molluscs has been reported on several occasions (wittke and renwrantz, 1984; merker and levine, 1986; hubert et al., 1997), but the natural target of these molecules has still not been clarified. it is conceivable that hemolyitic factors can be included in the humoral component of invertebrate innate immunity (hubert et al., 1997), but there is very little information about the relationship between hemolytic activity and immune efficiency in the mussel (malagoli and ottaviani, 2005). recently, the hemolytic activity of the bivalve mytilus galloprovincialis has been seen to be influenced by stressful and pathological conditions imposed either in laboratory aquaria (malagoli and ottaviani, 2005) or encountered in mussel farms (franchini et al., 2005). in order to take hemolytic activity as a valid parameter for evaluating whether the immune efficiency of mussels is compromised in particular circumstances, it is important to know how this activity changes during the year. this report provides data on fluctuations in hemolymph cytotoxic activity in mussels from the adriatic sea in italy during 2006. moreover, ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology via campi 213/d 41100 modena, italy e-mail: ottaviani.enzo@unimore.it comparison of the present results with data collected in 2005 (malagoli et al., 2005) and with seasonal variations in water temperature suggest that hemolymph cytotoxicity is a parameter subjected to seasonally-regulated fluctuations. materials and methods animals specimens of the bivalve mollusc mytilus galloprovincialis were obtained monthly from local fishermen in the cesenatico area (fc, italy). after their collection, 40 animals were used to obtain the hemolymph immediately, while the remaining specimens were maintained in the laboratory aquaria in artificial seawater (temperature 16 ± 1 °c, ph 8.0 ± 0.2 and salinity 35 ± 1 psu). after 14 days, a further 40 mussels were sacrificed and the hemolymph withdrawn. hemolymph preparation and cytotoxicity assay the detailed procedure for the hemolysis assay is described elsewhere (malagoli and ottaviani, 2005). in short, the hemolymph was collected by gently aspirating with a sterile syringe inserted between the mussel valves and filtered into sterile tubes using 0.2 μm sterile filters. hemolytic activity was evaluated by checking the cytolysis of human a positive erythrocytes obtained after washing the whole blood at least three times in 9 vol. of sterile nacl 0.9 %. subsequently, the erythrocytes were 10 fig. 1 course of mussel cytotoxicity during 2005 and 2006 in the of cesenatico area. please note that the value for february 2005 is missing (malagoli et al., 2005). re-suspended in sterile tbs (50 mm tris-hcl, 200 mm nacl, 10 mm cacl2, ph 8.5) at a final concentration of 2x109 cells/ml. five hundred μl of filtered hemolymph were added to 500 μl of erythrocyte suspension and then incubated for 1 h at 25 °c (hubert et al., 1997; malagoli and ottaviani, 2005). after incubation, samples were centrifuged at 3000xg for 5 min at 4 °c, and the optical density (od) of the supernatants was evaluated by measuring absorbance at 541 nm with a helios β spectrophotometer (spectronic unicam, cambridge, uk). samples with an od above the fixed od threshold level of 0,5 where considered cytotoxic (malagoli and ottaviani, 2005). the experiments were repeated twice in duplicate for each animal. all chemical reagents came form sigma-aldrich (st louis, mo, usa). results and discussion the monthly evaluation of the hemolytic activity revealed significant fluctuations in the percentage of cytotoxic animals during the year (fig. 1). even if the mean percentage of cytotoxic bivalves is 45 %, two peaks were registered during the year: one at the end of the spring and the second at the end of the summer. interestingly, the trends in hemolyitic activity in the second half of 2005 and 2006 almost overlapped (fig. 1). since no peculiar situations were reported for the area in which the mussels were reared, the comparable results indicate that cytotoxicity in mussel populations is normally subject to fluctuations during the year. temperature can be considered the most common variable in the mussel farm area assessed in this study. we therefore compared the time course of water temperature with that of cytoxicity in the mussel population (fig. 2). the two variables seem to be correlated, since the peak in hemolytic activity in the hemolymph corresponded to the two periods in which the temperature either started to rise or to fall (fig. 2). however, the comparison of cytotoxicity between animals sacrificed immediately after collection and those kept for two weeks in aquaria at a constant temperature indicates that temperature cannot be the main parameter in determining cytotoxicity (fig. 3). for each month, the percentage fig. 2 comparison between hemolymph cytotoxic activity and temperature variations during 2005 (a) and 2006 (b). 11 fig. 3 comparison between cytotoxicity immediately after collection (white bars) or after a period of conservation in the aquarium at 17 °c (black bars). of cytotoxic mussels was almost identical in the two groups of animals. the essentially unmodified cytotoxic activity following conservation in the aquarium is in agreement with observations in 2005 (malagoli et al., 2005), demonstrating that even when mussels are maintained at a constant temperature different to that of the seawater, the level of cytotoxicity among population does not change significantly. in previous articles, we have reported that sudden changes in aquarium temperature can modify the number of animals displaying significant cytotoxic activity (malagoli and ottaviani, 2005), but we have also found that when sudden modifications in environmental parameters do not intervene, cytotoxic activity is a relatively stable immune function (malagoli et al., 2005). further studies are required to establish whether the component influencing the time course of cytotoxicity is mainly environmental or rather connected to the mussel life cycle. the mussel’s cytotoxic response to seasonal changes would represent a classical example of ecoimmunology. evolutionary ecologists assume that immunological defences must be minimized in terms of metabolic cost, because there must be a trade-off between maintaining a normal immune response and facing the significant changes in life conditions. from an evolutionary point of view, this balance plays a key role in species survival (lochmiller and deerenberg, 2000). concluding, mussel cytotoxicity is an activity that changes over the year with a regular time course, meaning that normal levels can be predicted for a given period. our observations support the idea of using hemolymph cytotoxicity as a useful parameter in evaluating the immune efficiency of mussels at a specific point in time (malagoli and ottaviani, 2005). acknowledgment we are grateful to centro di ricerche marine (cesenatico, fc, italy) for financial support for this work. the authors wish also to thank the centro trasfusionale policlinico (modena) for providing the blood, arpa-er for giving us the access to temperature measurements and mr m marangoni who kindly provided the mussels. references franchini a, malagoli d, ottaviani e. investigation of the loss of byssus in mytilus galloprovincialis from mussel farms in the adriatic sea. cell biol. int. 29: 857-860, 2005. hubert f, cooper el, roch p. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloproíincialis. biochim. biophys. acta 1361: 29–41, 1997. lochmiller rl, deerenberg c. trade-offs in evolutionary immunology: just what is the cost of immunity? oikos 88: 87-98, 2000. malagoli d, ottaviani e. cytotoxicity as a marker of mussel health status j. mar. biol. ass. uk 85: 359-362, 2005. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005. inv. surv. j. 3: 1-3, 2006. merker mp, levine l. a protein from the marine mollusc aplysia californica that is hemolytic and stimulates arachidonic acid metabolism in cultured mammalian cells. toxicon 24: 451465, 1986. wittke m, renwrantz l. quantification of cytotoxic hemocytes of mytilus edulis using a cytotoxicity assay in agar. j. invertebr. pathol. 43: 248-253, 1984. 12 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj106.pdf isj 2: 124-131, 2005 issn 1824-307x research report wound repair in the marine worm sipunculus nudus (sipunculidae) g d’ancona lunetta institute of histology and embryology university of palermo, italy accepted september 14, 2005 abstract the cells and molecules involved in the wound healing of sipunculus nudus were studied. an incision, 5 mm in length, was cut longitudinally at a site opposite the anus and 10 mm from the introvert. the histological study performed at different times showed an involvement of both type i and type ii granulocytes in the process of healing. the former were capable of extracellular digestion and they were immunoreactive to anti-il-4, -il-10 and -epidermal growth factor (egf) antibodies (abs); the latter were involved in the synthesis of connective tissue from 24 h after the incision, thereby causing the initial closing of the wound. after 70 h, a continuous layer of type ii granulocytes was found on the sides of the wound where the future muscle tissue would be formed; many of these granulocytes had been partially degranulated. it was not possible to establish any existing relationship between the functions of type i granulocytes and their reactivity to anti-il-4, -il-10 and -egf abs. kew words: marine worm; sipunculus nudus; wound repair; cytokines introduction the process of wound healing has been the subject of intensive research, mainly in vertebrates (redd et al., 2004; harvey, 2005; whitney, 2005). with regard to invertebrates, kindred (1924) observed that the removal of a fragment of the body wall in echinoderms led to the healing of the wound, by the proliferation and infiltration of cells from the surrounding connective tissue. in asterias the aggregation of coelomocyte and of amoebocytes from adjacent tissue was seen to contribute to the wound healing (anderson, 1962, 1965). in the sea cucumber stichopus tremulus numerous morula cells, found in proximity of the incision, were supposed to play a significant role in healing wounds, and to be homologous with the vertebrate mastocytes (rollefsen, 1965). in contrast, in stichopus badionotus, after superficial cutaneous incisions cowden (1968) observed a rapid repair with complete fibrogenesis without the intervention of morula cells. corresponding author: d’ancona lunetta g istituto di istologia ed embriologia, dipartimento di biologia, università di palermo, viale delle scienze, 90123 palermo, italy e-mail: dancona@unipa.it the migration of epidermal and pigment cells from the periphery of the wound margin in thyone briareus caused re-epithelialization in the absence of evident mitotic activity, an morula cells seemed to be involved in this migration although their precise role is unknown (menton and eisen, 1974). fibroblast-like cells, pigment cells and numerous coelomocytes were the first cells to arrive at wound sites in the holothuria polii and the newly formed collagen fibres were synthesized by type ii spherula cells. cutaneous lesions performed after antigenic stimulation healed more slowly than controls since type ii spherula cells were also actively involved in the immune reaction by forming brown bodies in cooperation with the amoebocytes (d’ancona, unpublished). consequently, in this case, a lower number of type ii spherula cells are available for the synthesis of connective components (canicattì and d’ancona, 1989). recently, franchini and ottaviani (2000) have studied the effects of platelet-derived growth factor (pdgf-ab) and transforming growth factor (tgf-β)1 in the repair mechanism of wounds in the mollusc limax maximus. the main repair stages include an initial infiltration phase in which the hemocytes migrate and stratify at wound margins, actively phagocitize cell debris and damaged tissue and were immunoreactive to anti-il-1α, -il-8 and -tumor necrosis factor (tnf)-α antibodies (abs). this was followed by the formation of granulation tissue, the synthesis and depositing of 124 extracellular matrix components, such as fibronectin, collagen fibres and reticular fibres. finally, reepithelialization of the wound occurred. the exogenous administration of pdgf-ab and tgf-β1 stimulated the tissue healing process though a general acceleration of the activities involved. in the coelomic fluid of s. nudus two types of granulocytes (type i and type ii), are distinguishable on the basis of their morphology and chemical composition. type i granulocytes do not have a phagocytic capability, even though they contain lytic enzymes; type ii granulocytes show phagocytic activity. furthermore, haemerythrocytes, signet-ring cells, urna cells complexes, empty vesicles, vesicle fragments, laminar structures, aggregates of stem cells and brown bodies were also found (d’ancona et al., 2004). in the present paper the mechanism of wound repair in sipunculus nudus was studied. furthermore, it was also examined if cells reactive to anti-il-4, -il-10 and -egf abs were present and involved in the wound healing of the marine worm. materials and methods twenty adult specimens of sipunculus nudus were incised with a scalpel in the frontal part of the body, producing a wound in a site opposite to the anus and 10 mm from the introvert. the incision, 5 mm in length, was performed longitudinally and it involved the integument and the circular muscle structure below. the animals were sacrificed and fixed in bouin mixture at different times after the incision (3, 15, 18, 22, 24, 71 and 96 h) and unwounded specimens were used as controls. fragments including the wound and 2 mm of surrounding healthy tissue were included in paraffin and 7 µm thick sections were stained with gomori triple staining (the connective tissue resulted green and the type i granulocytes red) and alcian blue-pas (type ii granulocytes stained green and pink) (ganter and jòlles, 1969; mazzi, 1977). the immunocytochemical reactions were performed by incubating sections for 1 h at room temperature (rt) in mouse primary monoclonal abs (euroclon, celbio, italy), raised against il-4, il-10 and egf, diluted 1:100 in phosphate-buffered saline (pbs) (1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4 and 0.065 m na2hpo4), rapidly washed in pbs, incubated for 30 min at rt in biotinylated goat anti-mouse immunoglobulins (kit dako cytomation, denmark), washed in pbs, incubated in streptavidin peroxidase conjugate for 30 min at rt. after washing, sections were stained for 15 min in the chromogen aminoethylcarbamate. sections incubated with normal non-immune rabbit serum were used as controls. results in physiological conditions the body walls of s. nudus consist of a cubic epithelium, secreting an outer cuticle, a derma, a layer of circular muscle, a layer of longitudinal muscle and the peritoneum. the derma contains fine fibres, connective cells and coelomatic, longitudinal canals, which communicate with each other and with the general coelom (hyman, 1959). as far as the wounded specimens are concerned, 3 h after the incision, the wound did not show any signs of healing, the layers of muscle and connective tissue were still damaged and the cuticle was missing. on the outer part of the wound, coelomocytes were present, most of which were acidophilic type i granulocytes (fig. 1). furthermore, type ii granulocytes and a few haemerythrocytes were observed. three h after the incision, an intense immunoreaction with anti-egf ab was observed in type i granulocytes, including those which were present in the coelomatic cavity (fig. 2). type ii granulocytes and haemerythrocytes were negative. no changes in the response were observed up to 15 h after the incision, but for an increase in exocytised acidophilic material. eighteen h after incision, new muscle fibres were not observed but fine connective fibres were evident. both partially degranulated type i granulocytes and type ii granulocytes were present, in addition to transparent spherical cells on the outer part and the sides of the wound. twenty-two h after incision, the wound had been externally closed up by fine collagen fibres and internally by numerous coelomocytes (haemerythrocytes, type i granulocytes and various type ii granulocytes). at this time, egf-like material was found in all type i granulocytes, while other coelomocytes were negative (fig. 3). twenty-four h after incision, the wound had closed: on the internal part of the wound, acidophilic type i granulocytes were present and the circular, muscle tissue was interrupted towards the central part of the wound. in its place there was newly synthesized connective tissue with numerous type i and type ii granulocytes nearby. type i granulocytes were highly reactive to the anti-il-4 ab, while type ii granulocytes, were less numerous and negative (figs 4, 5). in incisions of the entire animal wall, both the derma and muscle tissue were interrupted and the circular muscle fibres displayed rounded extremities, which were covered by connective tissue (fig. 6). the wound had been closed by various thin, connective lamina distributed over 2-3 layers at the extremities. each lamina contained type ii granulocytes and a few transparent cells (fig. 7). some type ii granulocytes start to loose their basophilic core, while others devoid of the basophilic granules, were amoeboid in shape with a flattened nucleus and were involved in collagen fibre production (fig. 8). twenty-four h after the incision, large spaces containing degranulating type i granulocytes were highlighted near the tissue on the internal part of the wound, and these degranulated cells flattened and formed a sort of barrier that trapped haemerythrocytes and type ii granulocytes (fig. 9). type i granulocytes located towards the coelomatic cavity were almost degranulated with a weak reaction to anti-il-10 ab. type ii granulocytes did not react with this ab, while degranulated material resulted highly immunopositive (fig. 10). ninety-six h after the incision, the walls of s. nudus had been reconstituted but the connective tissue was not always well compacted and the muscle fibres did not show their typical circular arrangement (fig. 11). in the lateral parts of the wound, the muscle fibres were interrupted and a great number of “spongy” type ii granulocytes, 125 fig. 1 histological section 3 h after incision, stained with gomori triple staining. note, on the outer part of the wound (op), coelomocytes (←) with acidophilic material and degranulated acidophilic material (*). the connective tissue and muscle tissue (**) are still damaged and the cuticle (c) is also missing. coelomatic cavity (cc). fig. 2 immunocytochemical reaction with anti-egf ab 3h after incision. positive type i granulocytes (g1); negative type ii granulocytes (g2) and haemerithrocytes (h) in the coelomatic cavity. fig. 3 immunocytochemical reaction with anti-egf ab 22 h after incision. positive type i granulocytes (g1) and degranulated material (dm); negative haemerithrocytes (h). outer part of the wound (op), cuticle (c). 126 fig. 4 immunocytochemical reaction with anti-il-4 ab 24 h after incision. positive type i granulocytes (g1) are present in the connetive tissue (ct). coelomatic cavity (cc); longitudinal muscle (lm), outer part of the wound (op). fig. 5 detail of fig. 4. type i granulocytes (g1) positive and type ii granulocytes (g2) negative to anti-il-4 ab. collagen fibres (cf). fig. 6 histological section 24 h after wound, stained with gomori triple staining. the incision concerned the entire thickness of the animal wall. the wound is closed by connective lamina (cl). longitudinal canal (lc); coelomocytes and acidophilic material (c); circular muscle fibres (cm); coelomatic cavity (cc); longitudinal muscle (lm). 127 fig. 7 detail of fig. 6. note longitudinal canal (lc); degranulated acidophilic material (*); coelomocytes (c); connective lamina distributed over 2-3 layers (cl); circular muscle fibres with rounded extremities (cm); coelomatic cavity (cc); longitudinal muscle (lm); outer part of the wound (op). fig. 8 detail of fig. 6. external surface of the wound. note type i granulocyte (g1); connective tissue (ct) where type ii granulocytes (g2a) displayed their basophilic core and type ii granulocytes shaped like a triangle (g2) are producing thin bundles of collagen fibrils (cf). fig. 9 histological section stained with gomori triple staining 24 h after a deep incision. note a barrier made up of flattened type i granulocytes (g1), which have trapped haemerythrocytes (h), type ii granulocytes (g2) and degranulated material (dm). 128 fig. 10 immunocytochemical reaction of section with anti-il-10 ab 24h after incision. partially degranulated type i granulocytes (g1) were moderately reactive whilst the degranulated material (dm) was highly reactive. negative type ii granulocyte (g2). note type i flattened granulocytes (fg1); connective tissue (ct); outer part of the wound (op); coelomatic cavity (cc). fig. 11 histological section 96 h after incision stained with gomori triple staining. the wound is partially healed. note cuticle (c); granulocytes; poorly compacted collagen fibrils (cf); circular muscle fibres (cmf) being formed; coelomic cavity (cc); longitudinal muscle (lm). fig. 12 detail of fig. 11 in the lateral parts of wound. note the interrupted muscle fibres (mf) and a great number of spongy type ii granulocytes (g2), separated by connective fibres (cf). 129 separated by connective material, formed a base onto which the muscle fibres were formed (fig. 12). elongated type i granulocytes, with varying amounts of acidophilic granules, were found between the forming muscle fibres. discussion the histological study performed 3-96 h after the incision in s. nudus revealed that type i and type ii granulocytes from the coelomatic cavity are responsible of the wound repair. type i granulocytes are the first cells to be activated and they degranulated thereby causing the transfer of their material to the surrounding area and, in particular, on type ii granulocytes. the contribution of the coelomocytes was not always the same. in most cases, the coelomocytes migrated from the coelomic cavity to the lesion zone, due to the probable presence of various chemotactic substances produced in situ (abercrombie, 1972; postletwaite, 1976). type i granulocytes contain lysosomal enzymes (matozzo et al., 2001; d’ancona et al., 2004) that could act in the digestion of cell debris and damaged tissue. the urna cell complexes present in the coelomatic liquid of s. nudus (d’ancona et al., 2004) and endowed with phagocytic activity (bang and bang, 1962) did not participate in wound repair process. unlike the hemocytes found in the wound repair of l. maximus (franchini and ottaviani, 2000), type i granulocytes do not take part directly by means of phagocytosis but they secrete enzymes into the extracellular environment. type i granulocytes also have an important hemostatic function. indeed, they move from the coelomatic cavity towards the wound in great amounts in order to create an aggregate that can prevent the passage of coelomatic material towards the outer and of pathogens towards the inner side of the body. these cells flattened after degranulation, stuck along the lower margins of the wound and form a lamina which entrap other cells, especially haemerythrocytes. many of these, in turn, flocked around the wound with the aim of supplying oxygen, which is necessary for metabolic activity (steins et al., 2001). rollefsen (1965) hypothesized that morula cells, present in the hydrovascular system and dermal connective tissue of s. tremulus, can be involved in the production of the intercellular substance of connective tissue, as they are both metachromatic and pas positive. morula cells may form fibres (endean, 1966) which are responsible for connective tissue growth and repair (smith, 1981). in other echinoderms polysaccharides and proteins are present in the granular inclusions of spherula cells (endean, 1958; hetzel, 1963; johnson, 1969; d’ancona and canicattì, 1990). type ii granulocytes in s. nudus showed the same cytological characteristics of the morula cells of s. tremulus and h. poli type i spherula cells (d’ancona and canicattì, 1990). they also have the same histochemical affinity of connective tissue and it can, therefore, be hypothesized that type ii granulocytes may be the main cell producers of connective material. the granulation tissue, typical of wound healing in vertebrates, was also found in l. maximus (franchini and ottaviani, 2000). in this mollusc the main cell type involved in the different phases of repair process is the hemocyte. indeed, this cell was immunoreactive to cytokines, was able to phagocytize cell debris and damaged tissue and showed fibroblastlike activity, as found by sminia et al. (1973) in l. stagnalis. in s. nudus numerous type i granulocytes are found everywhere and they continuously exocytise acidophilic material between other cells and muscle fibres. moreover these cells contain hydrolytic enzymes, able to remove cell debris and damaged tissue, and were also immunoreactive to egf-, il4 and il10 abs. in marine worm it can be supposed, as in vertebrates, that in the first hours after incision, an inflammatory response occurs, involving cellular proliferation of granulocytes (d’ancona et al., 2004). in mammals and some invertebrates, growth factors such as pdgf and tgf-β, play an important role in healing wounds as they are chemotactic and proliferative factors (seppa et al., 1982; deuel et al., 1982; senior et al., 1983; postlethwaite et al., 1987; wahl et al., 1987; clark et al., 1997; ottaviani et al., 1997; franchini and ottaviani, 2000). the presence of egf-, il-4and il-10-like material in s. nudus does not exclude the possibility that other cytokines or other factors, found in other invertebrates, could also be present in type i granulocytes, and that they could be directly involved in the process of wound healing. however, it is so far impossible to attribute a specific function to egf-, il-4and il-10-like material in the process of the healing of wounds in s. nudus. references abercrombie m. behavior of cells toward one another. in: montagna w, billingham re (eds), advances in biology of skin. v. wound healing, academic press inc, london, pp 95-112, 1972. anderson jm. studies on visceral regeneration in sea-stars. i. regeneration of pyloric caeca in henricia leviscula (stimpson). biol. bull. 122: 321-342, 1962. anderson jm. studies on visceral regeneration in sea-stars. ii. regeneration of pyloric caeca in asteriidae, with notes on the source of cells in regenerating organs. biol. bull. 128: 1-23, 1965. bang fb, bang bg. studies on sipunculid blood: immunological properties of coelomic fluid and morphology of “urn cell“. cahiers biol. mar. 3: 363-374, 1962. canicattì c, d’ancona g. cellul ar aspects of holothuria polii immune response. j. invert. pathol. 53: 152-158, 1989. clark ra, mccoy ga, folkvord jm, mcpherson jm. tgf-â 1 stimulates cultured human fibroblasts to proliferate and produce tissue-like fibroplasia: a fibronectin matrixdependent event. j. cell physiol. 170: 69-80, 1997. cowden rr. cytological and histochemical observations on connective tissue cells and cutaneous wound healing in the sea cucumber stichopus badionotus. j. invert. path. 10: 151-159, 1968. d’ancona g, canicattì c. the coelomocytes of holothuria polii (echinodermata). ii. cytochemical staining properties. bas. appl. histochem. 34: 209-218, 1990 d’ancona g, farina e, manione r. sipunculus nudus: particulate components of the coelomic fluid and its relationship with brown bodies. ital. j. zool. 71: 191-199, 2004. deuel tf, senior rm, huang js, griffin gl. chemotaxis of monocytes and neutrophils to platelet-derived growth factor. j. clin. invest. 69: 1046-1049, 1982. endean r. the coelomocytes of holothuria leucospilata. quart. j. microsc. sci. 99: 47-60, 1958. 130 endean r. the coelomocytes and coelomic fluids. in: boolootian ra (ed), physiology of echinodermata, j. wiley (interscience), london, pp 301-328, 1966. franchini a, ottaviani e. repair of molluscan tissue injury: role of pdgf and tgf-β. tissue cell 32: 312-321, 2000. ganter p, jòlles g. histochimie normale et pathologique. gauthier-villars, paris, 1969. harvey c. wound healing. orthop. nurs. 24:143-57, 2005. hetzel hr. studies on holothurian coelomocytes. i. a survey of coelomocyte types. biol. bull. 125: 289-301, 1963. hyman le. the invertebrates: smaller coelomate groups. vol. 5, mcgraw hill book co, new york, 1959. johnson pt. the coelomic elements of sea urchin. histochemia 17: 213-231, 1969. kindred je. the cellular elements in the perivisceral fluid of echinoderms. biol. bull. 50: 147-154, 1924. matozzo v, perin l, cima f, ballarin l. phagocytic and enzymatic activities of cells and urn cell complexes in the coelomic fluid of the marine worm sipunculus nudus (sipunculida). ital. j. zool. 68: 273-280, 2001. mazzi v, manuale di tecniche istologiche e istochimiche. piccin editore, pavia, 1977. menton dn, eisen az. cutaneous wound healing in the sea cucumber thyone briareus. j. morph. 141: 185-204, 1974. ottaviani e, franchini a, kletsas d, bernardi m, genedani s. involvement of pdgf and tgf-β in cell migration and phagocytosis in invertebrate and human immunocytes. anim. biol. 6: 91-95, 1997. postlethwaite ae, snyderman r, kang ah. the chemotactic attraction of human fibroblasts to a lymphocyte-derived factor. j. exp. med. 144: 1188-1203, 1976. redd mj, cooper l, wood w, stramer b, martin p. wound healing and inflammation: embryos reveal the way to perfect repair. philos. trans. r. soc. lond. b biol. sci. 359:777-784, 2004. rollefsen i. studies on the mast cell-like morula cells of the holothurian sticopus tremulus. arb. univ. bergen. mat. naturv. s.8. oslo. 1-19, 1965. senior rm, griffin gl, huang js, walz da, deuel tf. chemotactic activity of platelet alpha granule proteins for fibroblast. j. cell biol. 96: 382-385,1983. seppa h, grotendorst g, seppa s, schiffmann e, martin gr. platelet-derived growth factor is chemotactic for fibroblasts. j. cell biol. 92: 584-588,1982. sminia t, pietersma k, scheerboom jem. histological and structural observations on wound healing in the freshwather pulmonate limnaea stagnalis. z. zellforsch. 141: 561-573, 1973. smith vj. the echinoderm. in: ratcliffe na, rowley af (eds), invertebrate blood cells. vol. 2, academic press, london, pp 513-562, 1981. steins a, hahn m, junger m. venous leg ulcers and microcirculation. clin. hemorheol. microcir. 24: 147-153, 2001. wahl sm, hunt da, wakefield lm, mccartney-francis n, wahl lm, roberts ab, sporn mb. transforming growth factor type â induces monocyte chemotaxis and growth factor production. proc. natl. acad. sci. usa 84: 57885792, 1987. whitney jd. overview: acute and chronic wounds. nurs. clin. north am. 40:191-205, 2005. 131 the fast evolutionary dynamics of ascidian mitochondrial genome: an exception to the general deuterostome evolutionary trend isj 6: s21-s28, 2009 issn 1824-307x review evolutionary mitogenomics of chordata: the strange case of ascidians and vertebrates c gissi, f griggio, f iannelli dipartimento di scienze biomolecolari e biotecnologie, università di milano, milano, italy accepted march 13, 2009 abstract the availability of almost one thousand complete mitochondrial genome (mtdna) sequences of chordates provides an almost unique opportunity to analyse the evolution of this genome in the phylum chordata, and to identify possible divergent evolutionary trends followed by the three chordate subphyla: vertebrata, cephalochordata and tunicata. here, we review some genome-level features of mtdna, such as genetic code, gene content, genome architecture and gene strand asymmetry, mostly focusing on differences existing between tunicates and remaining chordates. indeed, tunicate mtdnas show a surprisingly high variability in several genome-level features, even though the current tunicate taxon sampling is absolutely insufficient and is focused mainly on the class ascidiacea. on the contrary, a stabilization of the mtdna structural and evolutionary features is observed in both cephalochordates and vertebrates, where genome-level features are almost invariant. thus, different evolutionary dynamics, probably related to divergent functional constraints, have modelled the overall mtdna structure and organization of the three chordate subphyla. key words: mitochondrial genome; evolution; chordates; tunicates; ascidians introduction the mitochondrial genome (mtdna) is used both as a model system for studying genome evolution, and as a molecular marker to reconstruct animal phylogeny, both at high and low taxonomic levels (saccone et al., 1999; saccone et al., 2002). this dual interest in mtdna constitutes an advantage in phylogenetic studies, since the understanding of the evolutionary peculiarities of a given character greatly facilitates the evaluation of its reliability as a phylogenetic marker. for phylogenetic purposes, the sequences of one or more mitochondrial genes, or of the fast-evolving control region of vertebrates, are commonly analysed with traditional molecular evolutionary methods. besides the sequence itself, genome-level features have been also used for phylogenetic reconstructions: gene order, gene content, and changes in the genetic code are all examples of mt genome-level features, used to clarify controversial phylogenetic relationships, especially at high taxonomic levels (boore, 2006). ___________________________________________________________________________ corresponding author: carmela gissi dipartimento di scienze biomolecolari e biotecnologie università di milano via celoria 26, 20133 milano, italy e-mail: carmela.gissi@unimi.it to date, the entire mtdna sequence has been determined for 1206 metazoan species (gissi et al., 2008) but the taxon sampling is highly biased towards vertebrates and arthropods (genembl, september 2008). the taxon sampling of chordata is also quite heterogeneous, as it is almost exhaustive for vertebrates and cephalochordates (lancelets) but absolutely inadequate for tunicates, where sampling is almost exclusively derived from representatives of the class ascidiacea (fig. 1). in spite of the scarce data, it is evident that ascidian mtdnas possess several unusual features compared to other chordates, such as a fast nucleotide substitution rate (yokobori et al., 1999, 2005), gene orders that are extremely variable both within the class and compared to other metazoans (gissi et al., 2004; yokobori et al., 2005; iannelli et al., 2007a), and a variable number of trna genes (gissi and pesole, 2003; gissi et al., 2004; iannelli et al., 2007a). thus, several clues point to the existence of an almost opposite mtdna evolutionary trend between tunicate and remaining chordates, resulting in a strong variability of mt genome features in ascidians/tunicates versus a relative stability in vertebrates and cephalochordates (gissi et al., 2008). the “strange case” of ascidian mtdna is even more intriguing when we consider the controversies on the phylogenetic position of s21 mailto:carmela.gissi@unimi.it tunicates within chordates and the debated phylogenetic relationships among the three tunicate classes. traditional morphological data indicate that tunicates should constitute the basal chordate subphylum clustering cephalochordates and vertebrates in the euchordata group (rowe, 2004). however, recent molecular phylogenetic analyses based on a large number of nuclear genes strongly support a sister relationship between tunicates and vertebrates (olfactores clade) and a basal position of cephalochordates within chordates (bourlat et al., 2006; delsuc et al., 2006). in reality, the position of tunicates in molecular phylogenetic analyses is quite unstable and varies according to which genes or molecules (nuclear or mitochondrial dna) are used (zrzavy et al., 1998; cameron et al., 2000; winchell et al., 2002; oda et al., 2004; bourlat et al., 2006; delsuc et al., 2006). with regard to phylogenetic relationships within tunicata, the analyses of morphological and/or molecular characters have given rise to distinct phylogenetic hypotheses most of which suggest that larvacea is the sister group to the rest of tunicates, and that thaliacea is the sister group to phlebobranch ascidians, thus rendering ascidiacea a paraphyletic group (see references in stach and turbeville, 2002; zeng and swalla, 2005). in this review we will provide an overview of some structural and evolutionary peculiarities of the chordate mtdna, investigated through the analysis of a carefully revised dataset of 865 complete mtdnas (obtained from gissi et al., 2008). in particular, we will compare four genome-level features: genetic code, gene content, genome architecture and gene strand asymmetry, among the three subphyla of vertebrata, cephalochordata and tunicata, highlighting both differences and similarities. our synthesis should stimulate further comprehensive analyses of these and other mitochondrial genomic features within a phylogenetic framework. genetic code the three chordate subphyla use each their own modified mitochondrial genetic code (table 1), differing only in the meaning of agr codons. the cephalochordate genetic code is quite controversial, as the agr codons were initially assigned to glycine (spruyt et al., 1998) and then to serine (boore, 1999) based on the analysis of a single lancelet mtdna. further studies, carried out on a large number of mtdnas, and comparing the conservation within deuterostomes of amino acid sites with agr codons in lancelets, have confirmed the hypothesis that agr encodes serine (nohara et al., 2005a). it should be noted that the codon usage of lancelets is characterized by the absence or extremely low number of agg codons, which occur only 4 times in a total of 14 available complete mtdnas (see notes of table 1), thus the meaning of agg codon remains uncertain. these data leave the evolutionary history of the chordate mt genetic code an open question, particularly in the light of the recently reported basal phylogenetic position of lancelets compared to vertebrates and tunicates (bourlat et al., 2006; delsuc et al., 2006). in this fig. 1 number of available complete mitochondrial genomes of deuterostomes, corresponding to deuterostome mtdna dataset analysed in this review. the three taxonomic classes of tunicates are shown as a polytomy, due to phylogenetic controversies. deuterostome phylogeny is in accordance with bourlat et al. (2006). respect, it is interesting that the genetic codes of both vertebrates and tunicates are unique among metazoans, while the genetic code of lancelets is shared by many protostome and deuterostome phyla (i.e., annelida, arthropoda, mollusca, nematoda and xenoturbellida). thus, the data on genetic code seem to support the basal position of cephalochordates compared to remaining chordates. gene content within vertebrates, the most frequent mitochondrial gene content consists of 37 genes encoding for 13 protein subunits of the oxidative phosphorylation complexes, two ribosomal rnas, and 22 trnas necessary for the translational machinery located within mitochondria. among the 865 analysed chordates, there are no cases of rrna gene loss/acquisition, while only four vertebrate species lose/acquire one or two protein-coding genes (table 2). this is in accordance with observations carried out on metazoan mtdnas, where the number of proteincoding and rrna genes changes rarely (gissi et al., 2008). on the contrary, the trna gene content is quite variable between different metazoan phyla and, in general, it depends on the genetic code employed and the taxonomic group (gissi et al., 2008). according to the relaxed wobble rules in codon-anticodon recognition and to the genetic code observed (table 1), the mt translation machinery of chordata should require a minimum set of 22 trnas in vertebrates and lancelets, and 23 trnas in tunicates. in fact two trnas are needed to decode each of the six-fold degenerate amino acids (leucine and serine in all chordates, plus glycine in tunicates), while only one trna is needed to decode each remaining amino acid. this trna number, predicted on basis of the genetic code, holds for s22 table 1 mitochondrial genetic code of deuterostomes compared to the universal code. “codtab” indicates the number of the table containing the entire genetic code, available at the web site “http://www.ncbi.nlm.nih.gov/taxonomy/utils/wprintgc.cgi”. the genetic code of hemichordates is reported as codtab 9, although exceptions in the meaning of the aaa codon indicate that this is a new mt genetic code uga aua aga agg aaa codtab universal stop ile arg arg lys 1 mitochondrial chordata vertebrata trp met stop stop = 2 tunicata trp met gly gly = 13 cephalocordata a trp met ser ser/ absent = 5 hemichordata b trp = ser ser = new balanoglossus trp = ser absent absent 9 saccoglossus trp = ser ser = new echinodermata trp = ser ser asn 9 xenoturbellida trp met ser ser = 5 a: agg codons have been found only in epigonichthys maldivensis (2 occurrences) and in branchiostoma belcheri (1 occurrence in both the ab078191 and ab083383 mtdna sequences), while they are absent in the mtdna of remaining branchiostoma species and of asymmetron genus (7 and 4 mtdna sequences, respectively). b: the ac number of the two complete mtdna sequences of hemichordates are: af051097 (balanoglossus carnosus) and ay336131 (saccoglossus kowalevskii). vertebrates and lancelets but not for tunicates, whose most frequent (i.e. “standard”) trna gene number is 24 and includes an additional trna-met. in fact, all tunicates encode for the common trnamet with cat anticodon and for a trna-met with the unusual tat anticodon, probably acting as initiator and elongator trna-met, respectively (yokobori et al., 1999, 2003, 2005; gissi et al., 2004; iannelli et al., 2007a, 2007b). this additional trna-met(tat) gene is shared only with some mollusc bivalves, thus this feature has arisen independently in two distant animal lineages (gissi et al., 2008). variations from the standard trna gene content have been found less frequently in vertebrates and lancelets than in tunicates. as shown in table 2, only 1.6 % of the 849 vertebrate mtdnas analysed show loss/acquisition of trna genes. moreover, the loss of trna genes is an uncommon event compared to trna acquisition, as it has been found only in three of 849 vertebrates (table 2), suggesting that trna loss can not be easily compensated by an analogous function encoded by the nuclear genome. this may be due to differences in trna structure or difficulties in mitochondrial import of trnas. in addition, differences in trna content mainly concern amphibian species, which show acquisition of additional trna genes as results of duplication of mt regions and/or extensive gene order rearrangements (liu et al., 2005; mueller and boore, 2005; zhang et al., 2005; kurabayashi et al., 2006; san mauro et al., 2006). among lancelets, only branchiostoma floridae exhibits an additional trna gene (spruyt et al., 1998), although the modified cloverleaf structure and the presence of a non-canonical tct anticodon suggest that this is a trna-like secondary structure rather than a functional trna. finally, in tunicates the situation is extremely variable (see table 2): about half of the seven available tunicate mtdnas show differences from the expected trna gene number, moreover loss and acquisition of trna genes seem to be equally tolerated. although the tunicate taxon sampling is still insufficient to draw definitive conclusions, the current data suggest that the tunicate trna gene number could vary in a species-specific manner. genome architecture to compare the overall structure of mt genomes, we have introduced a new genomic feature, named genome “architecture” (ar). the genome ar takes into account both gene content and gene order, and it is defined as the order of the entire set of functional mt-encoded genes, including duplicated genes. therefore, two mtdnas with different architecture may show differences in gene content and/or gene order. the three chordate subphyla show different level of variability in genome ar, indeed genome ar is quite stable in vertebrates, moderately conserved in cephalochordates and highly variable in tunicates. in vertebrates, 80 % of the analysed species show the same genome ar as the human mtdna (hereafter named std, “standard”), while remaining species exhibit one of the 42 additional genome ars (table 3). in particular, the std architecture is shared by all eutherian and prototherian mammals, as well as by almost all fishes (except for few neopterygian species), and it is lost in all species belonging to metatheria, sauropsida (aves plus crocodylidae), and hyperoartia (table 3). in addition, non-standard ars are highly similar to the standard ar, as in most cases they can be converted into the standard ar by the translocation of few genes, most of which are located near to the mtdna replication origins (boore, 1999). s23 table 2 chordate taxa with a non-standard mitochondrial gene content (different from the expected number of 37 genes in vertebrates and lancelets; 39 genes in tunicates). the ac number of the corresponding mtdna sequences is also reported. the minus symbol indicates genes encoded by the minor strand. gsa: gene strand asymmetry, calculated as described in table 4 taxon (n° available mtdnas) ac number proteins trna gsa additional lost additional lost vertebrata (849) amphibia (82) polypedates megacephalus nc_006408 atp8, nad5 0.49 rhyacotriton variegatus nc_006331 trnt 0.53 plethodon elongatus nc_006335 trnt 0.53 mantella madagascariensis nc_007888 trnm 0.53 gegeneophis ramaswamii nc_006301 trnf 0.50 fejervarya limnocharis nc_005055 trnm 0.53 aneides hardii nc_006338 trnf, -trne, trnl(uur), -trnp 0.46 aves (73) diomedea melanophris a nc_007172 -nad6 trnt, -trnp, -trne 0.41 testudines (19) malacochersus tornieri b nc_007700 trnf 0.58 lepidosauria (44) cordylus warreni c nc_005962 trnt, -trnp 0.49 sphenodon punctatus nc_004815 nad5 trnk trnh, trnt 0.49 neopterygii (374) chionodraco rastrospinosus dq526431 nad6 trne 0.60 bathygadus antrodes nc_008222 trnl(uur) 0.53 cephalochordata (9) branchiostoma floridae y16474 trn(tct) d 0.51 tunicata (7) halocynthia roretzi ab024528 trnf 1 phallusia fumigata am292602 trni, trnx trnd 1 phallusia mammillata am292320 trnd 1 a: duplication of the region: +trnt-trnp-nad6-trne+cr, where cr corresponds to the control region (gibb et al., 2007) b: duplication of the region: +trnf+cr (parham et al., 2006), where cr corresponds to the control region c: tandem duplication of the region: +trnt-trnp (kumazawa, 2004) d: non-conventional trna with tct anticodon found in the b. floridae entry y16474 [first published as branchiostoma lanceolatum (spruyt et al., 1998), this sequence actually belongs to b. floridae (spruyt et al., 1998; nohara et al., 2005b)] in cephalochordates, the genome ar is slightly less conserved, as there are three different genome ars in 9 distinct species, and the most represented ar has been found in 55 % of the sampled species. finally, tunicates show an extreme ar variability, as each species has its own specific genome ar, with no ar shared by two or more mtdnas. this peculiarity appears even more surprising when considering that the seven sampled tunicates derive from a narrow taxonomic range and also include several congeneric ascidian species. additional mtdna sequences of ascidians produced in our laboratory (unpublished data) confirm this extreme ar variability. we have analysed the complete mtdna of ten additional ascidians, sampled both as closely (congeneric) and distantly related species, and no genome ar is present in more than one species. moreover, gene order variability at intra-genus level has been observed in all ascidian orders (aplousobranchiata, stolidobranchiata and phlebobranchiata). a comparative analysis among all chordates shows that no ar is shared by different subphyla when all genes are taken into account. on the contrary, the exclusion of trna genes makes the genome architectures (ar-trna) much more similar and sometimes identical between different subphyla. for example, the ar-trna of lancelets is identical or very similar to the standard vertebrate ar-trna depending on the genus (identical in branchiostoma plus epigonichthys; very similar in asymmetron). similarly, in vertebrates the exclusion of trna genes drastically reduces the number of different non-standard architectures (from 42 ar to 12 ar-trna; table 3) and increases the number of taxonomic groups showing a std architecture. in particular, excluding the trna genes, the std architecture is acquired by metatheria, crocodylidae and hyperoartia species, as well as by many lepidosaurians and amphibians (compare ar and ar-trna in table 3). these observations indicate that the extant genome ars of vertebrates and cephalochordates arose by modest rearrangements of the same ancestral ar, with modifications mainly concerning trna genes. s24 table 3 distribution of the standard (std) and non-standard (other) mtdna architecture (ar) between vertebrate taxonomic groups. ar: genome architecture calculated for all mt genes. ar(-trna): genome architecture calculated excluding the trna genes. std: standard ar, identical to the human mtdna architecture. nother : number of species showing a non-standard mtdna architecture. other_fishes: fish group including polypteridae, chondrostei, chondrichthyes, coelacanthiformes and dipnoi taxon mtdna n° n° ar n° ar(-trnas) std other a nother std other vertebrata 849 1 42 173 1 12 eutheria 197 1 1 prototheria 3 1 1 metatheria 22 1 22 1 testudines 19 1 2 2 1 1 lepidosauria 44 1 10 26 1 2 b aves 73 2 73 2 b crocodylidae 8 1 8 1 amphibia 82 1 16 26 1 4 b neopterygii 374 1 13 14 1 6 b other_fishes 23 1 1 hyperoartia 2 1 2 1 hyperotreti 2 1 1 a: four non-standard ar are shared by vertebrates belonging to different groups: ar-1 is shared by two neopterygians and two amphibians; ar-2 is shared by all metatherians and one amphibian; ar-3 is shared by one neopterygian and all crocodiles; ar-4 is shared by 72 birds and one lepidosaurian. b: the same non-standard genome ar(-trna) is shared by the following species: 72 aves, 2 neopterygii, 2 lepidosauria and 2 amphibia on the contrary, the genome architecture of tunicates does not show any similarity to that of remaining chordates. even the exclusion of trna genes, neither significantly reduces the number of different tunicate genome architectures (6 different ar-trna against 7 different ar when considering all genes) nor makes the tunicate mtdnas more similar in genome architecture to that of remaining chordates. these data remain valid also considering our enlarged and unpublished ascidian dataset. overall, these observations suggest a fast rate of mtdna rearrangements in the tunicate subphylum, with changes equally involving trna and other mitochondrial genes, while the strong similarity in ar-trna between vertebrates, cephalochordates, and remaining deuterostomes (excluding tunicates) (data not shown) supports the conservation of mtdna architecture during deuterostome diversification. gene strand distribution a peculiarity of metazoan mtdna is the asymmetric distribution of genes between the two strands, indeed a major and a minor strand are commonly recognized based on the number of encoded genes. this asymmetric gene distribution can be quantified using the gsa formula (gene strand asymmetry) (gissi et al., 2008), calculated as the absolute value of the difference in gene number between the two strands, divided by the total gene number. gsa values range from 0 to 1, with values close to zero indicating an almost equal number of genes encoded by the two strands, and values higher than 0.5 corresponding to the presence of at least 75 % of the genes on the major strand. the overall data on gene strand distribution in metazoans show a prevalence of high gsa values, while a symmetric gene distribution is very rare and it has been observed only in 17 phylogenetically distant species over 1206 complete metazoan mtdnas (gissi et al., 2008). among chordates, the gsa value is high and almost invariant within each subphylum. in vertebrates, the gsa is equal to 0.51 in almost all taxa, and the minor strand encodes only for one protein-coding gene, nad6, and 8 trna genes (table 4). the few exceptions (15 over 849 species) to this structure are related to inversion of a single trna gene (only two cases, table 4) or to changes in gene content (table 2). in the last cases, all additional duplicated genes are present on the same strand as the ancestral gene that gave rise to the duplication (table 2). in cephalochordates, the gsa ranges from 0.41 to 0.51, this modest variability being due to the different number of trnas encoded by the minor strand (from 8 to 10 trna genes, plus only one protein-coding gene, nad6 or nad5 depending on the species) (table 4). thus, vertebrates and lancelets exhibit a very similar gene strand asymmetry, as consequence of the similar genome architecture (see previous section), but they strongly differ in the tendency to switch the sense strand of a gene. indeed, lancelets show frequent gene inversions, both of large mt regions or single genes, while vertebrates show only two cases of gene inversion over a total of 849 analysed species (table 4). in particular, a strand switch of a 2.5 kb region including four genes (trnl(cun), nad5, trng, and nad6) has been found in the asymmetron genus compared to other lancelets and to the standard genome ar of vertebrates (nohara et al., 2005b; kon et al., 2007). s25 table 4 gene strand asymmetry (gsa) of chordate mtdnas, including data on number and name of genes encoded by the minor strand. for vertebrates, only species with the common mt gene content (37 genes) are reported in this table, and the minor strand genes are indicated as differences from the “standard” vertebrate situation. numbers in brackets refer to the number of species showing a given gsa taxon (n° species) gsa a minor strand genes n° trna cds vertebrata standard (833) 0.51 9 trnq, trna, trnn, trnc, trny, trns(ucn), trne, trnp nad6 neopterygii carapus bermudensis 0.46 10 standard plus trnm nad6 lepidosauria calotes versicolor 0.57 8 standard except trnp nad6 cephalochordata asymmetron inferum 0.46 10 trnn, trna, trnc, trny, trns(ucn), trng, trnl(cun), trne, trnp nad5 cephalochordata asymmetron lucayanum (3) 0.41 11 trna, trnc, trny, trnq, trnn, trns(ucn), trng, trnl(cun), trne, trnp nad5 cephalochordata branchiostoma (4) 0.51 9 trnq, trnn, trna, trnc, trny, trns(ucn), trne, trnp nad6 cephalochordata epigonichthys maldivensis 0.51 9 trnq, trnn, trna, trnc, trny, trns(ucn), trne, trnp nad6 tunicata (7) 1 0 a: the gsa is calculated as the absolute value of the difference in gene number between the two strands, divided by the total gene number. in addition, the inversion plus transposition of the trnq gene has been observed between asymmetron inferum and asymmetron lucayanus (kon et al., 2007). in vertebrates, the two observed cases of gene inversion concern a single trna gene (table 4) adjacent or translocated close to the control region (trnp and trnm, respectively), thus gene inversions have taken place near to the “hot spot” region of vertebrate genome rearrangements (boore, 1999). moreover, these rare events have occurred in two phylogenetically-distant species, a lizard and a fish: the inversion of trnp, found in the lizard calotes versicolor and shared by all south asian draconine agamids, appears incompatible with a trna remoulding process, and it has been explained by a homologous dna recombination helped by accidentally formed inverted repeats (amer and kumazawa, 2007); no hypothesis has yet been published to explain the translocation/inversion of trnm in the bone fish carapus bermudensis (see genembl accession number ap004404; (miya et al., 2003)). in general, gene inversion can not be explained by the tandem duplication/random gene loss model, the mechanism commonly invoked for mtdna rearrangements in metazoans, but can be easily explained by homologous or illegitimate recombination. thus, it has been proposed that the sporadic events of gene inversion observed in vertebrates are the result of rare dna recombination processes, while dna recombination is significantly high in the mtdna of cephalochordates, thus promoting frequent gene inversions in this subphylum (amer and kumazawa, 2007; kon et al., 2007). although the existence of recombination has been traditionally excluded in the mitochondria of metazoans, there is growing evidence for the occurrence of dna recombination in several taxonomic groups (lunt and hyman, 1997; hoarau et al., 2002; shao et al., 2005; tsaousis et al., 2005; guo et al., 2006) and for the presence of homologous enzymatic recombination activities in vertebrates (thyagarajan et al., 1996; kraytsberg et al., 2004). differently from other chordates, tunicates have a gsa equal to one, i.e. they show the most extreme gene strand asymmetry. thus, all genes of tunicates are encoded on the same strand, rendering the “minor” strand devoid of genes. this feature is shared by all tunicate species, including our unpublished dataset of ascidian mtdnas, making the gene strand asymmetry the only “invariant” mitochondrial feature currently found in tunicates. therefore, we can suppose that strong functional constraints have forced the tunicate mtdna to maintain all genes on the same strand, in spite of the numerous changes in genome ar and rearrangements of gene order: the overall transcription mechanism or processes regulating the level of transcription could be the most obvious functional constraints for the maintenance of all genes on the same strand. conclusions the data here summarized underline the existence of a significant variability between the three subphyla of chordata in several genome-level features. the genetic code exhibits a different specificity in each of the three subphyla, with cephalochordates retaining a genetic code more similar to that of remaining deuterostomes (table 1). similarities in genome architecture, i.e. in structural features such as gene content, gene order and gene strand distribution, cluster the vertebrates and cephalochordate subphyla to the exclusion of tunicates. in fact, tunicates are characterized by the s26 most extreme gene strand asymmetry, and a strong variability in genome ar, which is different between species, even when we analyse our unpublished dataset of ten additional ascidian species. on the contrary, cephalochordates are characterized by moderate rearrangements of genome ar, frequent gene inversions, and a gene order and gene strand asymmetry that can be easily traced back to that of vertebrates. finally, the genome ar of vertebrates is almost stable but not so invariant as previously reported: while gene inversions are very rare in all vertebrates, changes in gene content and gene order are quite frequent in amphibia and lepidosauria (see table 2 and table 3), and sporadic cases of genome rearrangements have been also observed in a few bony fishes, birds and turtles. thus, the initial dogma of a frozen genome ar in vertebrates has been broken by the identification of several deviations from the “standard” ar in some vertebrate groups (table 3). many of these nonstandard genome ars involve gene duplications or changes in the order of genes located close to the control region or surrounding the l-strand replication origin, suggesting that errors in the mtdna replication have given rise to these rearrangements. moreover, the trna genes are the most common gene category implicated in genome ar changes. although these differences in chordate mtdna evolutionary dynamics are already manifest, the extent of the surprising features of tunicate mtdna needs to be further confirmed through the analysis of a large number of ascidian mtdnas (our research field) and the sampling of the remaining tunicate classes of thaliacea and larvacea, for which mtdna sequences are almost absent. abbreviations cox1, cox2, cox3: cytochrome oxidase subunit i, ii, and iii protein genes; cob: cytochrome b gene; atp6, atp8: atp synthase subunit 6 and 8 genes; nad1, nad2, nad3, nad4, nad4l, nad5, nad6: nadh dehydrogenase subunit 1–6, 4l genes; rrns, rrnl: ribosomal rrna of the small and large subunit, respectively. transfer rna genes are named as trn followed by the one-letter code of the specified amino acid and, in brackets, the recognized codon or the used anticodon. acknowledgements this work was supported by the “italian ministry of university and research” and the university of milan (cofin-2006 and first projects to cg). we thank g pesole and d horner for valuable suggestions on the manuscript. references amer sa, kumazawa y. the mitochondrial genome of the lizard calotes versicolor and a novel gene inversion in south asian draconine agamids. mol. biol. evol. 24: 1330-1339, 2007. boore jl. animal mitochondrial genomes. nucleic acids res. 27: 1767-1780, 1999. boore jl. the use of genome-level characters for phylogenetic reconstruction. trends ecol. evol. 21: 439-446, 2006. bourlat sj, juliusdottir t, lowe cj, freeman r, aronowicz j, kirschner m, et al. deuterostome phylogeny reveals monophyletic chordates and the new phylum xenoturbellida. nature 444: 8588, 2006. cameron cb, garey jr, swalla bj. evolution of the chordate body plan: new insights from phylogenetic analyses of deuterostome phyla. proc. natl. acad. sci. usa 97: 4469-4474, 2000. delsuc f, brinkmann h, chourrout d, philippe h. tunicates and not cephalochordates are the closest living relatives of vertebrates. nature 439: 965-968, 2006. gibb gc, kardailsky o, kimball rt, braun el, penny d. mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations. mol. biol. evol. 24: 269-280, 2007. gissi c, iannelli f, pesole g. complete mtdna of ciona intestinalis reveals extensive gene rearrangement and the presence of an atp8 and an extra trnm gene in ascidians. j. mol. evol. 58: 376-389, 2004. gissi c, iannelli f, pesole g. evolution of the mitochondrial genome of metazoa as exemplified by comparison of congeneric species. heredity 101: 301-320, 2008. gissi c, pesole g. transcript mapping and genome annotation of ascidian mtdna using est data. genome res. 13: 2203-2212, 2003. guo x, liu s, liu y. evidence for recombination of mitochondrial dna in triploid crucian carp. genetics 172: 1745-1749, 2006. hoarau g, holla s, lescasse r, stam wt, olsen jl. heteroplasmy and evidence for recombination in the mitochondrial control region of the flatfish platichthys flesus. mol. biol. evol. 19: 2261-2264, 2002. iannelli f, griggio f, pesole g, gissi c. the mitochondrial genome of phallusia mammillata and phallusia fumigata (tunicata, ascidiacea): high genome plasticity at intra-genus level. bmc evol. biol. 7: 155, 2007a. iannelli f, pesole g, sordino p, gissi c. mitogenomics reveals two cryptic species in ciona intestinalis. trends genet. 23: 417-422, 2007b. kon t, nohara m, yamanoue y, fujiwara y, nishida m, nishikawa t. phylogenetic position of a whale-fall lancelet (cephalochordata) inferred from whole mitochondrial genome sequences. bmc evol. biol. 7: 127, 2007. kraytsberg y, schwartz m, brown ta, ebralidse k, kunz ws, clayton da, et al. recombination of human mitochondrial dna. science 304: 981, 2004. kumazawa y. mitochondrial dna sequences of five squamates: phylogenetic affiliation of snakes. dna res. 11: 137-144, 2004. kurabayashi a, usuki c, mikami n, fujii t, yonekawa h, sumida m, et al. complete nucleotide sequence of the mitochondrial genome of a malagasy poison frog mantella madagascariensis: evolutionary implications on mitochondrial genomes of higher anuran s27 groups. mol. phylogenet. evol. 39: 223-236, 2006. liu zq, wang yq, su b. the mitochondrial genome organization of the rice frog, fejervarya limnocharis (amphibia: anura): a new gene order in the vertebrate mtdna. gene 346: 145151, 2005. lunt dh, hyman bc. animal mitochondrial dna recombination. nature 387: 247, 1997. miya m, takeshima h, endo h, ishiguro nb, inoue jg, mukai t, et al. major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial dna sequences. mol. phylogenet. evol. 26: 121-138, 2003. mueller rl, boore jl. molecular mechanisms of extensive mitochondrial gene rearrangement in plethodontid salamanders. mol. biol. evol. 22: 2104-2112, 2005. nohara m, nishida m, miya m, nishikawa t. evolution of the mitochondrial genome in cephalochordata as inferred from complete nucleotide sequences from two epigonichthys species. j. mol. evol. 60: 526-537, 2005a. nohara m, nishida m, nishikawa t. new complete mitochondrial dna sequence of the lancelet branchiostoma lanceolatum (cephalochordata) and the identity of this species' sequences. zoolog sci. 22: 671-4, 2005b. oda h, akiyama-oda y, zhang s. two classic cadherin-related molecules with no cadherin extracellular repeats in the cephalochordate amphioxus: distinct adhesive specificities and possible involvement in the development of multicell-layered structures. j. cell. sci. 117: 2757-2767, 2004. parham jf, macey jr, papenfuss tj, feldman cr, turkozan o, polymeni r, et al. the phylogeny of mediterranean tortoises and their close relatives based on complete mitochondrial genome sequences from museum specimens. mol. phylogenet. evol. 38: 50-64, 2006. rowe t. assembling the tree of life. oxford university press, oxford, uk, 2004. saccone c, gissi c, reyes a, larizza a, sbisà e, pesole g. mitochondrial dna in metazoa: degree of freedom in a frozen event. gene 286: 3-12, 2002. saccone c, de giorgi c, gissi c, pesole g, reyes a. evolutionary genomics in metazoa: the mitochondrial dna as a model system. gene 238:195-209, 1999. san mauro d, gower dj, zardoya r, wilkinson m. a hotspot of gene order rearrangement by tandem duplication and random loss in the vertebrate mitochondrial genome. mol. biol. evol. 23: 227-234, 2006. shao r, mitani h, barker sc, takahashi m, fukunaga m. novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtdna recombination in the chigger mite, leptotrombidium pallidum. j. mol. evol. 60: 764-773, 2005. spruyt n, delarbre c, gachelin g, laudet v. complete sequence of the amphioxus (branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates. nucleic acids res. 26: 3279-3285, 1998. stach t, turbeville jm. phylogeny of tunicata inferred from molecular and morphological characters. mol. phylogenet. evol. 25: 408-428, 2002. thyagarajan b, padua ra, campbell c. mammalian mitochondria possess homologous dna recombination activity. j. biol. chem. 271: 27536-27543, 1996. tsaousis ad, martin dp, ladoukakis ed, posada d, zouros e. widespread recombination in published animal mtdna sequences. mol. biol. evol. 22: 925-933, 2005. winchell cj, sullivan j, cameron cb, swalla bj, mallatt j. evaluating hypotheses of deuterostome phylogeny and chordate evolution with new lsu and ssu ribosomal dna data. mol. biol. evol. 19: 762-776, 2002. yokobori s, oshima t, wada h. complete nucleotide sequence of the mitochondrial genome of doliolum nationalis with implications for evolution of urochordates. mol. phylogenet. evol. 34: 273-283, 2005. yokobori s, ueda t, feldmaier-fuchs g, paabo s, ueshima r, kondow a, et al. complete dna sequence of the mitochondrial genome of the ascidian halocynthia roretzi (chordata, urochordata). genetics 153: 1851-1862, 1999. yokobori s, watanabe y, oshima t. mitochondrial genome of ciona savignyi (urochordata, ascidiacea, enterogona): comparison of gene arrangement and trna genes with halocynthia roretzi mitochondrial genome. j. mol. evol. 57: 574-587, 2003. zeng l, swalla bj. molecular phylogeny of protochordates: chordate evolution. can. j. zool. 83: 24-33, 2005. zhang p, zhou h, liang d, liu yf, chen yq, qu lh. the complete mitochondrial genome of a tree frog, polypedates megacephalus (amphibia: anura: rhacophoridae), and a novel gene organization in living amphibians. gene 346: 133-143, 2005. zrzavy j, mihulka s, kepka p, bezdek a, tietz d. phylogeny of the metazoa based on morphological and 18s ribosomal dna evidence. cladistics 14: 249-285, 1998. s28 genetic code gene content amphibia (82) nc_007172 -nad6 nc_007700 nc_005962 cephalochordata (9) tunicata (7) gene strand distribution conclusions isj 5: 103-xxx, 2008 isj 5: 103-109, 2008 issn 1824-307x visions and perspectives specificity, learning and memory in the innate immune response m brehélin1, p roch2 1 ecologie microbienne des insectes et interactions hôte-pathogène, université de montpellier 2, inra, france 2 ecosystèmes lagunaires, université de montpellier 2, cnrs, ifremer, france accepted july 29, 2008 summary immunity in invertebrates was for long analyzed in terms of the overall response; this resulted in misunderstandings concerning specificity and memory. recent reports of maternal transmission of immunity, and the discovery of the high diversity of receptors-effectors, have required the status of innate immunity to be reconsidered. there are few examples of obvious specificity towards some pathogens, but this cannot be generalized to all invertebrate species. the existence of memory is even more controversial. here, we suggest looking for immune memory by quantifying key molecular effectors (i) within single individuals following first and second exposures to a pathogen and (ii) in primed mother and her offspring. key words: innate immunity; invertebrates; vertebrates; drosophila introduction although invertebrates are able to develop immune reactions, it has long been assumed that their immune systems are non adaptive (not anticipatory) and respond identically to multiple challenges. in other words, it was believed that they lack specificity and memory, and this conviction was strongly supported by the fact that, unquestionably, invertebrates do not possess antibodies, or t or b cells. however, this dogma that adaptive (anticipatory) immunity is absent from invertebrates is now cracking, not because the long search for antibodies has succeeded, but because at least some invertebrates possess functional equivalents of the acquired responses of vertebrates (see kvell et al., 2007 for a review). since the work of carton et al. (1992), diverse examples of strongly specific immune responses in invertebrates against potential parasites or pathogens have been reported (little et al., 2003; pham et al., 2007). in addition, the wide diversity of receptors and effectors, including fibrinogen-related proteins (frep) in snail (adema et al., 1997),toll-like ___________________________________________________________________________ corresponding author: philippe roch equipe pathogènes et environnement cnrs-um2-ifremer ecolag université de montpellier 2 cc 093, place eugène bataillon f-34095 montpellier cedex 5, france e-mail: philippe.roch@univ-montp2.fr receptors (tlr) in drosophila (tauszig et al., 2000) and in sea urchin (hibino et al., 2006), the gene family 185/333 in sea urchin (buckley and smith, 2007), antimicrobial peptide (amp) in shrimps (padhi et al., 2007) and in mussels (padhi and verghese, 2008; pallavicini et al., 2008), the latest presumably generated by gene duplication and positive darwinian selection, argues in favour of the existence of a sharp specificity in some of the invertebrate immune responses. in most studies on invertebrates, and in the present paper as well, what was called “specificity” referred to the overall result of the immune reaction, whereas in mammal immunity studies, “specificity” referred to the process of antigen recognition. concerning memory, various studies concluded that previous experience of a pathogen can provide an individual invertebrate, or its descendant, with enhanced immunity (cooper and roch, 1986; kurtz and franz, 2003; little et al., 2003). this process of enhanced immunity following previous encounter, can be divided in two steps. in the first step (referring to "learning"), the host has met the pathogen and has learnt something from this meeting: this is a behavioural or dynamic feature. in the second step (referring to "memory") the host remembered what he has learnt: this is a physiological or static feature. learning can happen without memory (the lesson was lost) but memory cannot exist without previous learning. in other words, it is evident that memory is inseparable from 103 mailto:philippe.roch@univ-montp2.fr host d. melanogaster susceptible strain: s resistant strain: r virulent strain: v no capsule no capsule avirulent strain: av no capsule capsule p a r a s it o ïd l . b ou la rd i fig. 1 the cellular immune reaction of d. melanogaster against the parasitoïd l. boulardi is called a capsule. it is a multilayer sheath built by hemocytes around the parasitoïd egg which is killed. depending on the combination between susceptible (s) or resistant (r) strains of d. melanogaster and virulent (v) or avirulent (av) strains of l. boulardi, the capsule is built or not. the result of the interaction is predictable [adapted from carton and nappi (2001)]. learning and that "learning-memory" is different from specificity. curiously, this obviousness has been forgotten in some recently published papers (kurtz and franz, 2003). although the specificity of some immune responses in invertebrates has been reported as being much stronger than that generally allocated to innate immunity, there is still no direct evidence for memory in invertebrates. to this end, we suggest quantifying key molecular effectors to discriminate between what is related to transfer of effectors (see below) and what is indeed memory. what was tested when looking for specificity? studies with populations of drosophila melanogaster parasitized by the parasitoïd leptopilina boulardi demonstrated that the immune response was strain specific: some strains of the host fly can kill (encapsulate) some but not all strains of this parasitoïd species (fig. 1) (carton and nappi, 2001). similarly, different daphnia clones have been reported to be differently protected against diverse strains of the same pathogenic bacteria (little et al., 2003); thus, different individuals within the same species seemed to respond differently, and this corresponds to a level of specificity not far from that observed in vertebrate adaptive immunity. specificity is generally understood to be the recognition of a foreign body by the host (kurtz, 2005). for instance in d. melanogaster, both alternative splicing of peptidoglycan recognition protein (pgrp) can play a role in the immune response (werner et al., 2003) and also as many as 18,000 isoforms of the receptor down syndrome cell adhesion molecule (dscam) can be generated (watson et al., 2005). consequently, the observed specificity of d. melanogaster immune responses may be related to the selection of specific isoforms of receptors (agaisse, 2007), as also suggested for molluscs (zhang et al., 2004), or to synergism between receptors (schulenburg et al., 2007). in the d. melanogaster-l. boulardi model described by carton and nappi (2001) (fig. 1), the different steps of the defence reaction can be dissected because this system predicted the outcome of the immune response of selected host strains to particular parasitoïd strains. the immune reaction triggered in d. melanogaster larvae against parasitoïd eggs is called encapsulation and involves four main steps (russo et al., 1996, 2001): (i) an increase in the number of circulating hemocytes, (ii) differentiation of a particular hemocyte type, the lamellocytes, (iii) the activation of the phenoloxidase cascade and (iv) formation of the capsule by accumulation of lamellocytes. the egg must first be recognized as foreign for the reaction to occur and, as stated by several authors (carton et al., 1992; poirie et al., 2000; schmidt et al., 2001), the host-parasitoïd relationships in the d. melanogasterl. boulardi system resembles the "gene-for-gene" recognition model of plants. possibly, the absence of capsule formation results from non recognition of the parasitoïd egg by the host; however, various observations are inconsistent with this possibility. for instance, whether or not the capsule is formed (step iv), the first two steps, involving modifications of blood constituent, are in all cases completed (russo et al., 2001). in addition, when the same larvae of resistant d. melanogaster strain are parasitized both by a virulent l. boulardi strain (no capsule formation) and by an avirulent strain (capsule formation), both types of egg were protected from encapsulation (this reaction was termed "cross protection") (labrosse et al., 2003). if 104 only the avirulent strain were recognised, capsule formation would have been triggered; however, there was no capsule formation. finally, active inhibition of the drosophila defence response by the parasitoïd has been described (labrosse et al., 2003). all these data clearly demonstrated that in the four possible combinations of host-parasitoïd strains (fig. 1), the immune response was always triggered, but then actively inhibited by the parasitoïd in three of the combinations. whatever the combination, i.e. whatever the reaction outcome, parasitoïd eggs were always recognized as foreign bodies. therefore, the specificity of the reaction does not depend only on the recognition process, but also, and in this case mainly, on a putative depressive effect of the parasitoïd. note that in numerous studies performed on invertebrates, with the exception of some works in drosophila (lemaitre and hoffmann, 2007) and in molluscs (zhang et al., 2004), specificity refers to the overall result of the response and not only to the process that triggered the reaction. for instance, carton and nappi (2001) looked for the formation of a capsule around the parasitoïd egg, little et al. (2003) analyzed the fitness of daphnia after exposure to pathogen, kurtz and franz (2003) counted the percentages of copepods infected, moret and siva-jothy (2003) assessed the survival and global antibacterial activity of tenebrio, pham et al. (2007) tested for the protection of drosophila against pathogen; in all these studies what was called "immune specificity" was in fact the product of both (i) recognition (reviewed in du pasquier, 2005) and (ii) complex interactions between the host and the pathogen/parasite. putative supports of specificity there are clearly multiple targets for inhibiting factors in the d. melanogaster-l. boulardi system. once the potential parasite/pathogen has been recognized, signal pathways are triggered. these signal pathways involve various enzymes, especially kinases, phosphatases, hydrolases and gtpases. each one of these molecules is a potential target for immunosuppressive factors. for example, the entomopathogenic bacterium photorhabdus luminescens can prevent its phagocytosis by insect macrophages by inhibiting the activity of the rho and rac gtpases in these cells (brugirard-ricaud et al., 2005). indeed, there are tens or even hundreds of possible targets that could be exploited to abort an elicited defence reaction. each of the inhibitor/target complexes could display a very high specificity, as is characteristic of enzyme-substrate or enzymeinhibitor relationships. in addition, the putative inhibitor from l. boulardi and its putative target in d. melanogaster may be subject to mutations, which could affect the binding affinity. also, alternative splicing or other genetic diversification mechanisms (schulenburg et al., 2007) could greatly increase the efficiency of the combinations. specificity could also be improved by the combination of different inhibitors with different targets in the same hostparasite model. therefore, active inhibition could result in an overall immune response with stronger specificity than that expected from the recognition mechanisms alone. as a consequence, the dogma of the weak specificity of innate immunity led to misinterpretation of those invertebrate immune responses that display strong specificity. is there a need for immune memory in invertebrates? the first line of evidence involved graft transplantation assays and concluded that there was recognition of foreign antigens in annelids (cooper, 1969; valembois, 1963). it was suggested that so-called “anti-graft immunity” was mediated by particular leukocytes which are stimulated during rejection (cooper and roch, 1984; valembois and roch, 1977). anti-graft immunity can be transferred to naïve earthworms by transferring stem cells from a grafted individual, suggesting the existence of memory (roch, 1973). the earthworm’s anti-graft immunity was described as having three characteristics: accelerated rejection, weak specificity and short-term memory (less than 10 days). the same grafting technology applied to scleratinian coral (hildemann et al., 1977), the sea urchin lytechinus pictus (coffaro and hinegardner, 1977), and the marine sponge callyspongia diffusa (bigger et al., 1982) lead to similar conclusions. curiously, and despite repeated efforts in insects, a specific short-term memory has only been found in the american cockroach, periplaneta americana (karp and rheins, 1980; rheins et al., 1980; hartman and karp, 1989). in our example of the d. melanogaster-l. boulardi system (see above), the capsule formation process did not show any memory as the specific interaction is dependent on genetic and not phenotypic adaptation. however, there are an increasing number of reports of the existence of memory in invertebrate immune responses. even if the term memory was used by the authors, its short-term duration was destroying their efforts to demonstrate the existence of a true immune memory. immune memory is advantageous only if there is a chance of being exposed to a previously encountered pathogen; there is therefore a direct relationship with the lifespan of the species. most invertebrates will have died before a secondary exposure is likely, and long-lived invertebrates, such as the cephalopod mollusc nautilus, the horseshoe crab limulus polyphemus, and some insect species (up to 17 years for a north american cicada), seem more likely than for instance does a rotifer as candidates for immunological learning-memory. indeed, little and kraaijeveld (2004) suggested that the lifespan relative to the delay between exposures is probably important. even in short-lived invertebrates, several cycles of infections may occur according to the life cycle of the pathogen. additionally, the same individual may be repeatedly or persistently exposed to similar types of pathogen in their natural environment. consequently, specific immune memory might exist in short-lived as well as in long-lived invertebrates. 105 time intensity 1-m 1-o mothers offspring time intensity 1-m 1-o mothers offspring a b time intensity 1 2 time intensity 1 2 c d fig. 2 how to test the hypothesis of learning through transmission of memory from mothers to offspring (a-b), or within the same individual subjected to two different exposures to the same pathogen (c-d). evolutions of the intensity of the immune reaction according to time and reproduction (vertical bar) following a first exposure of the mothers to the pathogen (1-m), and a first exposure of offspring issued from the challenged mothers to the same pathogen (1-o). a: hypothesis of transmission of immunity by transfer of effectors leading to an increased response, i.e. no learning. b: hypothesis of learning after exposure of the mothers and transmission of the memory to offspring leading to an accelerated immune response. evolutions of the intensity of the immune reaction according to time in the same individual following a first exposure to the pathogen (1), and a second exposure to the same pathogen (2). c: hypothesis of the persistence of high concentration of immune effectors leading to an increased response after the second exposure, i.e. no learning. d: after the first challenge, the immune response return to baseline and the accelerated immune response observed after the second exposure is due to learning-memory resulting from the first exposure. the importance of the concept of immune learning the existence of long-lasting cells, known to be the support of memory in vertebrates, has never been fully demonstrated in invertebrates. however, in addition to graft rejection assays, there are several examples of a second response being modified by a previous encounter, suggesting the existence of memory derived from learning. in particular, experiments in shrimp, (huang and song, 1999) and daphnia (little et al., 2003) provide strong arguments in favour of immune learning and memory: in these studies, larvae were specifically protected against one pathogen after "vaccination" of their mothers. to explain their results, the authors suggested that mothers impregnated their eggs with immune peptides. it is also possible that there was a kind of learning and memory that allowed better protection. this is exactly what differentiates between innate and adaptive immunity and has led some authors to speculate on adaptive aspects of immune responses in invertebrates (flajnik and du pasquier, 2004; agaisse, 2007; kurtz, 2005; kvell et al., 2007; pham et al., 2007). invertebrates have no antibodies, or t and b cells, so there must be a 106 completely different system that supports learning and memory. before dissecting the molecular basis of such a putative system, we must be sure that the shrimp or daphnia (or other invertebrates) are indeed capable of immune learning. to achieve this goal, we suggested continuously measuring protection between two exposures to the same pathogen; surprisingly, reports of such studies are rare, if any. if the protection (expressed as the level of one particular effector, for instance) remained almost the same between the two exposures, the hypothesis of a transmission of immunity from mothers to eggs (by transfer of the effector) remains valid (fig. 2a). however, in contrast to the persistence of specific b cells in vertebrates, this cannot be called memory but only transfer of molecular effectors, as observed for antibodies between mothers and offspring through the placenta in mammals, for instance. in contrast, if there was a decrease in protection between mothers and offspring, followed by a large increase after exposure of the offspring (fig. 2b), this would argue for existence of immune learning and transmission of memory from mothers to offspring. however, this phenomenon has not been found in the adaptive immunity of vertebrates. the situation is somewhat different in the case of multiple exposures of one individual to different strains of a pathogen species. exposure of the copepod macrocyclops albidus to its tapeworm parasite, schistocephalus solidus, reduces the chances of re-infection of the same host by siblings of the infecting tapeworm but not by unrelated parasites (kurtz and franz, 2003); this clearly evidences immune specificity at the level of the strain, as observed in the d. melanogaster-l. boulardi system or in daphnia. but can we call this phenomenon memory? two different models may apply to these observations. if the protection (expressed as previously as the level of one particular effector) remained almost the same between the two exposures, the persistence of the molecular effector in itself could account for the improved protection at the second exposure (fig. 2c). however, if protection decreased after the first exposure, then was substantially accelerated on the second exposure, it would be good evidence for learning and memory induced by the first exposure (fig. 2d). to confirm that true memory is established, referring to a kind of “vaccination”, it would be relatively simple to test whether the level of protection just before the second challenge was returning to baseline. to do that, several prerequisites included at least established optimum breeding conditions and minimum knowledge of immune capacities of the species under investigation. existence of memory remains to be demonstrated when considering the global immune response generally (and not only the recognition process), it is evident that specificity is very accurate, at least in some invertebrates (carton et al., 1992; kurtz and franz, 2003; little et al., 2003; pham et al., 2007). the specificity of some innate immune responses is very probably also present in vertebrates where it might have been obscured by the strength of adaptive immunity. it seems likely, given the extreme diversity of invertebrate (and of vertebrate) species, that there is not one single universal system underlying specificity. we have shown that the inhibiting effect triggered by the parasitoïd could explain the strong specificity of d. melanogaster-l. boulardi relationships. the recently discovered extended genomic diversity of immune effectors (imler and bulet, 2005; padhi et al., 2007), and receptor pgrps (royet et al., 2005), dscam (watson et al., 2005) and tlrs (takeda et al., 2003), is still not understood but may also account for the specificity of innate immune responses. finally, the existence of a true memory remains to be clearly demonstrated in invertebrates. we agree with hauton and smith (2007) that before there can be a "theory", facts about invertebrate immune responses must be established for a wide range of taxa. but unlike these authors, we recommend establishing the existence of memory in an invertebrate species before looking for its biochemical and molecular supports, also suggested by little et al. (2005). we proposed a change in the paradigm of a weak specificity for innate immunity and, considering the overall response, to allow for the possibility of sharp specificity to some of the innate immune responses, both in invertebrates and in vertebrates. meanwhile, on the basis of currently available facts and referring to the learning-memory concept, the dogma that adaptive immunity is absent from invertebrates must be maintained. acknowledgements the authors are grateful to the anonymous reviewers who helped in improving the paper. references adema cm, hertel la, miller rd, loker es. a family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection. proc. natl. acad. sci. usa 94: 8691-8696, 1997. agaisse h. an adaptive immune response in drosophila? cell host microbe 1: 91-93, 2007. bigger ch, jokiel pl, hildemann wh, johnston is. characterization of alloimmune memory in a sponge. j. immunol. 129: 1570-1572, 1982. brugirard-ricaud k, duchaud e, givaudan a, girard pa, kunst f, boemare n, et al. sitespecific antiphagocytic function of the photorhabdus luminescens type iii secretion system during insect colonization. cell. microbiol. 7: 363-371, 2005. buckley km, smith lc. extraordinary diversity among members of the large gene family, 185/333, from the purple sea urchin, strongylocentrotus purpuratus. bmc mol. biol. 8: 68, 2007. carton y, frey f, nappi a. genetic determinism of the cellular immune reaction in drosophila melanogaster. heredity 69: 393-299, 1992. carton y, nappi aj. immunogenetic aspects of the cellular immune response of drosophila 107 against parasitoids. immunogenetics 52: 157164, 2001. coffaro ka, hinegardner rt. immune response in the sea urchin lytechinus pictus. science 197: 1389-1390, 1977. cooper el. specific tissue graft rejection in earthworms. science 166: 1414-1415, 1969. cooper el, roch p. earthworm leukocyte interactions during early stages of graft rejection. j. exp. zool. 232: 67-72, 1984. cooper el, roch p. second-set allograft responses in the earthworm lumbricus terrestris. kinetics and characteristics. transplantation 41: 514-520, 1986. du pasquier l. immunology. insects diversify one molecule to serve two systems. science 309: 1826-1827, 2005. flajnik mf, du pasquier l. evolution of innate and adaptive immunity: can we draw a line? trends immunol. 25: 640-644, 2004. hartman rs, karp rd. short-term immunologic memory in the allograft response of the american cockroach, periplaneta americana. transplantation 47, 920-922, 1989. hauton c, smith vj. adaptive immunity in invertebrates: a straw house without mechanistic foundation. bioessays 29: 11381146, 2007. hibino t, loza-coll m, messier c, majeske aj, cohen ah, terwilliger dp, et al. the immune gene repertoire encoded in the purple sea urchin genome. dev. biol. 300: 349-365, 2006. hildemann wh, raison rl, cheung g, hull cj, akaka l, okamoto j. immunological specificity and memory in a scleractinian coral. nature 270: 219-223, 1977. huang cc, song yl. maternal transmission of immunity to white spot syndrome associated virus (wssv) in shrimp (penaeus monodon). dev. comp. immunol. 23: 545-552, 1999. imler jl, bulet p. antimicrobial peptides in drosophila: structures, activities and gene regulation. chem. immunol. allergy 86: 1-21, 2005. karp rd, rheins la. induction of specific humoral immunity to soluble proteins in the american cockroach (periplaneta americana). ii. nature of the secondary response. dev. comp. immunol. 4: 629-639, 1980. kurtz j. specific memory within innate immune systems. trends immunol. 26: 186-192, 2005. kurtz j, franz k. innate defence: evidence for memory in invertebrate immunity. nature 425: 37-38, 2003. kvell k, cooper el, engelmann p, bovari j, nemeth p. blurring borders: innate immunity with adaptive features. clin. dev. immunol. 2007: 83671, 2007. labrosse c, carton y, dubuffet a, drezen jm, poirie m. active suppression of d. melanogaster immune response by long gland products of the parasitic wasp leptopilina boulardi. j. insect physiol. 49: 513-522, 2003. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. little tj, hultmark d, read af. invertebrate immunity and the limits of mechanistic immunology. nat. immunol. 6: 651-654, 2005. little tj, kraaijeveld ar. ecological and evolutionary implications of immunological priming in invertebrates. trends ecol. evol. 19: 58-60, 2004 little tj, o'connor b, colegrave n, watt k, read af. maternal transfer of strain-specific immunity in an invertebrate. curr. biol. 13: 489-492, 2003. moret y, siva-jothy mt. adaptive innate immunity? responsive-mode prophylaxis in the mealworm beetle tenebrio molitor. proc. biol. sci. 270: 2475-2480, 2003. padhi a, verghese b. molecular diversity and evolution of myticin-c antimicrobial peptide variants in the mediterranean mussel, mytilus galloprovincialis. peptides 29: 1094-1101, 2008. padhi a, verghese b, otta sk, varghese b, ramu k. adaptive evolution after duplication of penaeidin antimicrobial peptides. fish shellfish immunol. 23: 553-566, 2007. pallavicini a, del mar costa m, gestal c, dreos r, figueras a, venier p, et al. high sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host-pathogen interactions. dev. comp. immunol. 32: 213-226, 2008. pham ln, dionne ms, shirasu-hiza m, schneider ds. a specific primed immune response in drosophila is dependent on phagocytes. plos pathog 3, e26, 2007. poirie m, frey f, hita m, huguet e, lemeunier f, periquet g, et al. drosophila resistance genes to parasitoids: chromosomal location and linkage analysis. proc. biol. sci. 267: 14171421, 2000. rheins la, karp rd, butz a. induction of specific humoral immunity to soluble proteins in the american cockroach (periplaneta americana). i. nature of the primary response. dev. comp. immunol. 4: 447-458, 1980. roch p. contribution à l'étude du transfert de l'immunité antigreffe chez le lombricien eisenia foetida sav. c. r. acad. sci. paris, série d 276: 1369-1372, 1973. royet j, reichhart jm, hoffmann ja. sensing and signaling during infection in drosophila. curr. opin. immunol. 17: 11-17, 2005. russo j, brehelin m, carton y. haemocyte changes in resistant and susceptible strains of d. melanogaster caused by virulent and avirulent strains of the parasitic wasp leptopilina boulardi. j insect physiol 47: 167-172, 2001. russo j, dupas s, frey f, carton y, brehelin m. insect immunity: early events in the encapsulation process of parasitoid (leptopilina boulardi) eggs in resistant and susceptible strains of drosophila. parasitology 112: 135142, 1996. schmidt o, theopold u, strand m. innate immunity and its evasion and suppression by hymenopteran endoparasitoids. bioessays 23: 344-351, 2001. 108 schulenburg h, boehnisch c, michiels nk. how do invertebrates generate a highly specific innate immune response? mol. immunol. 44: 3338-3344, 2007. takeda k, kaisho t, akira s. toll-like receptors. annu. rev. immunol. 21: 335-376, 2003. tauszig s, jouanguy e, hoffmann ja, imler jl. tollrelated receptors and the control of antimicrobial peptide expression in drosophila. proc. natl. acad. sci. usa 97: 10520-10525, 2000. valembois p. recherches sur la nature de la réaction antigreffe chez le lombricien eisenia foetida savigny. c. r. acad. sci. paris, série d 257: 3489-3490, 1963. valembois p, roch p. identification par autoradiographie des leucocytes stimulés à la suite de plaies ou de greffes chez un ver de terre. biologie cellulaire 28: 81-82, 1977. watson fl, puttmann-holgado r, thomas f, lamar dl, hughes m, kondo m, et al. (2005) extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 1874-1878, 2005. werner t, borge-renberg k, mellroth p, steiner h, hultmark d. functional diversity of the drosophila pgrp-lc gene cluster in the response to lipopolysaccharide and peptidoglycan. j. biol. chem. 278: 26319-26322, 2003. zhang sm, adema cm, kepler tb, loker es. diversification of ig superfamily genes in an invertebrate. science 305: 251-254, 2004. 109 cell death and the immune response of the sipunculid worm themiste petricola isj 7: 239-250, 2010 issn 1824-307x review cell death and the immune responses of the sipunculan worm themiste petricola ga blanco department of immunology, idehu-national research council (conicet), school of pharmacy and biochemistry, university of buenos aires (uba), buenos aires, argentina accepted october 25, 2010 abstract we have recently studied the role of cell death in the immune system of the sipunculan worm themiste petricola. typical biochemical and morphological changes of apoptosis were induced in celomocytes of these marine worms after in vitro exposure of cells to hydrogen peroxide. apoptosis was time and dose dependent, and required several hours to become apparent. surprisingly, in unexposed samples a subtype of granulocyte was observed to undergo homotypic aggregation, extensive cytoskeletal changes, and degranulation followed by cell death. this spontaneous response ending in cell death occurred in a divalent cation-dependent manner, served to entrap foreign particles, and was blocked by edta-containing saline solutions. even though the mode of granulocyte cell death shares some features with apoptosis, it appears to be a different form of programmed cell death since it occurs within minutes and does not produce single cell-derived apoptotic bodies but transforms itself into one or several syncytial masses with haemostatic and immune purposes. since numerous granulocyte types and multicellular masses involved in cellular immunity have been described in sipunculan worms, the review also discusses the potential influence of activation of granulocytes by sea water in expanding the variety of morphological types and multicellular structures identified through morphological studies among sipunculan species. key words: sipuncula; immune responses; hemostasis; cell death; celomocytes; apoptosis; coagulation introduction recent studies on celomocyte death in the sipunculan worm themiste petricola have introduced some new perspectives of cellular immune responses of these worms. the finding that a specific cell type of celomic granulocytes demonstrates rapid cell death as part of a well orchestrated response with hemostatic and immune ___________________________________________________________________________ corresponding author: guillermo a blanco catedra de inmunologia idehu uba conicet junin 956 4to piso, capital federal (1113), argentina e-mail: gblanco@ffyb.uba.ar list of abbreviations: lha, large hyaline amebocyte; lgl, large granular leukocyte; sgl, small granular leukocyte; pgrp peptidoglycan recognition protein; pgrp-s, pgrpsmall; fsc, forward light scatter; ssc, side light scatter; ps, phosphatidylserine; tunel, terminaldeoxynucleotidyl-transferase-mediated-dutp-nickend-labeling; dapi, 4',6-diamidino-2-phenylindole; jc-1, 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolylcarbocyanine iodide purposes (blanco, 2007; blanco et al., 2008; cavaliere et al., 2010) has implications to the interpretation of celomic cell morphological categories and cellular immune responses described hitherto in sipunculans. the first part of this review will present a brief summary of sipunculan celomic cells and cellular immune responses, as commonly described in previous studies conducted in different species of the phylum. then the main findings on a peculiar mode of rapid cell death of celomic granulocytes in t. petricola will be presented, and its implications to the immune system and hemostasis of sipunculans will be discussed. sipunculan body plan and the absence of a true circulatory system sipuncula is a phylum of unsegmented vermiform celomates with a plump trunk and a slender introvert that ends in a mouth encircled by tentacular outgrowths (stephen and edmonds, 1972; gibbs and cutler, 1987; rice, 1993). the body wall enclosing the celom cavity consists of several layers that include cuticle, epidermis, dermis, circular muscle layer, longitudinal muscle 239 layer, and peritoneum (fig. 1). the dermis varies from quite thick to very thin stratum and consists of a mesh of fine fibres containing connective-tissue cells, pigment cells, multinucleate bodies and leukocytes (hyman, 1959). a definite circulatory system is lacking in sipunculans and celomic fluid operates mechanically as a hydroskeleton. however, in some thick-walled species the dermis contains celomic canals or spaces communicating with each other and with the general celom (hyman, 1959).in larger species of the genus siphonosoma canals take the form of celomic diverticula that penetrate into the dermis as blind sacs of irregular shape, branched, and somewhat transversely arranged (hyman, 1959). the canals are lined by peritoneum and contain the same elements as the celomic fluid. thus celomic cells can reach the dermis and they can be found interspersed between a felt of fine fibres and connective-tissue cells (fig. 1). the main muscles of the interior are the retractors of the introvert. the contraction of the retractors causes the invagination of the introvert, which is extruded again by the general contraction of the circular layer of the body wall acting to compress the celomic fluid. this is the main factor causing celomic fluid flow (zuckerkandl, 1950). the tentacles consist of a separate lumen that contains the same elements as the celom, and although there are no openings into the celom the celomocytes seem able to penetrate into the system (maiorova and adrianov, 2005; adrianov et al., 2006). all sipunculans are dioecious and the sex cells are shed into the celom at an immature stage and complete their maturation while floating in the celomic fluid. they are voided by way of the nephridia, which act as gonoducts (hyman, 1959; adrianov et al., 2002). celomic cell types and cellular immune responses in sipunculans hemerythrocytes the celomic cavity contains a variety of dissimilar cell types. unfortunately, nomenclature of cell types is far from being standardized making it difficult to compare studies from different species. hemerythrocytes, the most abundant cells in the celomic fluid, are found in all species and confer the celomic fluid a pinkish tint due to the iron-containing substance hemerythrin (hyman, 1959; valembois and boiledieu, 1980; dybas, 1981a; rice, 1993; matozzo et al., 2001; lunetta, 2004; meyer and lieb, 2010). most authors agree in the morphological description of hemerythrocytes as nucleated biconvex disks, varying in size among different species, and often having one or more acid vacuoles (ochi, 1970; ochi and ohnishi, 1971). it has been suggested that they are of some assistance in ridding the celom of foreign particles since injected dyes are taken into these vacuoles (towle, 1975). other main cell types in the celom are leukocytes, multicellular structures and gametes (ochi, 1970). hyaline leukocytes leukocytes encompass a heterogenous group of cells mostly involved in cellular immune responses. main types of leukocytes, not necessarily all present fig. 1 the body wall of sipunculan worms consists of an external cuticle everywhere underlain by an epidermis with numerous glands (g). in some genera the dermis contains longitudinal canals (lc) communicating with the general celom. the bodywall musculature consists of outer circular (cm) and inner longitudinal layers (lm). a thin diagonal layer (dm) exists between the other two in some sipunculans but is infrequently mentioned (modified from hyman, 1959). in the same species, include hyaline cells with fine or no granules, and granulocytes with coarse granules that may be acidophilic, neutrophilic, or basophilic (marcou and volkonsky, 1933; hyman, 1959; matozzo et al., 2001). the presence of granules, extension of pseudopodia or an ameboid cell shape is often used as a morphological criterion to distinguish leukocytes from the most abundant biconcave disc-shaped hemerythrocytes. amebocytes are often referred to as phagocytic cells, with a hyaline cytoplasm having few or no granules and having one or several large lysosomic vacuoles often similar to that of hemerythrocytes (ochi and ohnishi, 1971). granular leukocytes most authors coincide in the occurrence of granulocytes among celomic leukocytes and their involvement in cellular immune responses (rice, 1993; matozzo et al., 2001; lunetta, 2004). they are often described as having numerous granules masking details of the cytoplasm and the nucleus (dybas, 1981b; matozzo et al., 2001; lunetta, 2004). the few studies that have quantified the relative abundance of granulocytes within celomic cells indicate a range between 5 % and 20 % (towle, 1975; matozzo et al., 2001; lunetta, 2004) more recently lunetta (2004) has introduced a classification that separates two broad categories of granulocytes from sipunculus nudus designated type i and type ii, and has provided differential characteristics between these two categories at the biochemical, morphological and functional level. this classification criterion may be applicable to most species of sipunculans and the categories proposed by lunetta (2004) will be recalled while discussing results obtained in t. petricola. granules 240 fig. 2 phagocytosis of zymosan particles (arrows) by large hyaline amebocyte (lha) stained with giemsa (a), and with acridine orange (b). in panels c and d degradation of particles was inhibited by alkalinizing the lysosomes with ammonium chloride. zymosan is engulfed but not degraded, thus the cytoplasm becomes packed with basophilic zymosan particles (c). note also that when lysosome vesicles are alkalinized acridine orange no longer stains them red-fluorescent (d). a sequence of nuclear apoptotic changes induced in lhas by exposure to hydrogen peroxide during 2 to 6 hours is shown in (e). cells were stained with acridine orange and ethidium bromide; green fluorescence indicates that cell membrane permeability is preserved (apoptotic live), while red fluorescence indicates loss of membrane permeability (apoptotic dead). exposure of phosphatydilserine during apoptosis of lhas and hemerythrocytes detected by annexin v-fitc and propidium iodide is shown in (f). red fluorescence indicates loss of membrane permeability. in panel (g) lhas and hemerythrocytes were stained with the potentiometric dye jc-1 showing normal mitochondrial membrane potential (orange fluorescence), while in (h) mitochondria became depolarized as indicated by shift from orange to green fluorescence. bar = 15 μm (modified from blanco et al., 2005). 241 from both categories of granulocytes have lytic enzymes including peroxidase, acid esterases, alkaline and acid phosphatases, and lipase (lunetta, 2004, 2005). even though type i granulocytes have lytic enzymes they do not contain any foreign material and do not show any phagocytic capability (lunetta, 2005). degranulation of type i granulocytes and production of extracellular acidophilic material has been noted by several authors (lunetta, 2004). in addition type i granulocytes often show thin cytoplasmatic filaments or filopodia, oriented in all directions. a wide range of morphological descriptions that may well be included in the type i category of granulocytes has been introduced by different authors. descriptions include elongated granulocytes, fusiform or curious shaped cells projecting pseudopodia, which often appear to join with one another (towle, 1975). yet dybas (1975) has reported a paired non-phagocytic granulocyte, occurring as a cell within a cell at a frequency of less than 0.5 % of total celomic cells. a recent study in phascolosoma esculenta reported granulocytes with condensed and clumped chromatin, syncytial granulocytes, cell complexes of granulocytes with podocytes, granulocytes forming pseudopodia and devoid of granules at the pseudopodia protrusion, and finally granulocytes having a large nucleus and often found in association with hemerythrocytes (ying et al., 2010). it should be noted that all of these cytological studies have been conducted in live or fixed preparations without concurrent assessment of cell viability prior to fixation. in addition it has been a common practice to use sea water as saline solution to dilute celomic fluid during cytological studies, either for supravital studies or prior to fixation of cells in suspensions. as it will be discussed further, non-phagocytic granulocytes may be activated in contact with sea water or saline solutions containing ca++ and show rapid cytoskeletal changes before dying within minutes. in contrast, type ii granulocytes of the classification proposed by lunetta (2004), are motile cells, actively phagocytic with lytic enzymes involved in intracellular digestion, and contain engulfed particulate material and lipids. both categories of granulocytes are reported to accumulate in the vicinity of foreign particles trapped within the celom together with masses of acidophilic material (lunetta, 2004). as will be discussed further type ii granulocytes of the category proposed by lunetta (2004) correspond to cells that do not lose viability as part of the cellular defence reactions but remain motile and actively phagocytic engulfing remnants of dying type i granulocytes and foreign particles as well. multicellular bodies various types of multicellular bodies are reported to occur in the celomic fluid of different species of sipunculans (hyman, 1959; rice, 1993). the main criterion to define these bodies has been the morphological identification of cells by light and electron microscopy. several authors have noted that the multicellular bodies include adhered cells and variable degrees of amorphous material that is thought to entrap particles and aid in adhesion. these masses, often of sizes from 30 to 100 μm, have received several names such as cell plates, giant multinucleate corpuscles or more recently brown bodies (lunetta, 2004). experimentally they have been induced by the injection of foreign particles into the celom and it has been considered a main cellular immune response of sipunculans (hyman, 1959; lunetta, 2004). brown bodies and other similar multicellular structures entrapping foreign bodies have been also referred to as capsules (tripplet et al., 1958), although there is not a histological resemblance to capsules occurring in other invertebrates such as insects and crustaceans. more recently lunetta (2004) described the structure of brown bodies induced by mammalian red blood cell injection as made of a core of acidophil material, probably derived from degranulation of type i granulocytes. the function of acidophilic material would be to cement the various parts of the brown body and external layers containing type i and type ii granulocytes (lunetta, 2004). granulocytes in brown bodies were described as misshaped, containing little cytoplasm, and having a large quantity of granulated material (lunetta, 2004). free urns the free urns, described only in some species of sipunculans, are cell complexes that swim freely within the celomic cavity and play an important role in the sipunculan immune defence by releasing sticky, mucoid tails that promptly trap invading pathogens (bang and bang, 1975; nicosia, 1979; bang and bang, 1980; nicosia and sowinski, 1995). urns are composed of two cells, a vesicle cell and a saucer-shaped ciliated cell, fitted together like an acorn into its cap (dybas, 1976). a smaller and variable population of cells, often referred to as thirdtype cells, is frequently found in close juxtaposition to the ciliated cell. lunetta (2004) showed that third type cells are adhering granulocytes that reacted positively with chloroacetate esterase and were enmeshed in acidophilic material. celomocyte cell death in t. petricola apoptosis in celomic cells apoptosis has been a main focus of research during the last 20 years and there is no doubt that it is a crucial process in adaptive and innate immunity of higher animals (kerr et al., 1972; opferman and korsmeyer, 2003; wang et al., 2003). apoptosis occurs during normal t and b cell ontogeny, central tolerance development, and downregulation of t and b cell responses (feig and peter, 2007). cell death in neutrophils during inflammation was classically thought to occur exclusively by passive necrosis due to release of lytic enzymes and damage by microbial products (sendo et al., 1996). however, recent studies have shown that neutrophils undergo apoptosis to ensure complete destruction of intracellular microbes, facilitate uptake by macrophages, and limit proinflammatory stimuli protecting bystander self cells (everett and mcfadden, 1999; fadok and chimini, 2001; fadok et al., 2001). 242 fig. 3 activation, shape changes and cytoplasmic disintegration in large granular leukocytes (lgl).a resting lgl is shown in (a). green fluorescence corresponds to intracellular ca++ level as indicated by ca++ probe fluo-4. early activated lgl emitting filopodia is shown in (b). note the increase in intracellular ca++ levels as indicated by green fluorescence. extensive shape changes observed at later time points after activation are shown in (c). nuclei were stained with dapi in a paraformaldehyde-fixed preparation in (c). a giemsa stained preparation is shown in (d) where cytoplasmic disintegration yields microparticles (mp), and isolated nuclei (n). microparticles are observed to be engulfed in a lha. in live preparation shown in (e) dapi stained only dna present in nuclear remnants and dead cells because the dye is non-permeant to live cells. note that dna-containing remnants were engulfed in lhas and sgls and remained in the cytoplasm, while nuclei were not stained indicating that the phagocytic cells were viable. in panels (f) and (g) a live preparation was stained with acridine orange showing acid granules in an early activated round lgl (right-bottom), and partial loss of granules in a late activated lgl with extensive pseudopodia formation (centre). bar = 15 μm (modified from blanco, 2007; blanco et al., 2008; cavaliere et al., 2010). the role of apoptosis in innate immunity has been also recognized in invertebrate animals where programmed cell destruction can aid in degradation of intracellular pathogens preventing spread of infection beyond the cell boundary (wang et al., 2003; russo and madec, 2007; wang et al., 2008; sokolova, 2009; kiss, 2010). yet some pathogens appear to have the ability to modulate the induction of apoptosis to their own advantage (sunila and labanca, 2003; wang et al., 2003; terahara and 243 takahashi, 2008). other studies have focused on cell death induced by ecotoxicants, since exposure to these substances may pose a threat to wildlife invertebrates by their ability to induce apoptosis in several cell types including immune cells (sokolova et al., 2004; cima et al., 2008; matozzo et al., 2008). all these findings have fostered the interest in characterizing apoptosis mechanism of immune cells in several invertebrate groups (podrabsky and krumschnabel, 2010). it should be pointed out that apoptosis is no longer a synonym of programmed cell death since other forms, including autophagic cell death and programmed necrosis, have an increasing relevance in immune response, amongst many other fields of study (sperandio et al., 2000; yorimitsu and klionsky, 2005; samara et al., 2008; kroemer et al., 2009). our first study on cell death in sipunculan celomocytes was focused on identifying standard indicators of apoptosis after exposure to hydrogen peroxide and evaluating to what extent hydrogen peroxide-induced cell death was similar to what had been described in other animals (blanco et al., 2005). that study was conducted on cell suspensions from celomic fluid containing hemeryhtrocytes and large hyaline amebocytes (lha) as their main constituents. both cell types have large acid vacuoles and particularly lha are actively phagocytic (blanco et al., 1995), and were shown to migrate in vitro toward gradients of endogenous or exogenous chemoattractants (cabrera et al., 2002). the lha vacuole has an extraordinary capacity to degrade engulfed material. when exposed to zymosan particles lha engulf the particles, route them to the vacuole and degrade it in such an efficient manner that only a scant number of particles can be observed in the cytoplasm (figs 2a, b). when acid degrading enzymes were inhibited by elevating the ph of the vacuole with ammonium chloride the cytoplasm became densely packed with particles since they were phagocytosed but not degraded (figs 2c, d) (blanco et al., 2005). exposure of this suspension of hemerythrocytes and lhas to hydrogen peroxide induced morphological and biochemical changes that are known to be present in apoptotic cells (blanco et al., 2005; galluzzi et al., 2009). changes at the nuclear level included chromatin condensation and fragmentation, nuclear membrane ripples, and dna degradation to oligonucleosome-sizes (fig. 2e) (blanco et al., 2005). extranuclear changes consisted of loss of mitochondrial membrane potential, cell volume decrease, exposure of phosphatidylserine (ps) on the outer membrane leaflet, and cell membrane blebbing (figs 2f-h) (blanco et al., 2005). activation and cell death in a subtype of granulocytes the celomic cell suspension used in our first study was particularly depleted of granular cells. the cell suspension was obtained by harvesting, washing, and incubating cells in saline solutions or culture media containing ca++ (blanco et al., 2005). when celomic fluid was stained with acridine orange and propidium iodide immediately after harvesting without washing and observed by fluorescence microscopy, we noted that a celomic cell type having acid granules was present in the samples (blanco, 2007). these cells, that we designated large granular leukocyte (lgl), were about 8 % of celomic cells, showed extensive morphological changes within minutes emitting filopodia and pseudopodia upon contact with glass surface (figs 3a-c), aggregated to each other, loss the acid granules and became dead as indicated by red fluorescence of the nuclei stained with propidium iodide (figs 3f, 3g, 4g-j). these granulocytes are similar in description to type i granulocyte category proposed by lunetta (2004). when celomic fluid was harvested in edta-containing solutions, lgls did not show morphological changes, preserved their round shape densely packed with granules and remained viable (blanco, 2007; blanco et al., 2008). moreover, when sea water was added to celomic fluid harvested in ca++ free saline preparations, lgls started to form aggregates and demonstrated shape changes, being more noticeable the larger the amount of sea water dispensed (blanco, 2007) (figs 4a-d). since all changes were blocked by edta we concluded that sea water and ca++ containing solution caused activation and aggregation of lgls in multicellular masses. a surprising finding was that although cell death of lgls occurred very rapidly, several apoptotic-like features were still observed including chromatin condensation and fragmentation, nuclear membrane ripples, loss of mitochondrial membrane potential and ps exposure (blanco, 2007; blanco et al., 2008). however, other typical apoptotic cytoplasmic changes were absent, including cell volume decrease, membrane blebbing and formation of apoptotic bodies. instead, the cytoplasm of lgls was often seen to disintegrate in small particles unless the cells remain in clumps, in which case the cytoplasmic remnants were kept as integral part of a syncytial mass (cavaliere et al., 2010) (figs 3d, 4ek). a suspected hemostatic role: isolating the cellular clot by the pullout method activation and aggregation of lgls was to some extent reminiscent of platelet aggregation and thrombus formation (george, 2000; heemskerk et al., 2002; harrison, 2005). both platelets and lgls show a series of prominent cytoskeletal changes after activation and continue to form a cell-cell aggregate, with exposure of ps residues in the outer leaflet of the plasma membrane and loss of mitochondrial membrane potential (pereira et al., 1999, 2002; li et al., 2000). the end product is in both cases an insoluble mass. however activation and aggregation systematically followed by death is not an accepted concept in platelet physiology due to its non-nucleated condition (perrotta et al., 2003). programmed cell death in platelets is still a concept that raises several concerns to most authors (wolf et al., 1999; zhang et al., 2007). however our finding in lgls was also reminiscent of cell death and disintegration of coagulocytes as occurs during arthropod coagulation (theopold et al., 2004). coagulocytes have an accessory role in arthropod clot formation 244 fig. 4 celomic fluid harvested in ca++ free saline solution is shown in (a). the effect of exposure to increasing concentrations of sea water (sw) is shown in (b) and (c). multiple lgl aggregates are observed as dark spots. in panel (d) lgl activation by 50 % sw was blocked by 6mm edta. panel (e) shows a large clot entrapping magnetic beads obtained in vitro and (f) shows a magnified image of (e) where beads are observed (arrows). in (g) and (h) a small clot entrapping beads (arrows) in vitro was stained with propidium iodide. in (i) and (j) a small clot entrapping magnetic beads (arrows) was stained with acridine orange. note loss of acid granules indicated by green fluorescence at the clot core and preservation at peripheral areas indicated by orange fluorescence. panel (k) shows small clots formed in vivo after injection of magnetic beads into the celom. no massive clots as shown in (e) were formed in vivo. these small clots entrapping beads coincide with morphological description of brown bodies (multicellular masses demonstrated to entrap particles injected in vivo in other species of sipunculans). bar = 100 μm in (a) through (e) and bar = 30 μm in (f) through (k) (modified from blanco, 2007; blanco et al., 2008). since extracellular strands formed by hemolymph coagulation proteins form the main clot structure (theopold et al., 2002; scherfer et al., 2004; bidla et al., 2005). even though cell-free celomic fluid does not form strands we speculated that sipunculans could yet form a clot mass, to some extent reminiscent of a platelet thrombus, by means of activation, adhesion and rapid death of nucleated cells. scherfer (2004) introduced a method to isolate clots from drosophila that was based on creating a clot over a suspension of magnetic beads and further separating the clot with a magnet (scherfer et al., 2004). using this so called pullout method we could isolate a massive clot ex vivo formed by lgls entrapping beads that could be further studied regarding several aspects of clot formation and structure (figs 4e-f). the hemostatic purpose of this cellular reaction was inferred upon the rapid formation of an insoluble macroscopic mass. however immediate entrapment of particles within the clot was also a strong evidence of being a first line immune response (blanco et al., 2008). 245 our first aim was to demonstrate that the reaction was indeed hemostatic. since a system of celomic canals allows sipunculan celomic cells to reach the dermis (fig. 1), we speculated that a body wall injury would create a sudden contact of celomic cells with sea water, causing lgls to activate, further aggregate, and demonstrate a systematic and rapid death to create a hemostatic plug. to prove this hypothesis we harvested celomic fluid and allow it to flow through a glass tube with an open end in contact with sea water. a massive clot was formed at the open end in contact with sea water which stopped the flow of the liquid column. when observed by light microscopy the clot was formed by aggregated lgls (cavaliere et al., 2010). the immune significance of cellular clotting and lgl death in t. petricola the immune significance of clot formation was made evident not only due to entrapment of magnetic beads but of several dissimilar biotic particles including bacteria, yeast and leukemic cells (cavaliere et al., 2010). by eliciting smaller clots over particle suspensions with controlled amounts of sea water we could observe that clots were formed by aggregating lgls that extended filopodia and adhered to each other creating a mesh where particles became entrapped (figs 4g-j). dna degradation as indicated by supravital propidium iodide staining of small clots was observed to occur quite fast at the core of lgl aggregates, while the peripheral layers tend to remain viable for a longer time (figs 4g, h). staining with lysosomotropic dyes like acridine orange showed that acid granules of lgls were released at the clot core, while most peripheral lgls showed intact granules indicating that there was a gradient of granule content release from the centre to the periphery (figs 4i, j). ps exposure as indicated by annexin v binding followed a similar gradient pattern from the centre to the periphery (blanco et al., 2008). dna degradation within the clot centre was further evidenced by the dna nickend labelling method (tunel) (cavaliere et al., 2010). thus we concluded that almost immediate entrapment of potential pathogens such as bacteria or fungi within the clot was followed by formation of a hostile environment within the clot core consisting of degradative enzymes derived from acid granules, and potentially from other enzymes activated as part of programmed cell death such as caspases, calpains and dnases (pasquet et al., 1996; shcherbina and remold-o'donnell, 1999; wolf et al., 1999). invertebrate cellular responses often utilize pattern-recognition receptors in the hemolymph or on the immune cell surface to identify pathogenassociated molecular patterns (kurata, 2010). members of the peptidoglycan recognition protein (pgrp) family recognize diverse bacteria-derived peptidoglycans and initiate appropriate immune reactions (kurata et al., 2006; goto and kurata, 2006). to further explore the immune significance of lgl clotting as a first line immune response we evaluated the presence of pgrp (blanco et al., 2008). using antibodies raised against different human recombinant pgrp we explored the expression of these pattern recognition proteins as indicated by immunofluorescence detection finding that lgls stained positive to anti pgrp-s (blanco et al., 2008). by harvesting celomic fluid in edta-containing saline solutions we were able to isolate resting lgls and evaluate its light dispersion properties through flow cytometry (blanco et al., 2008). these cells form a cluster of high side light scattering (ssc) due to the high granule content (fig. 5a). a high expression of pgrp-s was observed in resting lgls when stained with anti pgrp-s and evaluated by flow cytometry (blanco et al., 2008), since the pullout method allowed us to isolate clots entrapping magnetic beads we explored the presence of pgrp-s within these clot masses and also in clot supernatants noting that both the clot and the supernatant had high pgrp-s content (blanco et al., 2008). altogether these findings reinforce the role of clot formation as a first line immune cellular response with a role in pathogen-associated molecular pattern recognition. clot formation and the second line cellular immune response clot formation is not a single and isolated response in t. petricola but it is connected to a second line cellular immune response. light microscopic preparations of small clots were suggestive of two cell types involved in phagocytosis of lgl remnants, namely lhas and a second type of granulocyte that we designated small granular leukocyte (sgl) (fig. 3e). these granulocytes remained live as evidenced with viability probes, did not release granule content, and were often seen at the clot edges actively phagocytosing self remnants and particles shed from the clot (cavaliere et al., 2010). cytoskeletal arrangement of sgls was often suggestive of a polarized motile cell with uropodia and lamellipodia. thus these cells were coincident with type ii category of sipunculan granulocytes in the classification proposed by lunetta (2004). through flow cytometry sgls are found in the same cluster of resting lgls (fig. 5a). the cluster disappears when cell suspension is exposed to sea water due to activation, aggregation, and death of lgls, while many sgls may be washout with elicited lgl clots. thus sea water presence in harvested celomic cell suspensions that are further filtered through a small pore mesh, yields suspensions almost depleted of lgls and sgls (fig. 5b). in addition the cluster of non-clotting cells formed mainly by lhas and hemerythrocytes decreases its forward light side scatter properties most probably owing to cell shape changes associated with activation (fig. 5b). clot formation induces shedding of cytoplasmic remnants of a number of disintegrated lgls that are not completely retained at the clot. these cytoplasmic remnants can be stained and observed by flow cytometry (cavaliere et al., 2010). in addition, labelled cytoplasmic remnants were demonstrated to be phagocytosed by sgls, often at the clot neighbourhood (cavaliere et al., 2010). lhas were also detected to phagocytose these remnants although they appear to be loaded in the large lysosomic vacuoles and digested more 246 a b fig. 5 (a) flow cytometry dot plot of front (fsc) vs side (ssc) light scatter of celomic fluid harvested in edtacontaining saline solution. q1 quadrant shows resting lgls while q4 quadrant shows lhas, and hemerythrocytes that together encompass the most abundant cell types. (b) celomic fluid harvested and washed in ca++ or sea water-containing saline solutions becomes depleted of lgls as observed in q1 quadrant, and contains mainly lhas and hemeryhtrocytes observed in q4 quadrant. a decrease in fsc of this cluster is also observed as compared to (a). percentages were determined after running 80,000 celomic cells (modified from blanco et al., 2008). efficiently as occurs with most targets phagocytosed by these cells (figs 3d, e). dna remnants were similarly stained by dna labels and demonstrated to be engulfed by sgls and lhas (fig. 3e). in addition the tunel method demonstrated fragmentation of dna remnants that were engulfed by sgls and lhas (cavaliere et al., 2010). when clots were formed over suspensions of labelled bacteria the fate of these targets was similar to self remnants. bacteria appeared entrapped within the clot and sgls could be seen at the clot edges phagocytosing bacteria. lhas were also found to phagocytose bacteria although in areas not in much proximity to clot edges (cavaliere et al., 2010). clot formation in vivo and the origin of brown bodies since a large clot was formed in vitro by exposing the whole content of celomic fluid to a bead suspension, we expected that beads injected directly within the celom would elicit a similar massive clot in vivo. however injected beads recovered from the whole celomic fluid of an injected worm by the pullout method after 24 h showed several small clots of about 50 to 150 μm that entrapped magnetic beads (blanco, 2007). these small clots resembled brown bodies described in sipunculus nudus (lunetta, 2004) or similar multicellular masses described in other sipunculans (hyman, 1959; rice 1993). granulocytes could be barely recognized at the outer layers, while the core of multicellular masses was formed by an amorphous mass with entrapped beads interspersed (fig. 4k). these findings are in agreement with lunetta (2004) who proposed that formation of brown bodies, which is considered an immune response of sipunculans, would be somehow related to type i granulocytes since they were found in peripheral areas of brown bodies. we can now assert that amorphous material may not be derived from secretion of live cells but from activation, aggregation and active death of lgls as part of a cellular defence reaction. conclusion cell death of lgls is a central process of clot formation in t. petricola which involves both a hemostatic and an immune purpose. cell death is elicited following activation, occurs rapidly, and participates in a sequence of well orchestrated events. when evaluated in lgl aggregates, cell death occurs first at the clot core and later at the periphery. lgl death within the clot core provides a hostile environment to entrapped pathogens while containment at the periphery preserves neighbouring bystander cells and tissues. activation followed by cell death is responsible of several morphological changes in granulocytes. since granulocyte activation, aggregation and death are elicited when sea water is present in harvested celomic cells, failure to recognize this fact in the past may have contributed to the plethora of granulocyte types and multicellular structures described in sipunculan species. however brown bodies formation appears to result from lgl clotting within the celom and is definitely a first line immune cellular response of sipunculans. thus programmed cell death in celomic cells may not only occur through classic apoptosis due to external insults such as oxidative stress or toxic substances, but 247 may have peculiar cell death mechanisms tailored to efficiently and simultaneously achieve a hemostatic and immune purpose. references adrianov av, maiorova as, malakhov vv. microscopic anatomy and ultrastructure of nephridium in the sipunculan thysanocardia nigra ikeda, 1904 from the sea of japan. russian j. mar. biol. 28: 19-29, 2002. adrianov av, malakhov vv, maiorova as. development of the tentacular apparatus in sipunculans (sipuncula): i. thysanocardia nigra (ikeda, 1904) and themiste pyroides (chamberlin, 1920). j. morphol. 267: 569-583, 2006. bang bg, bang fb. cell recognition by mucus secreted by urn cell of sipunculus nudus. nature 253: 634-635, 1975. bang bg, bang fb. the urn cell complex of sipunculus nudus: a model for study of mucusstimulating substances. biol. bull. 159: 571581, 1980. bidla g, lindgren m, theopold u, dushay ms. hemolymph coagulation and phenoloxidase in drosophila larvae. dev. comp. immunol. 29: 669-679, 2005. blanco ga. involvement of apoptosis-like cell death in coelomocytes of themiste petricola (sipuncula) in the formation of a cellular clot with haemostatic and immune functions. in: valentino rg (ed), new cell apoptosis research, nova biomedical books, new york, pp 121-160, 2007. blanco ga, alvarez e, amor a, hajos s. phagocytosis of yeast by coelomocytes of the sipunculan worm themiste petricola: opsonization by plasma components. j. invertebr. pathol. 66: 39-45, 1995. blanco ga, bustamante j, garcia m, hajos se. hydrogen peroxide induces apoptotic-like cell death in coelomocytes of themiste petricola (sipuncula). biol. bull. 209: 168-183, 2005. blanco ga, malchiodi el, de marzi mc. cellular clot formation in a sipunculan worm: entrapment of foreign particles, cell death and identification of a pgrp-related protein. j. invertebr. pathol. 99: 156-165, 2008. cabrera pv, blanco g, ernst g, alvarez e, cooper el, hajos s. coelomocyte locomotion in the sipunculan themiste petricola induced by exogenous and endogenous chemoattractants: role of a cd44-like antigen-ha interaction. j. invertebr. pathol. 79: 111-119, 2002. cavaliere v, papademetrio dl, alvarez em, blanco ga. haemostatic and immune role of cellular clotting in the sipunculan themiste petricola. cell tissue res. 339: 597-611, 2010. cima f, bragadin m, ballarin l. toxic effects of new antifouling compounds on tunicate haemocytes i. sea-nine 211 and chlorothalonil. aquat. toxicol. 86: 299-312, 2008. dybas l. cell within a cell or a circulating cell pair. nature 257: 790-791, 1975. dybas l. a light and electron microscopic study of the ciliated urn of phasolosoma agassizii (sipunculida). cell tissue res. 169: 67-75, 1976. dybas l. sipunculans and echiuroids. in: ratcliffe na, rowley af (eds), invertebrate blood cells, vol. i, academic press, new york, 1981a. dybas l. cellular defense reactions of phascolosoma agassizii, a sipunculan worm: phagocytosis by granulocytes. biol. bull. 161: 104-114, 1981b. everett h, mcfadden g. apoptosis: an innate immune response to virus infection. trends microbiol. 7: 160-165, 1999. fadok va, bratton dl, guthrie l, henson pm. differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases. j. immunol. 166: 6847-6854, 2001. fadok va, chimini g. the phagocytosis of apoptotic cells. semin. immunol. 13: 365-372, 2001. feig c, peter me. how apoptosis got the immune system in shape. eur. j. immunol. 37 (suppl. 1): s61-s70, 2007. galluzzi l, aaronson sa, abrams j, alnemri es, andrews dw, baehrecke eh, et al. guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. cell death differ. 16: 1093-107, 2009. george jn. platelets. lancet 355: 1531-1539, 2000. gibbs pe, cutler eb. a classification of the phylum sipuncula. bull. br. mus. nat. hist. (zool.) 52: 43-58, 1987. goto a, kurata s. the multiple functions of the pgrp family in drosophila immunity. inv. surv. j. 3: 103-110, 2006. harrison p. platelet function analysis. blood rev. 19: 111-123, 2005. heemskerk jw, bevers em, lindhout t. platelet activation and blood coagulation. thromb. haemost. 88: 186-193, 2002. hyman lh. phylum sipunculida. in: the invertebrates, vol. 5, mcgraw-hill book co., new york, pp. 610-696,1959. kerr jf, wyllie ah, currie ar. apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. br. j. cancer. 26: 239-257, 1972. kiss t. apoptosis and its functional significance in molluscs. apoptosis 15: 313-321, 2010. kroemer g, galluzzi l, vandenabeele p, abrams j, alnemri es, baehrecke eh, et al. classification of cell death: recommendations of the nomenclature committee on cell death 2009. cell death differ. 16: 3-11, 2009. kurata s. extracellular and intracellular pathogen recognition by drosophila pgrp-le and pgrp-lc. int. immunol. 22: 143-8, 2010. kurata s, ariki s, kawabata s. recognition of pathogens and activation of immune responses in drosophila and horseshoe crab innate immunity. immunobiology 211: 237-49, 2006. li j, xia y, bertino am, coburn jp, kuter dj. the mechanism of apoptosis in human platelets during storage. transfusion 40: 1320-1329, 2000. 248 lunetta gd. sipunculus nudus: particulate components of the coelomic fluid and its relationship with brown bodies. ital. j. zool. 71: 191-199, 2004. lunetta gd. wound repair in the marine worm sipunculus nudus (sipunculidae). inv. surv. j. 2: 124-131, 2005. maiorova as, adrianov av. ultrastructure of tentacles in the sipunculid worm thysanocardia nigra ikeda, 1904 (sipuncula). russian j. mar. biol. 31: 21-27, 2005. marcou mj, volkonsky m. les lignées leucocytaires des sipunculidés. arch. d´anat. micros. 29: 245-260, 1933. matozzo v, cima f, perin l, ballarin l. phagocytic and enzymatic activities of cells and urn cell complexes in the coelomic fluid of the marine worm sipunculus nudus (sipuncula). ital. j. zool. 68: 273-280, 2001. matozzo v, rova g, ricciardi f, marin mg. immunotoxicity of the xenoestrogen 4nonylphenol to the cockle cerastoderma glaucum. mar. pollut. bull. 57: 453-459, 2008. meyer a, lieb b. respiratory proteins in sipunculus nudus -implications for phylogeny and evolution of the hemerythrin family. comp. biochem. physiol. 155b: 171-177, 2010. nicosia sv. lectin-induced mucus release in the urn cell complex of the marine lnvertebrate sipunculus nudus (linnaeus). science 206: 698-700, 1979. nicosia sv, sowinski jm. cytological analysis of the urn cell complex of sipunculus nudus before and after serum-induced secretion. biol. bull. 188: 267-80, 1995. ochi o. an electron microscopic study on the coelomic cells of some japanese sipuncula. in: proceedings of the international symposium on the biology of sipuncula and echiura. i. rice me, todorovic m (eds), naunco delo press, belgrade, pp 219-227,1970. ochi o, ohnishi s. fine structures of the coelomic cells in the sipunculid phascolosoma scolops i. general observations. mem. ehime univ. sec. ii ser. b 6: 7-20, 1971. opferman jt, korsmeyer sj. apoptosis in the development and maintenance of the immune system. nat. immunol. 4: 410-5, 2003. pasquet jm, dachary-prigent j, nurden at. calcium influx is a determining factor of calpain activation and microparticle formation in platelets. eur. j. biochem. 239: 647-654, 1996. pereira j, palomo i, ocqueteau m, soto m, aranda e, mezzano d. platelet aging in vivo is associated with loss of membrane phospholipid asymmetry. thromb. haemost. 82: 1318-1321, 1999. pereira j, soto m, palomo i, ocqueteau m, coetzee lm, astudillo s, et al. platelet aging in vivo is associated with activation of apoptotic pathways: studies in a model of suppressed thrombopoiesis in dogs. thromb. haemost. 87: 905-909, 2002. perrotta pl, perrotta cl, snyder el. apoptotic activity in stored human platelets. transfusion 43: 526-535, 2003. podrabsky je, krumschnabel g. cell death beyond worms, flies and humans: unusual model systems for cell death research. apoptosis 15: 243-248, 2010. rice m. sipuncula. in: microscopic anatomy of invertebrates, vol.12, chapter 7, onychophora, chilopoda, and lesser protostomata, harrison fw, rice me (eds), wiley-liss, inc., new york, pp 237-325, 1993. russo j, madec l. haemocyte apoptosis as a general cellular immune response of the snail, lymnaea stagnalis, to a toxicant. cell tissue res. 328: 431-441, 2007. samara c, syntichaki p, tavernarakis n. autophagy is required for necrotic cell death in caenorhabditis elegans. cell death differ. 15: 105-112, 2008. scherfer c, karlsson c, loseva o, bidla g, goto a, havemann j, et al. isolation and characterization of hemolymph clotting factors in drosophila melanogaster by a pullout method. curr. biol. 14: 625-629, 2004. sendo f, tsuchida h, takeda y, gon s, takei h, kato t, et al. regulation of neutrophil apoptosis its biological significance in inflammation and the immune response. hum. cell 9: 215-222, 1996. shcherbina a, remold-o'donnell e. role of caspase in a subset of human platelet activation responses. blood 93: 4222-4231, 1999. sokolova im. apoptosis in molluscan immune defense. inv. surv. j. 6: 49-58, 2009. sokolova im, evans s, hughes fm. cadmiuminduced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition. j. exp. biol. 207: 3369-3380, 2004. sperandio s, de belle i, bredesen de. an alternative, nonapoptotic form of programmed cell death. proc. natl. acad. sci. usa 97: 14376-14381, 2000. stephen ac, edmonds sj. the phyla sipuncula and echiura. trustees of the british museum (natural history), london, 1972. sunila i, labanca j. apoptosis in the pathogenesis of infectious diseases of the eastern oyster crassostrea virginica. dis. aquat. organ. 56: 163-170, 2003. terahara k, takahashi kg. mechanisms and immunological roles of apoptosis in molluscs. curr. pharm. des. 14: 131-137, 2008. theopold u, li d, fabbri m, scherfer c, schmidt o. the coagulation of insect hemolymph. cell. mol. life sci. 59: 363-372, 2002. theopold u, schmidt o, soderhall k, dushay ms. coagulation in arthropods: defence, wound closure and healing. trends immunol. 25: 289294, 2004. towle a. cell types in the coelomic fluid of phascolosoma agassizii. in: proceedings of the international symposium on the biology of sipuncula and echiura. i. rice me, todorovic m (eds), naunco delo press, belgrade, pp 211218, 1975. 249 tripplet el, cushing je, durall gl. observations on some immune reactions of the sipunculid worm dendrostomum zosterticolum. am. nat. 92: 287-293, 1958. valembois p, boiledieu d. fine structure and functions of haemerythrocytes and leucocytes of sipunculus nudus. j. morphol. 163: 69-77, 1980. wang l, zhi b, wu w, zhang x. requirement for shrimp caspase in apoptosis against virus infection. dev. comp. immunol. 32: 706-715, 2008. wang x, wu yc, fadok va, lee mc, gengyo-ando k, cheng lc, et al. cell corpse engulfment mediated by c. elegans phosphatidylserine receptor through ced-5 and ced-12. science 302: 1563-1566, 2003. wolf bb, goldstein jc, stennicke hr, beere h, amarante-mendes gp, salvesen gs, et al. calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation. blood 94: 16831692, 1999. ying xp, sun x, wu hx, dahms hu, chullasorn s, zhang yp, et al. the fine structure of coelomocytes in the sipunculid phascolosoma esculenta. micron 41: 71-78, 2010. yorimitsu t, klionsky dj. autophagy: molecular machinery for self-eating. cell death differ. 12 (suppl. 2): 1542-1552, 2005. zhang h, nimmer pm, tahir sk, chen j, fryer rm, hahn kr, et al. bcl-2 family proteins are essential for platelet survival. cell death differ. 14: 943-951, 2007. zuckerkandl e. coelomic pressures in sipunculus nudus. biol. bull. 98: 161-173, 1950. 250 research report isj 4: 95-100, 2007 issn 1824-307x research report vertebrate interleukins originated in invertebrates? s gerber, p cadet, m sheehan, gb stefano, kj mantione neuroscience research institute, state university of new york college at old westbury, old westbury, ny 11568-0210, usa accepted october 30, 2007 abstract previous studies have demonstrated that invertebrate immune and neural tissues contain mammalian-like cytokines, which activate specific cellular functions. therefore, it was of interest to attempt to identify these molecules via applied biosystems human genome survey arrays. the array was used to analyze the transcriptional profiles of mytilus edulis rna samples. the applied biosystems human genome survey array contains 31,700 60-mer oligonucleotides probes representing a set of 27, 868 individual human genes and more than 1,000 control probes. we show interleukin-like and tumor necrosis factor-like genes among other cytokine-like genes significantly expressed in this invertebrate tissue with a signal to noise value greater than 2. in morphine treated tissue additional cytokine genes were expressed. these cytokine-like genes are directly related to previously discovered molecules in invertebrates, suggesting that they first appeared earlier in evolution. key words: mussel; mytilus edulis; cytokines; microarray introduction mytilus edulis neural tissues contain both immuneand neural-like signaling molecules found in mammals (see stefano, 1982, 1990a, 1992). in regard to catecholamines, the neural tissues of mytilus contain both dopamine and norepinephrine (stefano, 1982, 1990b). in reference to cytokine-like molecules, interleukin (il)-1-, il-6and tumor necrosis factor (tnf)-like molecules have been identified via radio-immune assay in mytilus ganglia and immune tissues (hughes et al., 1990, 1991a; hughes and chin, 1994; scharrer et al., 1996). thus, invertebrate ganglia and immune tissues appear to have the potential to respond like mammalian tissues to these signaling molecules. concerning the signaling of these molecules, invertebrate immune tissues, i.e., immunocytes, ___________________________________________________________________________ corresponding author: kirk j mantione neuroscience research institute state university of new york college at old westbury old westbury, ny 11568-0210, usa e-mail: kjmantione@sunynri.org respond to il-1 and il-6 by undergoing conformational changes indicative of becoming activated, i.e., amoeboid, including stimulating mobility (hughes et al., 1990, 1991a,b; hughes and chin, 1994; scharrer et al., 1996). thus, given their presence and action in invertebrate physiological systems it was of great interest to determine if human microarray chips would also show that they are present given the many biochemical and pharmacological similarities. materials and methods mytilus edulis were harvested from the shores of long island sound at mattituck, new york during the month of march. animals were then transported to the laboratory in chilled seawater (4-10 °c). in the laboratory, they were maintained as previously described in detail (stefano et al., 1994). m. edulis pedal ganglia (20 per array) were dissected and kept on ice until needed. in order to determine the presence of 95 table 1 interleukin-like, tumor necrosis factor-like, and other cytokine-like genes that were significantly expressed as analyzed by the human genome survey microarray (applied biosystems) with a signal to noise value greater than 2 in the untreated mytilus edulis pedal ganglia tissue. tumor necrosis factor (tnf)-like molecules present tnfrsf11a tumor necrosis factor receptor superfamily, member 11a, activator of nfkb tnfrsf25; kiaa0720 tumor necrosis factor receptor superfamily, member 25; putative nfkb activating protein interleukins-like molecules present il16 interleukin 16 (lymphocyte chemoattractant factor) il7 interleukin 7 il31ra interleukin 31 receptor a il15 interleukin 15 il11ra interleukin 11 receptor, alpha il18bp interleukin 18 binding protein il23r interleukin-23 receptor additional cytokine-like molecules present chemokine and chemokine receptor activity ccl23 chemokine (c-c motif) ligand 23 ccrl2 chemokine (c-c motif) receptor-like 2 ccl1 chemokine (c-c motif) ligand 1 ccr9 chemokine (c-c motif) receptor 9 cxcr3 chemokine (c-x-c motif) receptor 3 ccl13 chemokine (c-c motif) ligand 13 ccl15; ccl14 chemokine (c-c motif) ligand 15; chemokine (c-c motif) ligand 14 ccl19 chemokine (c-c motif) ligand 19 ccl24 chemokine (c-c motif) ligand 24 mgc12815; ccl3l1; ccl3 chemokine (c-c motif) ligand 3-like, centromeric; chemokine (c-c motif) ligand 3-like 1; chemokine (c-c motif) ligand 3 ccr6 chemokine (c-c motif) receptor 6 cxcl12 chemokine (c-x-c motif) ligand 12 (stromal cell-derived factor 1) cell growth and growth factor activity gdf8 growth differentiation factor 8 gdf2 growth differentiation factor 2 ebaf endometrial bleeding associated factor (left-right determination, factor a; transforming growth factor beta superfamily) bmp1 bone morphogenetic protein 1 myh11 myosin, heavy polypeptide 11, smooth muscle cytokinesis and other cell-cycle activity cdk6 cyclin-dependent kinase 6 cdc23 cdc23 (cell division cycle 23, yeast, homolog) ccnd3 cyclin d3 cdc25a cell division cycle 25a ccnc cyclin c ccnl1 cyclin l1 cdc14a cdc14 cell division cycle 14 homolog a (s. cerevisiae) pard6a par-6 partitioning defective 6 homolog alpha (c.elegans) pard3 par-3 partitioning defective 3 homolog (c. elegans) prc1 protein regulator of cytokinesis 1 stat1 signal transducer and activator of transcription 1, 91kda nedd5 neural precursor cell expressed, developmentally down-regulated 5 3-sep septin 3 dock1 dedicator of cytokinesis 1 immune activity lif leukemia inhibitory factor (cholinergic differentiation factor) osm oncostatin m pf4 platelet factor 4 (chemokine (c-x-c motif) ligand 4) 96 tlr2 toll-like receptor 2 ifnk interferon, kappa other dapk1 death-associated protein kinase 1 lats1 lats, large tumor suppressor, homolog 1 (drosophila) pin1 protein (peptidyl-prolyl cis/trans isomerase) nima-interacting 1 pin1l protein (peptidyl-prolyl cis/trans isomerase) nima-interacting 1-like mobk1b mob1, mps one binder kinase activator-like 1b (yeast) sdfr1 stromal cell derived factor receptor 1 c17 cytokine-like protein c17 epor erythropoietin receptor csf2rb colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) acvr2b activin a receptor, type iib nrp1 neuropilin 1 pbef1 pre-b-cell colony enhancing factor 1 obrgrp; lepr leptin receptor gene-related protein; leptin receptor tlt4 trem-like transcript 4 loc392255 similar to growth differentiation factor 16 aforementioned molecules upon stimulation with a neuroimmune effecter using microarray, ganglia were incubated at 4 °c in filtered seawater or treated with 1 μm morphine for 18 h. applied biosystems expression array analysis applied biosystems human genome survey arrays were used to analyze the transcriptional profiles of rna samples. the applied biosystems human genome survey array contains 31,700 60mer oligonucleotides probes representing a set of 27, 868 individual human genes and more than 1,000 control probes. sequences used for microarray probe design are from curated transcripts from the celera genomics human genome database (www.celeradiscoverysystem.com), refseq transcripts that have been structurally curated from the locuslink (http://ncbi.nlm.nih.bov/locuslink/refseq.html) public database, high-quality cdna sequences from the mammalian gene collection (mgc) (http://mgc.nci.nih.gov) and transcripts that were experimentally validated at applied biosystems. total rna from 20 m. edulis pedal ganglia was isolated with the rneasy mini kit (qiagen, valencia, ca, usa). the tissue was lysed in 600 µl buffer rlt and homogenized by passing the lysate 5 times through a 20-gauge needle fitted to a 3 ml syringe. the samples were then processed following the manufacturer's detailed instructions. in the final step, the rna was eluted with 50 µl of rnase-free water by centrifugation for 1 min at 10,000 rpm. quality of the rna was analyzed using agilent 2100 bioanalyzer (agilent, santa clara, ca, usa) using the total rna nanochip according to manufacturer’s protocol. digoxigenin-utp labeled crna was generated and linearly amplified from 1 µg of total rna using applied biosystems chemiluminescent rt-ivt labeling kit v 2.0 and manufacturer's protocol. array hybridization, chemiluminescence detection, image acquisition and analysis were performed using applied biosystems chemiluminescence detection kit and applied biosystems 1700 chemiluminescent microarray analyzer following manufacturer's protocol. to each chip, 15 µg of labeled crna targets were hybridized at 55 °c for 19 h. ab1700 expression system software was used to extract assay signal, and assay signal to noise ratio values from the microarray images. to select expressed genes, the gene list was further filtered by removing genes with a signal to noise value less than two. results the previously discovered invertebrate cytokine-like molecules include tumor necrosis factor-like molecules as well as il-1-, il-2-, il-4-, il6and il-10-like molecules. table 1 demonstrates interleukin-like and tumor necrosis factor-like genes among other cytokine-like genes that were significantly expressed as analyzed by the human gene survey microarray (applied biosystems) with a signal to noise value greater than 2 in the untreated m. edulis pedal ganglia tissue. with a signal to noise value greater than 2, all genes expressed are thus considered to have a strong significant presence. tumor necrosis-like factors were present in the untreated tissue. additionally, several interleukin-like molecules also were present. in the morphine treated tissue, however, several additional genes were expressed (table 2). among these genes expressed was il-10, an interleukin-like molecule previously demonstrated in 97 http://www.celeradiscoverysystem.com/ http://ncbi.nlm.nih.bov/locuslink/refseq.html http://mgc.nci.nih.gov/ table 2 interleukin-like, tumor necrosis factor-like, and other cytokine-like genes that were significantly expressed in mytilus edulis pedal ganglia after morphine treatment. genes with a signal to noise ratio greater than 2 as analyzed by the human genome survey microarray (applied biosystems) were listed. tumor necrosis factor (tnf)-like molecules present tnfrsf10a tumor necrosis factor receptor superfamily, member 10a tnfsf12tnfsf13; tnfsf12; tnfsf13 tumor necrosis factor (ligand) superfamily, member 12-member 13; tumor necrosis factor (ligand) superfamily, member 12; tumor necrosis factor (ligand) superfamily, member 13 interleukin-like molecules present il17rb interleukin 17 receptor b il10 interleukin 10 il23a interleukin 23, alpha subunit p19 il5 interleukin 5 (colony-stimulating factor, eosinophil) il31ra interleukin 31 receptor a il18 interleukin 18 (interferon-gamma-inducing factor) il15ra interleukin 15 receptor, alpha additional cytokine-like molecules present cytokinesis and other cell-cycle activity cdc25b cell division cycle 25b anapc5 anaphase promoting complex subunit 5 cdk7 cyclin-dependent kinase 7 (mo15 homolog, xenopus laevis, cdk-activating kinase) cdk4 cyclin-dependent kinase 4 ropn1 ropporin, rhophilin associated protein 1 socs2 suppressor of cytokine signaling 2 pnutl1; gp1bb peanut-like 1 (drosophila); glycoprotein ib (platelet), beta polypeptide spag5 sperm associated antigen 5 anapc4 anaphase promoting complex subunit 4 cdc6 cdc6 cell division cycle 6 homolog (s. cerevisiae) ube2c ubiquitin-conjugating enzyme e2c dock3 dedicator of cytokinesis 3 other cntfr ciliary neurotrophic factor receptor asb9 ankyrin repeat and socs box-containing 9 prei3 preimplantation protein 3 stat2 signal transducer and activator of transcription 2, 113kda gab3 grb2-associated binding protein 3 obrgrp; lepr leptin receptor gene-related protein; leptin receptor asb10 ankyrin repeat and socs box-containing 10 crlf3 cytokine receptor-like factor 3 11-sep septin 11 asb1 ankyrin repeat and socs box-containing 1 ikbkb inhibitor of kappa light polypeptide gene enhancer in b-cells, kinase beta 98 table 3 additional interleukin-like genes expressed in presence of morphine as analyzed by the human genome survey microarray (applied biosystems) with a signal to noise ratio between 1 and 2. gene name gene ontology il1f10 interleukin 1 family member 10 (theta) immune response, interleukin-1 receptor antagonist activity, extracellular space il1f5 interleukin 1 family member 5, delta immune response; interleukin-1 receptor antagonist activity; extracellular space il1rl2 interleukin 1 receptor-like 2 interleukin-1, type i, activating receptor activity, transmembrane receptor activity, integral to membrane il1rl1 interleukin 1 receptor-like 1 signal transduction, transmembrane receptor activity; receptor signaling protein activity;interleukin-1 receptor activity il1f9 interleukin 1 family member 9 cell-cell signaling; immune response; response to pest/pathogen/parasite; interleukin-1 receptor antagonist activity; extracellular space il6st interleukin 6 signal transducer (gp 130, oncostatin m receptor) extracellular space; integral to membrane; protein binding; glycogen metabolic process; positive regulation of cell proliferation; regulation of notch signaling pathway; signal transduction il4 interleukin 4 cholesterol metabolism; regulation of isotype switching; cell proliferation; b-cell differentiation; cellular defense response; t-helper 2 type immune response; connective tissue growth factor biosynthesis; chemotaxis, interleukin4 receptor binding; extracellular space invertebrates (stefano et al., 1999) and tnf-like molecules different from those expressed in the untreated tissue. proinflammatory cytokines and tnf play a major role in inflammation response. the immunosuppressive effect of morphine treatment is demonstrated by a significant presence in expression of the anti-inflammatory il-10-like molecule. additionally, the significant presence of tnf receptor-like molecules indicates the down regulation of proinflammatory tnf-like molecules. the additional newly discovered cytokine-like molecules detected by microarray in both the untreated and morphine treated tissue provide researchers with a multitude of possible subjects for future investigation. given the logarithmic analysis supplied by the spotfire for functional genomics program (spotfire, somerville, maine), any positive signal to noise value indicates gene presence is in greater amounts than background noise. furthermore, the gene sequence of the human transcript on the microarray chip is not identical to the gene sequence of the corresponding m. edulis transcript. however, the array hybridization, as well as the washes, used the same stringency as human nucleic acid assays and we were still able to detect approximately 5000 genes. it is thus important to note the presence of any additional neuro-immune significant interleukinlike signal molecules that were detected upon morphine treatment of m. edulis pedal ganglia tissue (table 3). these interleukin-like genes are directly related to previously discovered interleukin-like molecules in invertebrates. discussion as noted earlier, invertebrate ganglia, immunocytes, and microglia contain il-1and il-6like signaling molecules (beck and habicht, 1986; hughes et al, 1990, 1991a; paemen et al, 1992; stefano, 1992; stefano et al, 1992; hughes and chin, 1994; scharrer et al, 1996). based on these findings, one can surmise that an interleukin-like molecule secreted from these invertebrate cells may have the ability to release dopamine from neurons. recently, sawada and colleagues, as well as others, demonstrated that mammalian il-1 and il-2 and -4 have the ability to alter invertebrate neural ion channels in a stereoselective manner, further strengthening the hypothesis that these immunocyte-derived molecules can alter neural activities as well as stimulate them ( sawada et al., 1991; szucs et al., 1992; franchini et al., 1996; rozsa et al., 1997; kletsas et al., 1998). in previous and current research, measures are taken to confirm gene expression including taqman probes (applied biosystems) and molecular methods including western blotting. the ability of microarray to corroborate with and/or confirm an expanse of previous research is demonstrated in this study of cytokine-like molecules found in m. edulis. given the comprehensive nature of a single microarray chip and the accuracy and precision of the data expressed by these chips, this research indicates that the use of microarray could be independently sufficient for determining gene expression. 99 in summary, it appears that immune-neural communication does occur in invertebrate neural tissues. certainly, the opposite has also been shown, i.e., that neuropeptides can alter and direct invertebrate immune actions (see stefano et al., 1996). this research has been able to confirm such previous findings using microarray technology. acknowledgments this work was supported in part by the following grants: nimh 47392, and ncmhd 001429. kj mantione is supported by the new york state empire innovation program. references beck g, habicht gs. isolation and characterization of a primitive interleukin-1-like protein from an invertebrate, asterias forbesi. proc. natl. acad. sci. usa 83: 7429-7433, 1986. franchini a, kletsas d, ottaviani e. immunocytochemical evidence of pdgfand tgf-β-like molecules in invertebrate and vertebrate immunocytes: an evolutionary approach. histochem. j. 28: 599-605, 1996. hughes tk, chin r. interactions of neuropeptides and cytokines. in: scharrer b, smith em, stefano gb (eds), neuropeptides and immunoregulation, springer-verlag, berlin, pp 101-119, 1994. hughes tk, chin r, smith em, leung mk, stefano gb. similarities of signal systems in vertebrates and invertebrates: detection, action, and interactions of immunoreactive monokines in the mussel, mytilus edulis. adv. neuroimmunol. 1: 59-70, 1991a. hughes tk, smith em, cadet p, sinisterra ji, leung mk, shipp ma et al. interaction of immunoactive monokines (il-1 and tnf) in the bivalve mollusc mytilus edulis. proc. natl. acad. sci. usa 87: 4426-4429, 1990. hughes tkj, smith em, barnett ja, charles r, stefano gb. lps stimulated invertebrate hemocytes: a role for immunoreactive tnf and il-1. dev. comp. immunol. 15: 117-122, 1991b. kletsas d, sassi d, franchini a, ottaviani e. pdgf and tgf-β induce cell shape changes in invertebrate immunocytes via specific cell surface receptors. eur. j. cell biol. 75: 362366, 1998. paemen lr, porchet-hennere e, masson m, leung mk, hughes tk, stefano gb. glial localization of interleukin-1a in invertebrate ganglia. cell. mol. neurobiol. 12: 463-472, 1992. rozsa ks, rubakhin ss, szucs a, hughes tk, stefano gb. opposite effects of il-2 and -4 on gaba-induced inward currents in dialyzed lymnaea neurons. gen. pharmacol. 29: 73-77, 1997. sawada m, hara n, maeno t. ionic mechanism of the outward current induced by extracellular ejection of interleukin-1 onto identified neurons of aplysia. brain res. 545: 248-256, 1991. scharrer b, paemen lr, smith em, hughes tk, liu y, pope m, et al. the presence and effects of mammalian signal molecules in immunocytes of the insect leucophaea madarae. cell. tissue res. 283: 93-97, 1996. stefano gb. comparative aspects of opioiddopamine interaction. cell. mol. neurobiol. 2: 167-178, 1982. stefano gb. neurobiology of mytilus edulis. university of manchester press, manchester, 1990a. stefano gb. norepinephrine: presence and interaction with endogenous biogenic amines. in: stefano gb (ed), neurobiology of mytilus edulis, manchester university press, manchester, pp 93-103, 1990b. stefano gb. invertebrate and vertebrate immune and nervous system signal molecule commonalities. cell. mol. neurobiol. 12: 357366, 1992. stefano gb, kahoud j, hughes j. inhibition of microglial egress in excised ganglia by human interleukin 10: implications for its activity in invertebrates. acta biol. hung. 50: 247-256, 1999. stefano gb, paemen lr, hughes tk, jr. autoimmunoregulation: differential modulation of cd10/neutral endopeptidase 24.11 by tumor necrosis factor and neuropeptides. j. neuroimmunol. 41: 9-14, 1992. stefano gb, scharrer b, smith em, hughes tk, magazine hi, bilfinger tv et al. opioid and opiate immunoregulatory processes. crit. rev. immunol. 16: 109-144, 1996. stefano gb, teoh mb, grant a, reid c, teoh h, hughes tk. electric field exposure activates immunocytes: evidence for calcium dependency. electro-magnetobiol. 13: 123-136, 1994. szucs a, stefano gb, hughes tk, rozsa ks. modulation of voltage-activated ion currents on identified neurons of helix pomatia l. by interleukin-1. cell. mol. neurobiol. 12: 429-438, 1992. 100 review isj 5: 12-19, 2008 issn 1824-307x review norepinephrine and octopamine: linking stress and immune function across phyla sa adamo department of psychology, dalhousie university, canada accepted february 8, 2008 abstract in species from three widely divergent phyla (arthropoda, mollusca and chordata) tyrosine derivatives (norepinephrine or octopamine) mediate a response to acute stress. part of this response is a change in immune function that results in a decrease in resistance to pathogens. this decrease in disease resistance appears maladaptive. however, if the connections between norepinephrine/octopamine and immune function were maladaptive, they should have been selected against. none of the four commonly proposed adaptive explanations for acute stress-induced changes in immune function fit the available data for species from all three phyla. however, this result is probably due to the lack of information about acute stress-induced immunosuppression in invertebrates and a lack of ecologically valid studies in vertebrates. understanding why immune function and disease resistance changes during acute stress will require greater comparative study. key words: immunocompetence; immunosuppression; insect; mollusc; vertebrate; adaptive benefits introduction when responding to danger, animals from across the animal kingdom alter their physiology in order to optimize it for the performance of flight-orfight behaviours (wingfield, 2003). this reaction is called the acute stress response. species from at least three diverse phyla (chordata, mollusca and arthropoda) coordinate their acute stress response using chemically similar derivatives of the amino acid tyrosine (ottaviani and franceschi, 1996). vertebrates (cooper et al., 2003) and molluscs (lacoste et al. 2001a) release norepinephrine (ne) during acute stress, while insects release norepinephrine’s chemical cousin, octopamine (o) (orchard et al., 1993). in both vertebrates (charmandari et al., 2005) and invertebrates (roeder, 2005) ne and oa mediate a range of stress related responses. most of these responses prepare the animal for extreme physical exertion (charmandari et al., 2005; roeder, 2005). however, the acute stress response also has complex, but largely immunosuppressive effects in a wide range of animals (adamo and parsons, 2006). the acute stress response can influence immune function ___________________________________________________________________________ corresponding author: shelley a adamo department of psychology dalhousie university halifax, ns, b3h 4j1, canada e-mail: sadamo@dal.ca because immune cells in vertebrates (e.g. webster et al., 2002; madden, 2003), molluscs (lacoste et al., 2001b) and insects (gole et al., 1982; orr et al., 1985) have receptors for ne or oa. the consistent connection between acute stress, ne (vertebrates and molluscs) or oa (insects) and immune function suggests that modulating immune function during acute stress serves an important adaptive function. in this paper, i use a comparative approach to examine four adaptive explanations for the existence of acute stress-induced immunosuppression. two tyrosine derivatives: norepinephrine and octopamine oa and ne are both derived from the amino acid tyrosine, although via different pathways (cooper et al., 2003). oa is synthesized from tyramine, while ne is synthesized from dopamine (fig. 1). oa and ne are identical in structure, except for the number of hydroxyl groups on the benzene ring (fig. 1). molluscs use both ne and oa as a signaling molecule (e.g. ne: sloley et al., 1990, lacoste et al., 2001a, b; oa vehovszky et al., 2005). insects, on the other hand, use oa, but not ne as a signaling molecule (roeder, 1999). vertebrates make extensive use of ne and its metabolite, epinephrine as signaling molecules (cooper et al., 12 mailto:sadamo@dal.ca fig. 1. biosynthetic pathways for norepinephrine and octopamine. adapted from cooper et al. (2003). 2003), but make little, if any, use of oa (roeder, 1999; pflüger and stevenson, 2005; farooqui, 2007). most invertebrate oa receptors have substantial sequence homology with vertebrate adrenergic (e.g. ne) receptors (evans and maqueira, 2005; roeder, 2005). also, oa receptors in invertebrates have similar pharmacological profiles to vertebrate adrenergic receptors (e.g. farooqui, 2007; evans and maqueira, 2005). moreover, pharmcological studies of invertebrate oa receptors demonstrate that they show some affinity for ne (evans and maqueira, 2005). for example, in the aquatic snail lymnaea stagnalis the cloned oa receptor has high affinity for oa, but also exhibits some affinity for ne (gerhardt et al., 1997). similarly, human alpha-adrenergic receptors (subtypes 2a, b, c) have high affinity for ne, but they also show some affinity for oa (gerhardt et al., 1997). the similarity between oa and ne receptors suggests that both had a common origin millions of years ago (pflüger and stevenson, 2005). oa and ne transporters also seem to have had a common origin (caveney et al., 2006). interestingly, molluscs appear to lack both an oa and an ne transporter, even though they contain both compounds. some insects also lack an oa transporter (e.g. drosophila) and must deactivate oa enzymatically (caveney et al., 2006). the chemical similarity between oa and ne, the similarity in the enzymes involved in their synthesis, and the similarities in the sequences of their receptor and transporter molecules support the argument that oa and ne pathways arose from the same ancestral pathway (caveney et al., 2006). both oa and ne play a role in stress adaptation, suggesting that this is an ancient conserved function for these compounds (gerhardt et al., 1997; roeder, 1999). norepinephrine, octopamine and acute stress molluscs (bivalves) react to stressful stimuli by contracting the large muscles that hold the shell closed (moore, 2006). this is the bivalve equivalent of flight-or-fight behaviour. it also results in an increase in ne in the hemolymph (lacoste et al., 2001a). ne is released by chromaffin-like cells in the oyster heart (lacoste et al., 2001a). in vertebrates, the sympathetic nervous system (sns) is activated in response to flight-or-fight situations and releases ne into immune organs (nance and sanders, 2007). all primary and secondary immune organs receive noradrenergic innervation, as do all body surfaces that are potential sites of microbial invasion (e.g. skin, gut or oral mucosa) (nance and sanders, 2007). ne also increases in concentration in the plasma (sachser, 1987; matt et al., 1997). therefore ne can reach the entire vertebrate immune system. in insects, oa is released as a neurohormone during flight-or-fight behaviours (orchard et al., 1997; pflüger and stevenson, 2005; roeder, 2005). oa is released into the periphery by dorsal unpaired medial cells (dum cells) (roeder, 2005). dum cells are considered to be the insect equivalent of the vertebrate sns based on their anatomy and pattern of innervation (evans and maqueira, 2005; roeder, 2005). therefore ne, or its chemical cousin oa, is released by a wide range of animals in response to acute stress. these compounds are widely disseminated allowing ne (charmandari et al., 2005) and oa (orchard et al, 1993; roeder, 2005) 13 to affect the immune system as well as mediating other stress responses. norepinephrine, octopamine and immune function immune cells release ne and it appears to have a paracrine-like function in both molluscs (ottaviani et al., 1993; ottaviani and franceschi, 1996; lacoste et al., 2001b) and vertebrates (flierl et al., 2007). therefore, the role of ne as an immunoregulator may be very ancient (ottaviani and franceschi, 1996). in molluscs, acute stress transiently suppresses immune function and increases susceptibility to bacterial infection (table 1). this increased susceptibility to disease is caused, at least in part, by the release of ne during acute stress. molluscan hemocytes contain receptors for ne (lacoste 2001b), and ne has negative effects on hemocyte function (table 1). injections of ne result in decreased bacterial clearance and increased mortality in oysters challenged with a bacterial pathogen (lacoste et al., 2001c). acute stress results in a transient decline in resistance to bacterial infection in insects as well (crickets, adamo and parsons, 2006). some of this decrease in disease resistance may be mediated by oa. injections of oa prior to a bacterial challenge results in increased mortality (adamo and parson, 2006). however, oa also has immunoenhancing effects (brey, 1994; table 2). it can even increase resistance to infection when the pathogen is coincubated with oa (baines et al., 1992; baines and downer, 1992). as in insects, acute stress in vertebrates results in a mix of immunosuppressive and immunoenhancing effects (dhabhar, 2002; ortega, 2003; gleeson et al., 2004; glaser and kiecoltglaser, 2005; nance and sanders, 2007; ortega et al., 2007). despite this complex mix of positive and negative effects, acute stress increases susceptibility to pathogens (e.g. cao and lawrence, 2002). one bout of intense exercise in mice (e.g. davis et al., 1997) or humans (gleeson et al., 2004) leads to an increased risk of disease and/or mortality in response to a pathogen challenge. ne appears to be causally involved in the increase in disease susceptibility after acute stress (e.g. kohut et al., 1998; cao et al., 2003; emeny et al., 2007). the complexity of the effects of ne on vertebrate immune function has prevented a clear adaptive explanation for these changes (sternberg, 2006). madden (2003), maestroni (2005), and kin and sanders (2006) suggest that these complex effects are a result of ne playing a role in maintaining immune function homeostasis. ne and oa may play a similar role in invertebrates. in molluscs and insects, ne or oa are present in the hemolymph of resting animals. although this could be because it is difficult to take blood from animals without stressing them, it may also indicate that oa and ne are chronically present in the hemolymph. oa has a half-life of 15 min or less in insect hemolymph (goosey and candy, 1982), and, therefore it should not be detectable unless it is constantly being released. a background level of oa or ne in non-stressed animals would be consistent with the hypothesis that these compounds help maintain normal immune function in invertebrates. however, if oa or ne helps maintain immune homeostasis in a variety of animals, why do the levels of oa and ne increase dramatically during acute stress? in other words, how does pushing the ‘immune thermostat’ towards an extreme end benefit animals during acute stress? adaptive function of ne and oa effects on immune function during acute stress in molluscs, insects and vertebrates, the acute stress response results in a brief period during which the animal’s ability to fight off infection is reduced (fig. 2, however see below). ne or oa appear to mediate some of this immunosuppression (fig. 2). this effect of ne or oa on the immune system appears to be maladaptive. increasing susceptibility to disease seems likely to reduce survival and reproductive success, and such a response should be selected against. as dhabhar (2002) has pointed out, during fighting or fleeing, animals run a real risk of injury and, therefore, exposure to pathogens. although it might make good adaptive sense to delay copulation, digestion, and egg laying until the predator has passed, the immune response may not be dispensable during flight-or-fight behaviours because of the increased risk of injury (dhabhar, 2002). nevertheless, the fact that animals from three different phyla exhibit the same apparently maladaptive response suggests that, despite the costs, it provides some benefit. below i review some of the suggestions as to why animals display acute stress-induced immunosuppression. these hypotheses are not mutually exclusive. in particular i explore whether there are any explanations that might fit the evidence from animals across all three phyla. table 1 effects of norepinephrine on molluscan immune functions immune functions effects references susceptibility to bacterial infection ↑ lacoste et al., 2001c phagocytosis at physiological concentrations ↓ lacoste et al., 2002a production of reactive oxygen species induced by interleukin-1 ↓ lacoste et al., 2001d apoptosis ↑ lacoste et al., 2002b 14 i focus on evidence obtained from whole animal studies. immune values taken in vitro are often different when measured in vivo (nance and sanders, 2007). more importantly, as kohut et al. (2005) comments, there is often a lack of association between declines in various immune functions and actual disease resistance (also see adamo, 2004). from an evolutionary perspective, it is the change in disease resistance that is important. 1. the ‘energy crisis’ hypothesis. one common hypothesis for the existence of acute stress-induced immunosuppression is that it allows animals to channel more energy into flight-or-fight behaviour (e.g. see råberg et al., 1998; segerstrom, 2007). however, it is unclear whether immunosuppression would save energy. for example, some mechanisms of immunosuppression, such as apoptosis, require an increase in energy expenditure (dhabhar, 2002). at present there is little direct evidence supporting the ‘energy-crisis’ hypothesis (adamo and parsons, 2006). 2. the ‘resource crunch’ hypothesis. animals make a number of physiological changes in order to make flight-or-fight possible (wingfield, 2003; charmandari et al., 2005). the ‘resource crunch’ hypothesis suggests that some of these changes will result in resources being shifted away from the immune system in order to optimize the flight-orfight response. this hypothesis differs from the ‘energy-crisis’ hypothesis because it is not energy per se that is limiting, but specific molecules that are required for both immunity and some other physiological function. the ‘resource crunch’ hypothesis explains, at least in part, acute stress-induced immunosuppression in insects. in crickets, conflicts between immune function and lipid transport can lead to acute stress-induced immunosuppression (adamo et al., 2008). crickets release oa during flight-or-fight behaviours (adamo et al., 1995). for about an hour after flying or fighting, crickets become more susceptible to bacterial infection (adamo and parsons, 2006). oa, either directly and/or indirectly, induces the mobilization of lipid from the fat body in order to fuel flight-or-fight behaviours (orchard et al., 1993). as lipid levels in the hemolymph increase, the protein apolipophorin iii (apolpiii) changes its confirmation, and combines with high density lipophorin (hdlp) to form low density lipophorin (ldlp) which has an increased lipid carrying capacity (see weers and ryan, 2006, for review). however, in the unlipidated form, apolpiii acts as an immune surveillance molecule (weers and ryan, 2006). once apolpiii becomes part of ldlp, it appears to loose that ability, resulting in a decline in immune surveillance. the decline in immune surveillance probably explains the increase in disease susceptibility after flying and fighting (adamo et al., 2008). therefore, in crickets, intense activity leads to transient immunosuppression because apolpiii is co-opted into lipid transport and becomes unavailable as an immune surveillance molecule. therefore, crickets become immunosuppressed during flight-or-fight even if they have abundant energy stores (adamo et al., 2008). the ability of oa to mobilize lipid explains why oa can produce immunosuppression when injected into crickets. injecting oa results in the release of lipid (woodring et al., 1989), which leads to a decline in the immune surveillance molecule apolpiii as it combines with hdlp to form ldlp (weers and ryan, 2006). but why does oa also have immunoenhancing effects (table 2)? i hypothesize that oa also works to maintain immune system function as some of the components of the immune system are being siphoned off into lipid transport. in other words, oa helps liberate lipid stores (needed to fuel flight-orfight behaviour) while simultaneously reconfiguring the immune system to maintain maximal function under the new physiological conditions. i predict that without the effects of oa on immune function, disease resistance would decline even more precipitously during flying or fighting in crickets. this hypothesis explains why oa can have both immunosuppressive and immunoenhancing effects. why do crickets not make enough apolpiii to support both immune surveillance and increased lipid transport? first, it would be energetically expensive to do so. apolpiii is already a very abundant protein in the hemolymph of many adult insects (weers and ryan, 2006). to produce more of this protein would decrease the energy available for reproduction and other activities. furthermore, as apolpiii concentrations increase, apolpiii may begin to bind to the animal’s own molecules, initiating an inappropriate immune response. such autoimmunity could be costly (e.g. sadd and sivajothy, 2006). therefore, shuttling apolpiii between immune surveillance and lipid transport may be the most adaptive response, even though it results in transient immunosuppression during flying or fighting. table 2 effects of octopamine on insect immune functions immune functions effects references susceptibility to bacterial infection ↑ adamo and parsons, 2006 phagocytosis ↑ baines et al., 1992 nodule formation (insect immune response) ↑ baines et al., 1992 hemocyte locomotion ↑ dielh-jones et al., 1996 hemocyte number at low (physiological) doses ↓ dunphy and downer, 1994 hemocyte number at higher (pharmacological) doses ↑ dunphy and downer, 1994 15 fig. 2. schematic outline of the connections between ne, oa, acute stress and immune function in different phyla. dum cells, dorsal unpaired median cells; ne, norepinephrine; oa, octopamine; sns, sympathetic nervous system. see text for references it is unclear whether a similar scenario can explain acute-stress induced immunosuppression in molluscs. in molluscs, the known effects of ne are all negative (table 1). however, there have been few studies on acute stress-induced immunosuppression in molluscs. more data are needed to assess whether molluscs suffer from a ‘resource crunch’ during acute stress. in vertebrates a number of molecules are shared between the immune system and other physiological systems. for example, lipid metabolism and immune function are also intertwined in vertebrates (e.g. van elzen et al., 2005). mammalian lipoproteins transport lipid (e.g. cholesterol), but they also participate in innate immunity (khovidhunkit et al., 2004). lipid carriers such as very low density lipoprotein (vldl) bind to and neutralize viruses and other pathogen products (khovidhunkit et al., 2004). during infection, vldl levels increase (khovidhunkit et al., 2004). however, after a single bout of intense exercise, the total concentration of vldl particles in the blood declines by 38 % in humans (børsheim et al., 1999). whether changes in mammalian lipoprotein concentrations during intense activity results in acute stress-induced immunosuppression remains unknown. some studies in mammals indirectly support the ‘resource crunch’ hypothesis. for example, despite the immunosuppressive effects of ne, mice that were stressed by minor surgery, and then exposed to infectious agents, were more likely to die from infection if they received ß-adrenoreceptor blockers (schmitz et al., 2007). in another study, mice given ß-adrenoreceptor blockers prior to intense exercise were more likely to die after a viral challenge compared with controls (kohut et al., 2005). these studies suggest that the effects of ne on immune function result in increased disease resistance when they occur within the context of an acute stress response. these results are consistent with the hypothesis that ne works to reconfigure the immune system in order to maintain immune function during a ‘resource crunch’. however, other studies have found that blocking ß1-adrenergic receptors decreased acute stress-induced immunosuppression (cao et al., 2003). emeny et al. (2007) found that mice lacking adrenoreceptors on their immune cells cleared a bacterial infection (listeria monocytogenes) more quickly after acute stress than mice with immune cells capable of responding to ne. however, the relationship between the speed with which an animal can clear bacteria from liver and spleen and its ability to survive an infection was not stated in these studies (cao and lawrence, 2002; cao et al., 2003; emeny et al., 2007). when determining the effects of various drugs on disease resistance, keil et al. (2001) used an ld10 dose of l. monocytogenes and measured the effect on mortality, not on bacterial clearance. in drosophila melanogaster, the ability to clear bacteria from the hemocoel does not correlate with the ability to survive an infection (corby-harris et al., 2007). 3. the ‘over excitation’ hypothesis. acute stress-induced immunosuppression may be beneficial because it prevents the immune system from becoming too active and harming the animal. during intense exercise, tissues such as muscle suffer minor damage, increasing the risk of inflammation and an autoimmune reaction (råberg et al., 1998). therefore, the immune system shifts towards a less inflammatory state, (i.e. a shift from th1 to th2 responses, elenkov and chrousos, 2006). this shift leads to a decrease in inflammation, but also an increased susceptibility to bacterial and viral pathogens. the cost of the increased risk of infection is thought to be less than an autoimmune reaction or damage from an overactive immune response. however, this key assumption remains untested. the ‘over-excitation’ hypothesis does raise the question as to why the prevention of ‘overexcitation’ occurs to the point that animals are left susceptible to bacterial infections during acute stress. it would be more adaptive to prevent autoimmunity and over-inflammation while maintaining normal anti-microbial defenses, unless there is some physiological constraint that makes this impossible. the ‘over-excitation’ hypothesis does fit the available data on molluscs (table 1), but is not supported by the insect data (table 2). in insects, immune cell activity appears to be up-regulated during acute stress (table 2). animals run the risk of having an over-active immune system during both an acute stress response and during an immune challenge. in vertebrates, an immune challenge also activates the acute stress response (elenkov and chrousos, 2006). this indirectly supports the ‘over-excitation’ hypothesis. however, insects release oa during an 16 immune challenge too (dunphy and downer, 1994), although its source is uncertain (adamo, 2005). given that oa appears to increase immune cell activity (table 2), these data do not support the ‘over-excitation’ hypothesis. immunologists should be wary of putting too much confidence in this hypothesis without more supporting data. 4. the ‘shift in focus’ hypothesis. this hypothesis suggests that during flight-or-fight animals are not immunosuppressed per se, but that they have shifted the focus of their immune effort from protecting against systemic invaders, to protecting against opportunistic organisms that might gain entry during wounding (dhabhar, 2002). dhabhar (2002) has shown that acute stress can increase a delayed-type hypersensitivity in rodents and that catecholamines (e.g. ne and epinephrine) play a role in this enhancement. this change could result in increased protection from wound infection (dhabhar, 2002). however, tests with real bacteria have mixed results. restraint stress produced a decrease in wound healing and a decrease in the ability mice to clear bacteria introduced into a wound (rojas et al., 2002). however, the duration of the stress (mice were restrained for 12 h at a time for 8 days) is more typical of a chronic than an acute stress. campisi et al. (2002) found that after a series of tail shocks given over 2 h, acutely stressed rats recovered more quickly than unstressed rats from a subcutaneous injection of a relatively benign bacterium (there was no mortality) (campisi et al., 2002). however, this experiment does not convincingly show that acutely stressed rats are less likely to develop infected wounds. the ‘shift in focus’ hypothesis does not appear to apply to insects. although oa enhances hemolymph clotting in some arthropods (e.g. battelle and kravitz, 1978), in insects, flight-or-fight behaviour results in an increase in infection after wounding (adamo and parsons, 2006). there is no evidence available from the molluscs. conclusions the involvement of ne (or oa) in mediating stress-induced changes in immune function may be ancient (ottaviani and franceschi, 1996). regardless of whether these connections have been conserved over millions of years or have evolved independently in multiple lineages, their existence in animals from different phyla suggests that there is strong selection pressure for a change in immune function in response to acute stress. none of the four suggested adaptive functions reviewed here explains acute stress-induced immunosuppression in all species. in part this is due to a lack of information about acute stress-induced immunosuppression in invertebrates. all of the information presented here rests on a handful of studies. but it is also because the key experiments are missing in vertebrate studies. for example, despite work on the ‘shift in focus’ hypothesis for more than a decade, whether acute stress actually decreases susceptibility to opportunistic wound infections under real world conditions remains unknown. the lack of a real world test of the ‘shift in focus’ hypothesis highlights a general lack of ecological context in these studies. for example, acute stress is typically produced using highly artificial stimuli, such as restraint stress or tail shock. these stressors have an unknown connection to an ecologically relevant stressor (e.g. a predator). nor is the duration or intensity of the artificial stressors correlated with data about what the animal would experience in the field. studies using a more ecological perspective will be critical if we are to understand the adaptive significance of the changes in immune response that occur during acute stress. references adamo sa. how should behavioural ecologists interpret measurments of immunity? anim. behav. 68: 1443-1449, 2004. adamo sa. parasitic suppression of feeding in the tobacco hornworm, manduca sexta: parallels with feeding depression after an immune challenge. arch. insect biochem. physiol. 60: 185-197, 2005. adamo sa, linn ce, hoy rr. the role of neurohormonal octopamine during 'fight or flight' behaviour in the field cricket gryllus bimaculatus. j. exp. biol. 198: 1691-1700, 1995. adamo sa, parsons nm. the emergency life-history stage and immunity in the cricket, gryllus texensis. anim. behav. 72: 235-244, 2006. adamo sa, roberts jl, easy rh, ross nw. competition between immune function and lipid transport for the protein apolipophorin iii leads to stress-induced immunosuppression in crickets. j. exp. biol. 211: 531-538, 2008. baines d, desantis t, downer r. octopamine and 5-hydroxytryptamine enhance the phagocytic and nodule formation activities of cockroach (periplaneta americana) haemocytes. j. insect physiol. 38: 905-914, 1992. baines d, downer rgh. 5-hydroxytryptaminesensitive adenylate cyclase affects phagocytosis in cockroach hemocytes. arch. insect biochem. physiol. 21: 303-316, 1992. battelle ba, kravitz ea. targets of octopamine action in the lobster: cyclic nucleotide changes and physiological effects in hemolymph, heart and exoskeletal muscle. j. pharmacol. exp. ther. 205: 438-448, 1978. børsheim e, knardahl s, høstmark at. short-term effects of exercise on plasma very low density lipoproteins (vldl) and fatty acids. med. sci. sports exer. 31: 522-530, 1999. brey pt. the impact of stress on insect immunity. bull. inst. pasteur 92: 101-118, 1994. campisi j, leem th, fleshner m. acute stress decreases inflammation at the site of infection: a role for nitric oxide. physiol. behav. 77: 291299, 2002. cao l, hudson ca, lawrence da. acute cold/restraint stress inhibits host resistance to listeria monocytogenes via ß1-adrenergic receptors. brain behav. immun. 17: 121-133, 2003. 17 cao l, lawrence da. suppression of host resistance to listeria monocytogenes by acute cold/restraint stress: lack of direct il-6 involvement. j. neuroimmunol. 133: 132-143, 2002. caveney s, cladman w, verellen l, donly c. ancestry of neuronal monoamine transporters in the metazoa. j. exp. biol. 209: 4858-4868, 2006. charmandari e, tsigos c, chrousos g. endocrinology of the stress response. ann. rev. physiol. 67: 259-284, 2005. cooper jr, bloom fe, roth rr. the biochemical basis of neuropharmacology. 8th edition ed. oxford university press, new york, 2003. corby-harris v, habel ke, ali fg, promislow del. alternative measures of response to pseudomonas aeruginosa infection in drosophila melanogaster. j. evol. biol. 20: 526533, 2007. davis jm, kohut ml, colbert lh, jackson da, ghaffer a, mayer ep. exercise, alveolar macrophage function and susceptibility to respiratory function. j. app. physiol. 83: 14611466, 1997. dhabhar f. stress-induced augmentation of immune function-the role of stress hormones, leuckocyte trafficking, and cytokines. brain behav. immun. 16: 785-798, 2002. diehl-jones wl, mandato ca, whent g, downer rgh. monoaminergic regulation of hemocyte activity. j. insect physiol. 42: 13-19, 1996. dunphy gb, downer rgh. octopamine, a modulator of the haemocytic nodulation response of non-immune galleria mellonella. j. insect physiol. 40: 267-272, 1994. elenkov ij, chrousos gp. stress system organization, physiology and immunoregulation. neuroimmunodulation 13: 257-267, 2006. emeny rt, gao d, lawrence da. ß1-adrenergic receptors on immune cells impair innate defences against listeria. j. immunol. 178: 4876-4884, 2007. evans pd, maqueira b. insect octopamine receptors: a new classification scheme based on studies of cloned drosophila g-protein coupled receptors. invert. neurosci. 5: 111-118, 2005. farooqui t. octopamine-mediated neuromodulation of insect senses. neurochem. res. 32: 15111529, 2007. flierl ma, rittirsch d, nadeau ba, chen aj, sarma jv, zetoune fs, et al. phagocyte-derived catecholamines enhance acute inflammatory injury. nature 449: 721-726, 2007. gerhardt cc, bakker ra, piek gj, planta rj, vreugdenhil e, leysen je, et al. molecular cloning and pharmacological characterization of a molluscan octopamine receptor. mol. pharmacol. 51: 293-300, 1997. glaser r, kiecolt-glaser jk. stress-induced immune dysfunction: implications for health. nature rev. immunol. 5: 243-251, 2005. gleeson m, nieman dc, pedersen bk. exercise, nutrition and immune function. j. sports sci. 22: 115-125, 2004. gole jwd, downer rgh, sohi ss. octopaminesensitive adenylate cyclase in haemocytes of the forest tent caterpillar, malacosoma disstria hubner (lepidoptera: lasiocampidae). can. j. zool. 60: 825-829, 1982. goosey mw, candy dj. the release and removal of octopamine by tissues of the locust schistocerca americana gregaria. insect biochem. 12: 681-685, 1982. keil d, luebke rw, pruett sb. quantifying the relationship between multiple immunological parameters and host resistance: probing the limits of reductionism. j. immunol. 167: 45434552, 2001. khovidhunkit w, kim ms, memon ra, shigenaga jk, moser ah, feingold kr, et al. effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host. j. lipid res. 45: 1169-1196, 2004. kin nw, sanders vm. it takes nerve to tell t and b cells what to do. j. leukoc. biol. 79: 10931104, 2006. kohut ml, davis jm, jackson da, colbert lh, strasner a, essig da, et al. the role of stress hormones in exercise-induced suppression of alveolar macrophage antiviral function. j. neuroimmunol. 81: 193-200, 1998. kohut ml, martin ae, senchina ds, lee w. glucorticoids produced during exercise may be necessary for optimal virus-induced il-2 and cell proliferation whereas both catecholamines and glucorticoids may be required for adequate immune defense to viral infection. brain behav. immun. 19: 423-435, 2005. lacoste a, cueff a, poulet sa. p35-sensitive caspases, map kinases and rho modulate beta-adrenergic induction of apoptosis in mollusc immune cells. j. cell sci. 115: 761768, 2002b. lacoste a, jalabert f, malham s, cueff a, poulet s. stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (crassostrea gigas) to vibrio splendidus. appl. environ. microbiol. 67: 23042309, 2001c. lacoste a, malham sk, cueff a, jalabert f, gélébart f, poulet sa. evidence for a form of adrenergic response to stress in the mollusc crassostrea gigas. j. exp. biol. 204: 12471255, 2001a. lacoste a, malham sk, cueff a, poulet sa. noradrenaline modulates hemocyte reactive oxygen species production via beta-adrenergic receptors in the oyster crassostrea giga. dev. comp. immunol. 25: 285-289, 2001b. lacoste a, malham sk, cueff a, poulet sa. noradrenaline reduces the stimulatory effect of interleukin-1 alpha on reactive oxygen species production by oyster immunocytes. invert. biol. 120: 358-364, 2001d. lacoste a, malham sk, gelebart f, cueff a, poulet sa. stress-induced changes in the oyster crassostrea gigas. dev. comp. immunol. 26: 19, 2002a. 18 madden ks. catecholamines, sympathetic innervation. and immunity. brain behav. immun. 17: s5-s10, 2003. maestroni gj. adrenergic modulation of dendritic cells function: relevance for the immune homeostasis. curr. neurovasc. res. 2: 169173, 2005. matt ks, moore mc, knapp r, moore it. sympathetic mediation of stress and aggressive competition: plasma catecholamines in free-living male tree lizards. physiol. behav. 61: 639-647, 1997. moore j. an introduction to the invertebrates. 2nd edition. cambridge university press, cambridge, 2006. munford rs. detoxifying endotoxin: time, place and person. j. endotoxin res. 11: 69-84, 2005. nance dm, sanders vm. autonomic innervation and regulation of the immune system (1987-2007). brain behav. immun. 21: 736-745, 2007. orchard i, ramirez jm, lange ab. a multifunctional role for octopamine in locust flight. ann. rev. entomol. 38: 227-249, 1993. orr gl, gole jwd, downer rgh. characterization of an octopamine-sensitive adenylate cyclase in hemocyte membrane fragments of the american cockroach periplaneta americana l. insect biochem. 15: 695-701, 1985. ortega e. neuroendocrine mediators in the modulation of phagocytosis by exercise: physiological implications. exerc. immunol. rev. 9: 70-93, 2003. ortega e, giraldo e, hinchado md, martin l, garcia ll, de la fuente m. neuroimmunodulation during exercise: role of catecholamines as 'stress mediator' and/or 'danger signal' for the innate immune response. neuroimmunodulation 14: 206-212, 2007. ottaviani e, caselgrandi e, franchini a, franceschi c. crf provokes the release of norepinephrine by hemocytes of viviparus ater (gastropoda, prosobranchia): further evidence in favour of the evolutionary hypothesis of the 'mobile immune brain'. biochem. biophys. res. comm. 193: 446-452, 1993. ottaviani e, franceschi c. the neuroimmunology of stress from invertebrates to man. prog. neurobiol. 48: 421-440, 1996. pflüger hj, stevenson pa. evolutionary aspects of octopaminergic systems with emphasis on arthropods. arthropod struct. dev. 34: 379-396, 2005. råberg l, grahn m, hasselquist d, svensson e. on the adaptive significance of stress-induced immunosuppression. proc. r. soc. lond. b 265: 1637-1641, 1998. roeder t. octopamine in invertebrates. prog. neurobiol. 59: 1-31, 1999. roeder t. tyramine and octopamine: ruling behavior and metabolism. ann. rev. entomol. 50: 447-477, 2005. rojas ig, padgett da, sheridan jf, marucha pt. stress-induced susceptibility to bacterial infection during cutaneous wound healing. brain behav. immun. 16: 74-84, 2002. sachser n. short-term responses of plasma norepinephrine, epinephrine, glucorticoid and testosterone titres to social and non-social stressors in male guinea pigs of different social status. physiol. behav. 39: 11-20, 1987. sadd bm, siva-jothy mt. self-harm caused by an insect's innate immunity. proc. r. soc. lond. b 273: 2571-2574, 2006. schmitz d, wilsenack k, lendemanns s, schedlowski m, oberbeck r. ß-adrenergic blockade during systemic inflammation: impact on cellular immune functions and survival in a murine model of sepsis. resuscitation 72: 286294, 2007. segerstrom sc. stress, energy and immunity: an ecological view. curr. direc. psychol. sci. 16: 326-330, 2007. sloley bd, juorio av, durdan da. highperformance liquid-chromatographic analysis of monoamines and some of their gammaglutamyl conjugates produced by the brain and other tissues of helix aspersa (gastropoda). cell. mol. neurobiol. 10: 175-192, 1990. sternberg em. neural regulation of innate immunity: a coordinated nonspecific response to pathogens. nature rev. immunol. 6: 318-328, 2006. van den elzen p, garg s, leon l, brigl m, leadbetter ea, gumperz je, et al. apolipoprotein-mediated pathways of lipid antigen presentation. nature 437: 906-910, 2005. vehovszky a, szabo h, elliot cjh. octopamine increases the excitability of neurons in the snail feeding system by modulation of inward sodium current but not outward potassium current. bmc neurosci. 6: 70, 2005. webster ji, tonelli l, sternberg em. neuroendocrine regulation of immunity. ann. rev. immunol. 20: 125-163, 2002. weers pmm, ryan ro. apolipophorin iii: role model apolipoprotein. insect biochem. mol. biol. 36: 231-240, 2006. wingfield jc. control of behavioural strategies for capricious environments. anim. behav. 66: 807-816, 2003. woodring j, mcbride la, fields p. the role of octopamine in handling and exercise-induced hyperglycaemia and hyperlipaemia in acheta domesticus. j. insect physiol. 35: 613-617, 1989. 19 abstract isj 7: 146-148, 2010 issn 1824-307x visions and perspectives cytokine network in invertebrates: the very next phase of comparative immunology d malagoli department of biology, university of modena and reggio emilia, modena, italy accepted may 20, 2010 abstract information on invertebrate cytokines has been growing impressively in the past five years. however, molecular characterization of newly discovered cytokines has not proportionally improved our understanding of the main reason underpinning their conservation among metazoans. one possible explanation is that cytokines have been conserved for the fundamental processes they control, but in mammals a single cytokine can hardly be considered as controller of complex reactions. in mammals, cytokines constitute a network of communication, and only this network can be considered the real controller of the effects that cytokines exert on immune or developmental functions. in all, the capability of constituting a network could represent the principal reason for cytokine evolution and conservation through diversification of metazoans. key words: cytokines; innate immunity; invertebrates; evolution introduction in mammals, cytokines are described as molecules responsible for the regulation of the maturation, proliferation, differentiation and survival of lymphocytes, macrophages and dendritic cells, and are further classified as lymphokines, monokines, interleukins and chemokines based on their origin and function (corbellini, 2010). among invertebrates (paraphiletic term but useful for the purposes of the manuscript), cytokines are present but much less characterized. no more than 6 years ago, the knowledge on invertebrate cytokines was limited and based on indirect evidence (ottaviani et al., 2004), and the very existence of possible counterparts of vertebrate cytokines was disputed (beschin et al., 2004). recent experiments have introduced dramatic changes in this scenario, so that it could be said now we are moving into the “second age” of invertebrate cytokines. the “second age” of invertebrate cytokines: state of art and future perspectives in the last three years, several reports on invertebrate immunity have used the terms “putative cytokine”, “cytokine-like” etc. (malagoli et al., 2007; parrinello et al., 2008; roberts et al., 2008; zhang et al., 2008; de zoysa et al., 2009, 2010; schikorski et ___________________________________________________________________________ corresponding author: davide malagoli department of biology, university of modena and reggio emilia, via campi 213/d, 41125 modena, italy e-mail: davide.malagoli@unimore.it al., 2009), but the factors that are commonly accepted as full-title invertebrate cytokines at present are spätzle (ferrandon et al., 2004) and unpaired (upd)-3 (agaisse et al., 2003) in drosophila melanogaster, insect chemotactic peptide (icp) in the moth pseudaletia separata (nakatogawa et al., 2009) and astakine-1 in pacifastacus leniusculus (söderhall et al., 2005). these cytokines do not present traits of similarity with vertebrate cytokines, but seem to be widespread in different invertebrate models (an et al., 2010; hsiao et al., 2010), and their receptor and the signalling pathway they elicit appear to be conserved among metazoans (malagoli et al., 2010). interestingly, while the research for cytokine in invertebrates have been prompted mainly by evolutionary-aimed studies (beschin et al., 2004; ottaviani et al., 2004), very few considerations on the evolution of cytokines have been made after the discovery of full-title cytokines. it may appear a paradox, but more speculations on the possible history of cytokines can be realized starting from molecules that at present appear to be candidate cytokines. indeed, some of those molecules, namely drosophila helical factor (dhf) (malagoli et al., 2007), ciona intestinalis tumor necrosis factor (citnf) (parrinello et al., 2008) and hirudo medicinalis endothelial monocyte-activating polypeptide ii (hmemap-ii) (schikorski et al., 2009) promise to give a significant contribution for the understanding of the evolution of cytokines. dhf, or more simply hf as indicated in flybase (www.flybase.org), is a molecule with a predicted structure typical of the vertebrate helical 146 cytokines (huising et al., 2006). the discovery of hf (malagoli et al., 2007) represented a significant contribution supporting the hypothesis that the molecular structure is a component of equal importance to gene and protein sequences in evolutionary studies (malagoli and ottaviani, 2007). citnf is another molecule in which sequence conservation is limited to key region of the molecule (parrinello et al., 2008), and the same is true for hmemap-ii (schikorski et al., 2009). in these respects, it has to be remarked that differently from hf, there is still no information about the activity of citnf and hmemap-ii in ciona and hirudo, respectively, because their discovery has not been followed by a detailed functional analysis, yet. functional assays, based for instance on the utilization of native and recombinant molecules, are however necessary in order to unravel if the structure/sequence conservation corresponds to similar function. however, even among the speculations on the evolution of cytokines, there is a fundamental aspect raised by the discovery of cytokines in invertebrates that has been neglected by comparative immunologists. in their efforts to isolate and characterize cytokines in invertebrates, researchers have been keeping their focus on the specific molecule under study. this led to very detailed and complete characterizations (nakatogawa et al., 2009), but some studies seemed almost concluded with the discovery and characterization themselves. the risk for the next future is to have a long list of new cytokines well-characterized in terms of molecular aspects, but very limited information on their implications for evolutionary biology. the presence of cytokines in organisms with quite different evolutionary histories make it difficult to understand the basis of their conservation. the most obvious explanation is that these molecules are ancient signals that, regulating reactions fundamental for survival and homeostasis maintenance, have been conserved in structure and function. but if we step back to the principle reference of comparative biologists, i.e., mammals and, above all, humans, we can observe that the term "cytokine" indicates a soluble factor that necessarily acts together with many other cytokines in order to determine an effect (corbellini, 2010). in mammals, the importance of cytokines relies principally on their capability of constituting molecular networks, and the functional meaning of a single cytokine is almost absent if we do not consider that molecule acting within a specific molecular context with many other players. accordingly, human immunologists have articulated numerous and complex proposals to describe the interactions between different systems and mediators, e.g., the bow-ties (ottaviani et al., 2008). molecules identified as cytokines have been conserved in diverging metazoan taxa for hundreds million years, and it should be asked if this is due to their capacity to interact and constitute molecular networks, or to function as single signal molecules. the object of natural selection have been cytokines or cytokine networks? admittedly, only in d. melanogaster more than a cytokine has been identified at present, but if we consider the amount of data collected in the past five years and the possibilities offered today by bioinformatic approaches and the high throughput technologies, the discovery of several other invertebrate cytokines appears just a matter of time. now the complete molecular characterization of single factors is a reality in several invertebrate models, the very next phase of comparative immunology must be the identification of networks of cytokines as occurs in vertebrate species. the research for cytokine networks in invertebrate is a topic that directly involve comparative immunologists and evolutionary biologists as well. even though there are several clues on the conservation of structure, receptors and signaling activities of invertebrate cytokines, if these molecules fail to constitute complex networks, should they still be called cytokines? references agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell 5: 441-450, 2003. an c, jiang h, kanost mr. proteolytic activation and function of the cytokine spätzle in the innate immune response of a lepidopteran insect, manduca sexta. febs j. 277:148-162, 2010. beschin a, bilej m, magez s, lucas r, de baetselier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on a similar lectin-like activity. prog. mol. subcell. biol. 34:145-163, 2004. corbellini g. immunology: a historical perspective. in: andrea grignolio (ed), immunology today: three historical perspectives under three theoretical horizons, bononia press university, bologna, italy, pp 35-52, 2010. de zoysa m, nikapitiya c, oh c, whang i, lee js, jung sj, et al. molecular evidence for the existence of lipopolysaccharide-induced tnfalpha factor (litaf) and rel/nf-kb pathways in disk abalone (haliotis discus discus). fish shellfish immunol. 28: 754-763, 2010. de zoysa m, jung s, lee j. first molluscan tnfalpha homologue of the tnf superfamily in disk abalone: molecular characterization and expression analysis. fish shellfish immunol. 26: 625-631, 2009. ferrandon d, imler jl, hoffmann ja. sensing infection in drosophila: toll and beyond. semin. immunol. 16: 43-53, 2004. hsiao cy, song yl. a long form of shrimp astakine transcript: molecular cloning, characterization and functional elucidation in promoting hematopoiesis. fish shellfish immunol. 28: 7786, 2010. huising mo, kruiswijk cp, flik g. phylogeny and evolution of class-i helical cytokines. j. endocrinol. 189: 1-25, 2006. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. 147 malagoli d, sacchi s, ottaviani e. lectins and cytokines in celomatic invertebrates: two tales with the same end. inv. surv. j. 7: 1-10, 2010. malagoli d, ottaviani e. helical cytokines and invertebrate immunity: a new field of research. scand. j. immunol. 66: 484-485, 2007. nakatogawa s, oda y, kamiya m, kamijima t, aizawa t, clark kd, et al. a novel peptide mediates aggregation and migration of hemocytes from an insect. curr. biol. 19: 779785, 2009. ottaviani e, malagoli d, capri m, franceschi c. ecoimmunology: is there any room for the neuroendocrine system? bioessays 30: 868874, 2008. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34:1-25, 2004. parrinello n, vizzini a, arizza v, salerno g, parrinello d, cammarata m, et al. enhanced expression of a cloned and sequenced ciona intestinalis tnfalpha-like (citnf alpha) gene during the lps-induced inflammatory response. cell tissue res. 334: 305-317, 2008. roberts s, gueguen y, de lorgeril j, goetz f. rapid accumulation of an interleukin 17 homolog transcript in crassostrea gigas hemocytes following bacterial exposure. dev. comp. immunol. 32: 1099-1104, 2008. schikorski d, cuvillier-hot v, boidin-wichlacz c, slomianny c, salzet m, tasiemski a. deciphering the immune function and regulation by a tlr of the cytokine emapii in the lesioned central nervous system using a leech model. j. immunol. 183: 7119-7128, 2009. söderhäll i, kim ya, jiravanichpaisal p, lee sy, söderhäll k. an ancient role for a prokineticin domain in invertebrate hematopoiesis. j. immunol. 174: 6153-6160, 2005. zhang x, luan w, jin s, xiang j. a novel tumor necrosis factor ligand superfamily member (cstl) from ciona savignyi: molecular identification and expression analysis. dev. comp. immunol. 32: 1362-1373, 2008. 148 visions and perspectives isj 6: 1-6, 2009 issn 1824-307x visions and perspectives around the word stress: its biological and evolutive implications e ottaviani, d malagoli department of animal biology, university of modena and reggio emilia, modena, italy accepted january 7, 2009 abstract stress is a general adaptive reaction crucial for survival and basically positive that involves the neuroendocrine and the immune systems. in all bilaterian metazoans, the molecular mediators of the stress response, i.e., corticotrophin-releasing hormone, corticotrophin, catecholamines and glucocorticoids, have been preserved during evolution, even if the increased complexity of animals have corresponded to a more articulated stress response that, following the eco-immunology perspective, we speculate to be hierarchically organized along three levels. kew words: stressors; stress response; vertebrates; invertebrates; evolution eustress and distress, not simply “stress” among the general public, the word stress evokes a concept of negativity, which is maintained even among those that have, or should have, knowledge of biology. this situation becomes even more embarrassing when considering that the scientific concept of stress has had the good fortune to become very popular, but at the same time the misfortune to be insufficiently understood. moreover, the use of the term stress in the field of advertising has certainly not clarified its meaning. the present paper aims to provide a correct interpretation of the concept of stress, and especially to emphasize the importance of its positivity, i.e., the role played by stress response in the survival of all animal species on the earth and maintained during evolution. the phenomenon of stress was identified and conceptualized by hans selye who in 1936 published a paper, entitled: "a syndrome produced by different nocuous agents ". before describing the mechanisms of this phenomenon, we should underline some semantic details. stress is fundamentally characterized by two moments and aspects, i.e., the “stimulus” and the “response”. the word stress can indicate both, so creating a possible semantic ambiguity. selye (1978) suggested the word “stressors” (stressogenic ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it agent) to indicate the causal agent, while keeping the word “stress” and “stress response” (response to stress) to indicate the final outcome. moreover, according to selye (1978), the word “stress” has meaning only if related to specific biological situations. regarding the mechanisms of the response to stress, in mammals different organs belonging to the nervous and endocrine systems, such as the hypothalamus, the pituitary and adrenal glands, are involved (selye, 1978). the response triggers physiological processes that operate along two routes. the first is the nervous pathway involving the autonomic nervous system and the medullar portion of adrenal glands leading to the release of catecholamines (ca) (epinephrine and norepinephrine). these molecules provoke a very rapid response, inducing physiological changes, such as the degradation of glycogen to glucose and its increase in the blood, so improving the quality of the life. this situation is further improved with activation of the second track, the endocrine pathway, in which the cortex portion of adrenal glands is involved. schematically, the different stimuli that cause stress induce the release of the corticotrophin-releasing hormone (crh) by the hypothalamus. in turn, the crh provokes corticotrophin (acth) release from the pituitary. this hormone enters the bloodstream and binds specific receptors for acth present on the cells of the cortical portion of the adrenal glands and results in the release of steroid hormones such as glucorticoids (gc). these hormones (cortisol in humans and corticosterone in mice) have different 1 bilatera deuterostomia protostomia mollusca annelida arthropoda echinodermata urochordata cephalochordata vertebrata crh pomc-derived peptides gc ca ? ? ? ? ? ? ? ? fig. 1 presence of the molecules involved in the stress response in the most representative taxa of bilatera. effects; in particular they are involved in regulating the biosynthesis and release of ca. altogether, it emerges that the stress pathway involves molecules in the following order: crh, acth and ca. this complex mechanism that improves the quality of life by means of the release of ca, hormones and steroids is called “eustress” that means beneficial, positive stress. however, stress response must be of short duration. indeed, prolonged exposure to stressors leads to a sustained release of ca and cortisol associated with psychological, functional and pathological symptoms (including bleeding and ulcers) described by selye (1978). this overrun of the stress response is better defined as “distress” that means negative stress. the relationship between neuroendocrine and immune systems stressor and stress response, by one side, and antigens and immune response, on the other, have always been considered as two distinct phenomena, having been discovered and studied separately and, consequently, having become the topics of specific disciplines. however, this division is inconsistent with reality, and the distinction between stressor and antigen or stress and immune response, is to be considered only quantitative and semantic. this dualism was first overcome in experiments undertaken by hugo besedovsky and colleagues (1987). they showed that interleukin (il)-1, a classic mediator of the immune system, is able to activate the hypothalamus-pituitary-adrenal axis. this observation indicates that stressors that induce an immune response (bacteria, viruses, etc.) must also be inserted in the list of the stressogenic agents, suggesting that there is a deep correlation between the immune system and response to stress. edwin blalock and eric smith demonstrated that cells from the immune system, such as lymphocytes and macrophages may play a central role in the induction of stress (blalock and smith, 1985; blalock et al., 1985; blalock, 1989). indeed, lymphocytes and macrophages, well-known producers of cytokines, have also to be considered as neuroendocrine cells being able to synthesize a variety of hormones (i.e., classical molecules produced by the endocrine system) and neuropeptides (i.e., classical molecules produced by the nervous system). furthermore, lymphocytes and macrophages may, in turn, respond to hormones and neuropeptides produced by cells from the neuroendocrine system (blalock and smith, 1985; blalock et al., 1985; blalock, 1989). in summary, various levels of integration between the immune and neuroendocrine systems can be traced: • classical products from the immune system, i.e., cytokines, can act on cells from the neuroendocrine system, modifying the latter's functions; • immune stimuli and hypothalamic releasing factors induce immune cells to synthesize neuropeptides which, in turn, may influence the activity of the neuroendocrine system; • classical hormones and neurotransmitters bind to specific receptors on immune cells and modulate their activity; • cytokines and cytokine-like peptides that are potentially able to modulate immune cell activity are produced by cells from the nervous system. 2 these observations suggest that the three systems (immune, endocrine and nervous) should be considered as anatomically distinct components of a single integrated immuno-neuro-endocrine system involved in the maintenance of the body homeostasis, justifying the conclusion that the response to stress is essential for survival. accordingly, it should be underlined that this interplay between the immune and neuroendocrine systems is not restricted to mammals or other vertebrates, but can be retrieved also in invertebrates (ottaviani and franceschi, 1996), where the molecular cascade of stress response described in the previous paragraph has been observed in immune-competent cells. crh and acth crh has been isolated and characterized by hypothalamic extracts of sheep by vale’s group (1981). later searches showed the presence of crh also in not nervous tissue (seasholtz et al., 2002). a similar picture has been detected in cartilaginous and bony fish as well as in tetrapods, i.e., in all vertebrates (fig. 1) (sato and george, 1973; petrusz et al., 1983; waugh et al., 1985; panzica et al., 1986; roubos, 1997; lovejoy and balment, 1999; summers, 2001; engelsma et al., 2002; seasholtz et al., 2002; malagoli et al., 2004; huising et al., 2005). crh-like molecules were also found in the nervous system of different invertebrate taxa, such as molluscs (sonetti et al., 1986), annelids (rèmy et al., 1982) and insects (verhaert et al., 1984; malagoli et al., 2002), as well as in the immunocytes and hemolymph of molluscs (ottaviani et al., 1990). unfortunately, no data are at present available for echinoderms, urochordates and cephalochordates, that represent the most important invertebrate taxa sharing the deuterostomian lineage with vertebrates (fig. 1). acth is a small peptide enclosed within the pro-opiomelanocortin (pomc) precursor that was initially found in the human pituitary gland (phifer et al., 1974; eberle, 1988). subsequently, acth was also detected in mammalian extra-pituitary areas (ottaviani et al., 1997). as noted above for crh, acth-like molecules were also found in intraand extra-pituitary areas in other vertebrate taxa, namely fish, amphibians, reptiles and birds (fig. 1) (ottaviani et al., 1997; roubos, 1997; engelsma et al., 2002). also different invertebrate taxa (molluscs, annelids, insects, urochordates and cephalochordates) contain immunoreactive acth molecules (ottaviani et al., 1997). no data are available for echinoderms (fig. 1). gc, ca and cytokines in 1985, david norris identified the source of gc, in particular, of cortisol and corticosterone, in the cells of the adrenal cortex of mammals. nonmammalian vertebrates also produce gc (fig. 1) (summers, 2001; engelsma et al., 2002; wada, 2008), but the typical adrenal glands found in mammals are not present in these animals. fish present a group of cells homologue to adrenocortical and chromaffin mammalian tissue, and these two tissues are joined in various ways in tetrapods. the presence of gc-like molecules has also been detected in invertebrates, even if few studies are reported in literature. cortisol immunoreactive molecules were detected in immunocytes from molluscs using an immunocytochemical method (ottaviani et al., 1998), and cortisol and corticosterone have been recorded in the insect calliphora vicina by autoradiography (bidmon and stumpf, 1991). no further data are available for other invertebrate taxa. as far as the presence of ca is concerned, these molecules were detected in all vertebrates (leboulenger et al., 1984; korte et al., 1997; reid et al., 1998; summers, 2001; tsigos and chrousos, 2002). in invertebrates ca were found in molluscs (ottaviani and franceschi, 1996; lacoste et al., 2001; hooper et al., 2007; adamo, 2008), annelids (díaz-miranda et al., 1982; fleming, 1993), arthropods (murdock, 1971; klemm, 1983; adamo, 2008), echinoderms (huet and franquinet, 1981), urochordates (kimura et al., 2003) and cephalochordates (moret et al., 2004). finally, as for ca, cytokines have been observed in all vertebrate lineages (cohen and haynes, 1991; myers et al., 1992; abbas et al., 1994; scapigliati et al., 2000; engelsma et al., 2002; kaiser et al., 2004) and in some invertebrate taxa. with regard the latter, either cytokines or cytokinelike molecules were found in molluscs (ottaviani et al., 2004; de zoysa et al., in press), annelids (ottaviani et al., 2004), arthropods (morisato and anderson, 1994; agaisse et al., 2003; kauppila et al., 2003; söderhäll et al., 2005; ottaviani et al., 2004; lemaitre and hoffmann, 2007; malagoli et al., 2007) and urochordates (parrinello et al., 2008; zhang et al., 2008). a refined orchestra with the same players all the actors that play a role in the stress response must have appeared quite early in animals, since they can be retrieved in different bilaterian lineages (fig. 1). it should be underlined that the cascade of molecular events involved in the stress response is the same in all the bilaterians analyzed so far, i.e., crh, acth and ca. however, since invertebrates lack the organs usually related to vertebrate stress-response, i.e., hypothalamus, pituitary and adrenal glands, it remains to be established how invertebrate stress response can occur in such a simplified scenario. our experiments in molluscs let us to speculate that in less complex organisms the stress response involves the circulating and phagocytic immunocyte endowed with the same molecules that are released and act in the same order described above (ottaviani et al., 1997). however, if the primitive organization of stress response was restricted to single cells, how it came that it has been split up in different organs in vertebrates? in experiments in the catfish ameiurus nebulosus we have observed that fish exposed to lipopolysaccharide (lps) for 15 and 120 min showed an increase in procrh-like molecules in the brain after 15 min but not after 120 min, while the increase in procrh levels in the peripheral organs 3 such as the liver and head kidney persisted for the entire treatment. these findings suggest that stress response is hierarchicallyand time-regulated (malagoli et al., 2004). more precisely, the first and simpler level is the “cell” level by which circulating immunocytes and some cells in various organs, have maintained the capability to resume the stress response. the “cell” level can be taken to represent the persistence of the “ancestral” version of stress response in complex organisms. the second level is the “organ” level, representing a local stress response in which cells distributed within a whole organ are involved. in this case, other organs may not be interested by the stress response that is therefore managed by single components. finally, the third level is the “body” levels, involving different organs connected in a functional net, coordinating the entire system, as it is for the hypothalamuspituitary-adrenal gland axis (ottaviani et al., 1998). this level represent the most complex machinery in stress response, but not necessary its activity is overlapped to that of the other levels (malagoli et al., 2004). it may be surmized that during evolution of vertebrates, while circulating cells maintain their capability of promoting an immune-neuroendocrine response to stressor (“cell level”), some cells were specialized to respond to stressor within organs, thus constituting the “organ” level. the organization of a “system” or “body” level could derive form the constitution of a functional net between organs that were progressively specialized for the intertwined relations between increasingly complex nervous and endocrine systems. this concept of “hierarchy” is in agreement with the fundamental tenet of ecological immunology, i.e., to minimize the cost of biological responses (lochmiller and deerenberg, 2000). in this respect, the “organ” level described above represents a paradigmatic example. fish challenged with lps increased their expression of procrh-like molecules in the brain after 15 min but not after 120 min, while after 120 min the increase in procrh levels persisted in the liver and head kidney. in terms of energy expenditure, we can speculate that it is more convenient for the organism to face the stressor at first also with the “body” level, then, if the stressor does not change its intensity, the stress response is mainly transferred to the periphery and to the “organ” level, thus limiting the involvement of the central nervous system to just the first phase of the stress (malagoli et al., 2004; ottaviani et al., 2008). conclusions in their response to agents that are potentially able to alter their homeostasis and threaten their survival, living organisms exploit a complex and integrated mechanism involving the immuneneuroendocrine system and molecules that have been preserved during evolution, though differently located as a consequence of the increasing complexity of the organisms. stress is a general, adaptive reaction that is crucial for survival and basically positive. most of the negative effects reported in the literature derive from the general perception of stress and refer to extreme conditions of excessive stress. the most common stress of moderate magnitude must be considered physiological and, as seyle (1978) reported, represents “the spice of life”. references abbas ak, lichtman ah, pober js. cellular and molecular immunology, 2nd ed. saunders wb company, philadelphia, usa. adamo sa. norepinephrine and octopamine: linking stress and immune function across phyla. inv. surv. j. 5: 12-19, 2008. agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell 5: 441-450, 2003. berkenbosch f, van oers j, del rey a, tilders f, besedovsky h. corticotropin-releasing factorproducing neurons in the rat activated by interleukin-1. science 238: 524-526, 1987. bidmon hj, stumpf we. uptake, distribution and binding of vertebrate and invertebrate steroid hormones and time-dependence of ponasterone a binding in calliphora vicina. comparisons among cholesterol, corticosterone, cortisol, dexamethasone, 5 alpha-dihydrotestosterone, 1,25-dihydroxyvitamin d3, ecdysone, estradiol17 beta, ponasterone a, progesterone, and testosterone. histochemistry 96: 419-434, 1991. blalock je, harbour-mcmenamin d, smith em. peptide hormones shared by the neuroendocrine and immunologic systems. j immunol. 135 (2 suppl): 858s-861s, 1985. blalock je, smith em. the immune system: our mobile brain? immunol. today 6: 115-117, 1985. blalock je. a molecular basis for bidirectional communication between the immune and neuroendocrine systems. physiol. rev. 69: 132, 1989. cohen n, haynes l. the phylogenic conservation of cytokines. in: warr gw, cohen n (eds), phylogenesis of immune function, crc press, boca raton, pp 241-268, 1991. de zoysa m, jung s, lee j. first molluscan tnfalpha homologue of the tnf superfamily in disk abalone: molecular characterization and expression analysis. fish shellfish immunol. [in press]. díaz-miranda l, de motta ge, garcía-arrarás je. monoamines and neuropeptides as transmitters in the sedentary polychaete sabellastarte magnifica: actions on the longitudinal muscle of the body wall. j. exp. zool. 263: 54-67, 1982. eberle an. the melanotropins. karger, basel, 1088. engelsma my, huising mo, van muiswinkel wb, flik g, kwang j, savelkoul hf, et al. neuroendocrine-immune interactions in fish: a role for interleukin-1. vet. immunol immunopathol. 87: 467-479, 2002. fleming mw. catecholamines during development of the parasitic nematode, haemonchus contortus. comp. biochem. physiol. 104c: 333334, 1993. hooper c, day r, slocombe r, handlinger j, benkendorff k. stress and immune responses in abalone: limitations in current knowledge and investigative methods based on other models. fish shellfish immunol. 22: 363-379, 2007. 4 huet m, franquinet r. histofluorescence study and biochemical assay of catecholamines (dopamine and noradrenaline) during the course of arm-tip regeneration in the starfish, asterina gibbosa (echinodermata, asteroidea). histochemistry 72: 149-154, 1981. huising mo, metz jr, de mazon af, verburg-van kemenade bm, flik g. regulation of the stress response in early vertebrates. ann. ny acad. sci. 1040: 345-347, 2005. kaiser p, rothwell l, avery s, balu s. evolution of the interleukins. dev. comp. immunol. 28: 375394, 2004. kauppila s, maaty ws, chen p, tomar rs, eby mt, chapo j, et al. eiger and its receptor, wengen, comprise a tnf-like system in drosophila. oncogene 22: 4860-4867, 2003. kimura y, yoshida m, morisawa m. interaction between noradrenaline or adrenaline and the beta 1-adrenergic receptor in the nervous system triggers early metamorphosis of larvae in the ascidian, ciona savignyi. dev. biol. 258: 129-140, 2003. klemm n. monoamine-containing neurons and their projections in the brain (supraoesophageal ganglion) of cockroaches. an aldehyde fluorescence study. cell tissue res. 229: 379402, 1983. korte sm, beuving g, ruesink w, blokhuis hj. plasma catecholamine and corticosterone levels during manual restraint in chicks from a high and low feather pecking line of laying hens. physiol. behav. 62: 437-441, 1997. lacoste a, malham sk, cueff a, poulet sa. stressinduced catecholamine changes in the hemolymph of the oyster crassostrea gigas. gen. comp. endocrinol. 122: 181-188, 2001. leboulenger f, charnay y, dubois pm, rossier j, coy dh, pelletier g, et al. the coexistence of neuropeptides and catecholamines in the adrenal gland. research on paracrine effects on adrenal cortex cells. ann. endocrinol. (paris) 45: 217-227, 1984. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. annu. rev. immunol. 25: 697-743, 2007. lochmiller rl, deerenberg c. trade-offs in evolutionary immunology: just what is the cost of immunity? oikos 88: 87-98, 2000. lovejoy da, balment rj. evolution and physiology of the corticotropin-releasing factor (crf) family of neuropeptides in vertebrates. gen. comp. endocrinol. 115: 1-22, 1999. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. malagoli d, mandrioli m, ottaviani e. cloning and characterisation of a procorticotrophin-releasing hormone in the izd-mb-0503 immunocyte line from the insect mamestra brassicae. peptides 23: 1829-1836, 2002. malagoli d, mandrioli m, ottaviani e. procrh in the teleost ameiurus nebulosus: gene cloning and role in lps-induced stress response. brain behav. immun. 18: 451-457, 2004. moret f, guilland jc, coudouel s, rochette l, vernier p. distribution of tyrosine hydroxylase, dopamine, and serotonin in the central nervous system of amphioxus (branchiostoma lanceolatum): implications for the evolution of catecholamine systems in vertebrates. j. comp. neurol. 468: 135-150, 2004. morisato d, anderson kv. the spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsalventral pattern of the drosophila embryo. cell 76: 677-688, 1994. murdock ll. catecholamines in arthropods: a review. comp. gen. pharmacol. 2: 254-274, 1971. myers tj, lillehoj hs, fetterer rh. partial purification and characterization of chicken interleukin-2. vet. immunol. immunopathol. 34: 97-114, 1992. norris do. vertebrate endocrinology, 2nd ed. lea and febiger, philadelphia, usa, 1985. ottaviani e, franceschi c. the neuroimmunology of stress from invertebrates to man. prog. neurobiol. 48: 421-440, 1996. ottaviani e, franchini a, franceschi c. presence of immunoreactive molecules to crh and cortisol in invertebrate haemocytes and lower and higher vertebrate thymus. histochem. j. 30: 61-67, 1998. ottaviani e, franchini a, franceschi c. proopiomelanocortin-derived peptides, cytokines, and nitric oxide in immune responses and stress: an evolutionary approach. int. rev. cytol. 170: 79-141, 1997. ottaviani e, malagoli d, capri m, franceschi c. ecoimmunology: is there any room for the neuroendocrine system? bioessays 30: 868974, 2008. ottaviani e, malagoli d, franceschi c. common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidence to in silico approaches. trends immunol. 28: 497-502, 2007. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. ottaviani e, petraglia f, montagnani g, cossarizza a, monti d, franceschi c. presence of acth and beta-endorphin immunoreactive molecules in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata) and their possible role in phagocytosis. regul. pept. 27: 1-9, 1990. panzica gc, viglietti-panzica c, fasolo a, vandesande f. crf-like immunoreactive system in the quail brain. j. hirnforsch. 27: 539547, 1986. parrinello n, vizzini a, arizza v, salerno g, parrinello d, cammarata m, et al. enhanced expression of a cloned and sequenced ciona intestinalis tnfalpha-like (citnfalpha) gene during the lps-induced inflammatory response. cell tissue res. 334: 305-317, 2008. petrusz p, merchenthaler i, maderdrut jl, vigh s, schally av. corticotropin-releasing factor (crf)-like immunoreactivity in the vertebrate endocrine pancreas. proc. natl. acad. sci. usa 80: 1721-1725, 1983. 5 phifer rf, orth dn, spicer ss. specific demonstration of the human hypophyseal adrenocortico-melanotropic (acth-msh) cell. j. clin. endocrinol. metab. 39: 684-692, 1974. sonetti d, vacirca f, fasolo a. localization of sustance p (sp)-. neuropeptide y (npy)and corticotropin-releasing factor (crf)-like immunoreactive cells in the cns of the freshwater planorbis corneus. neurosci. lett. suppl. 26: 322, 1986. reid sg, bernier nj, perry sf. the adrenergic stress response in fish: control of catecholamine storage and release. comp. biochem. physiol. 120c: 1-27, 1998. summers ch. mechanisms for quick and variable responses. brain. behav. evol. 57: 283-292, 2001. rémy c, tramu g, dubois mp. immunohistological demonstration of a crf-like material in the central nervous system of the annelid dendrobaena. cell tissue res. 227: 569-575, 1982. tsigos c, chrousos gp. hypothalamic-pituitaryadrenal axis, neuroendocrine factors and stress. j. psychosom. res. 53: 865-871, 2002. roubos ew. background adaptation by xenopus laevis: a model for studying neuronal information processing in the pituitary pars intermedia. comp. biochem. physiol. 118a: 533-550, 1997. vale w, spiess j, rivier c, rivier j. characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. science 213: 1394-1397, 1981. sato m, george jc. diurnal rhythm of corticotrophin-releasing factor activity in the pigeon hypothalamus. canad. j. physiol. pharmacol. 51: 743-747, 1973. verhaert p, marivoet s, vandesande f, de loof a. localization of crf immunoreactivity in the central nervous system of three vertebrate and one insect species. cell tissue res. 238: 4953, 1984. scapigliati g, bird s, secombes cj. invertebrate and fish cytokines. eur. cytokine netw. 11: 354-61, 2000. wada h. glucocorticoids: mediators of vertebrate ontogenetic transitions. gen. comp. endocrinol. 156: 441-453, 2008. seasholtz af, valverde ra, denver rj. corticotropin-releasing hormone-binding protein: biochemistry and function from fishes to mammals. j. endocrinol. 175: 89-97, 2002. waugh d, anderson g, armour kj, balment rj, hazon n, conlon jm. a peptide from the caudal neurosecretory system of the dogfish scyliorhinus canicula that is structurally related to urotensin i. gen. comp. endocrinol. 99: 333339, 1995. selye h. a syndrome produced by diverse nocuous agents. nature 138: 32, 1936. selye h. the stress of life. mcgraw-hill book co., new york, usa, 1978. zhang x, luan w, jin s, xiang j. a novel tumor necrosis factor ligand superfamily member (cstl) from ciona savignyi: molecular identification and expression analysis. dev. comp. immunol. 32: 1362-1373, 2008. söderhäll i, kim ya, jiravanichpaisal p, lee sy, söderhäll k. an ancient role for a prokineticin domain in invertebrate hematopoiesis. j. immunol. 174: 6153-6160, 2005. 6 antifungal peptides in marine invertebrates isj 7: 53-66, 2010 issn 1824-307x review antifungal peptides in marine invertebrates n fusetani faculty of fisheries sciences, hokkaido university, hakodate, 041-8611, japan accepted december 21, 2009 abstract a majority of terrestrial and marine organisms use to fend off a wide range of microorganisms, including bacteria and fungi by employing “antimicrobial peptides (amps)” that are ribosomally synthesized from proteinogenic amino acids. amps are a primary component of innate immune mechanisms in marine invertebrates. in contrast, marine sponges seem to contain no amps, but often contain nonribosomal peptides consisting of unusual amino acids that exhibit potent cytotoxic and antifungal activity. most of these peptides are considered to be of symbiotic bacterial origin. similarly opisthobranch molluscs sequester unusual bioactive nonribosomal peptides from their prey organisms, cyanobacteria. however, roles of these peptides are unknown. key words: nonribosomal peptide; antifungal activity; antimicrobial peptide; innate immunity; marine invertebrate introduction an increasing number of disease outbreaks have been recorded in marine invertebrates from viral, bacterial and fungal infections, which are largely influenced by environmental conditions, such as pollution and climate warming. perhaps the most well-known example is “coral breaching” which is partly caused by bacterial and fungal infections (mydlarz et al., 2006). immunological mechanisms in marine invertebrates are different from vertebrate immune system; they rely solely on innate immune systems that include both humoral and cellular responses, the former of which is performed by antimicrobial peptides contained in the blood and plasma. cellular immunity also involves antimicrobial peptides secreted into the hemolymph by hemocytes (tincu and taylor, 2004). naturally-occurring peptides are either synthesized by ribosomal machinery from 20 proteinogenic amino acids or by large enzymes and enzyme complexes called nonribosomal peptide synthases (mcintosh et al., 2009). antimicrobial peptides (amps) involved in marine invertebrate immunity are ribosomal peptides (gene-encoded peptides) and classified into: a) linear α-helical peptides, b) peptides with intramolecular disulfide _________________________________________________________________________ corresponding author: nobuhiro fusetani faculty of fisheries sciences hokkaido university, hakodate 041-8611, japan e-mail: anobu@fish.hokudai.ac.jp bridges, c) β-sheet and small proteins, and d) peptides with one or two predominant amino acids (bulet et al., 2004; tincu and taylor, 2004; hancock et al., 2006; jenssen et al., 2006). the majority of amps are amphiphilic and cationic, containing both hydrophilic and hydrophobic surfaces. they show antimicrobial activity by forming pores in microbial membranes or disrupting membranes (yeaman and yount, 2003; brogden, 2005; jenssen et al., 2006). a total of 1,518 amps are listed in the second version of antimicrobial peptide database, among which 442 peptides are antifungal (wang et al., 2009). nonribosomal peptides found in sponges, molluscs and tunicates are composed of unusual amino acids including d-amino acids and contain organic acids in addition to amino acids as cases of depsipeptides. they exhibit a wide range of biological activities, such as antimicrobial, cytotoxic, and enzyme inhibitory. a majority of them are considered to be derived either from microbial symbionts or cyanobacteria on which opisthobranch molluscs prey as mentioned later. surprisingly, a very limited number of peptides found in marine invertebrates have been reported to be antifungal; obviously much more peptides would be antifungal if tested. this review describes only the peptides reported to be antifungal. porifera sponges are the oldest metazoans and share a common ancestor with other metazoans. they 53 fig. 1 structure of discodermin a possess key molecules which are found in mammalian innate and adaptive immune systems (müller et al., 1999). even a marine sponge was reported to produce a perforin-like antibacterial protein (thankur et al., 2003). no antifungal peptides classified as amps have been identified in sponges, although antifungal cyclic peptides of ribosomal origin were reported from marine sponges as mentioned later. instead, sponges contain a large variety of bioactive compounds, including cytotoxc and antimicrobial (blunt et al., 2009), many of which are considered as microbial symbiont origin (piel, 2004). a number of bioactive unusual nonribosomal peptides were also isolated from sponges, some of which are antifungal (fusetani and matsunaga, 1993; matsunaga and fusetani, 2003; blunt et al., 2009). discodermin a (fig. 1), the first bioactive sponge peptide isolated from discodermia kiiensis, contains a large number of d-amino acids and such unusual amino acids as tert-leucine (t-leu), cysteinoic acid (cya), and sarcosine (sar) (fusetani, 1988; li et al., 1998). it possesses a wide range of bioactivities, including inhibition of various enzymes, antifungal (growth inhibition of candida albicans at 20 μg/disk). a number of its congeners were later isolated from various sponges (li et al., 1998). jaspamide (= jasplakinolide) (fig. 2), a cyclic depsipeptide comprising of such unsual amino acids as n-methyl-2-bromo-d-tryptophan (me-brtrp) and l-β-tyrosine (l-βtyr) isolated from fijian sponges of the genus jaspis, was fungicidal against c. albicans with both an mic and a minimal lethal concentration of 25 μg/ml (scott et al., 1988). it is known to promote actin polymerization (bubb et al., 1994). similar peptides have been reported from various sponges (li et al., 1998; molinski, 2004). marine sponges of the genus theonella are prolific in bioactive metabolites possessing unusual structures (bewley and faulkner, 1998). theonellamide f (fig. 3) is a bicyclic peptide isolated from a japanese theonella sp. containing several unusual amino acids, e.g. histidinoalanine, 3-methyl-p-bromophenylalanine, (2s,4r)-2-amino-4-hydroxyadipic acid (l-ahad), and (3s,4s,5e,7e)-3-amino-4-hydroxy-6-methyl-8 (p-bromophenyl)-5,7-octadienoic acid (aboa). it inhibited the growth of c. albicans with an mic 6.3 μg/ml (fusetani and matsunaga, 1993; li et al., 1998). a glycosylated peptide of the theonellamide family named theonegramide (fig. 4) with antifungal activity against c. albicans at 10 μg/disk was reported from t. swinhoei collected from palau (bewley and faulkner, 1996). fig. 2 structure of jaspamide 54 fig. 3 structure of theonellamide f fig. 4 structure of theonegramide cyclolithistide a (fig. 5) is another class of cyclic peptide isolated from a papua new guinean collection of t. swinhoei and contains unusual amino acids, including 4-chloroisoleucine (cl-ile), 2-amino-pentanoic acid (d-ape), and 4-amino-3,5-dihydroxyhexanoic acid (adha). it showed antifungal activity at 20 μg/disk (clark et al., 1998). microsclerodermin a (fig. 6), a highly unusual cyclic hexapeptide isolated from a palauan thenonella sp., contains new amino acids, e.g., (2s,3r,4s,5s,6s,11e)-3-amino-6-methyl-12-(p-me thoxyphenyl)2,4,5-trihydroxydodec-11-enoic acid (ammtd), (3r)-4-amino-3-hydroxylbutyric acid (gabob), and 3-hydroxy-4-amino-5-vinylpyrrolidone. it inhibited the growth of c. albicans at 1.5 μg/disk (bewley et al., 1994). several congeners have been reported from lithistid sponges (molinski, 2004). these peptides were suggested to be produced by a new δ-proteobacterium found in theonella sp. (bewley and faulkner, 1998). 55 fig. 5 structure of cyclolithistide a fig. 6 structure of microsclerodermin a callipeltin a (fig. 7) was originally isolated as an anti-hiv cyclodepsipeptide comprising of many unusual amino acids including (2r,3r,4s)-4-amino-7-guanidino-2,3-dihydroxyhep tanoic acid (agdhe) from a new caledonian lithistid sponge callipelta sp. it exhibited a 30 mm inhibitory zone at 100 μg/disk (zampella et al., 1996). more sponge nonribosomal peptides were reported to be antifungal (li et al., 1998; molinski 2004; blunt et al., 2009), but modes of antifungal activity of these nonribosomal peptides mentioned above have not been fully elucidated. antifungal ribosomal peptides have not been isolated from marine sponges, except for hymenamides, pro-rich cyclic heptapeptides isolated from hymeniacidon sp. collected in okinawa (kobayashi et al., 1993). hymenamide a (fig. 8) was antifungal against c. albicans with an mic 33 μg/ml as well as cytotoxic. cnidaria although antifungal activity has been detected in some gorgonian species, no antifungal peptides have been isolated. however, a cytotoxic pentapeptide named gymnangiamide (fig. 9) similar to dolastatin 10 (fig.10), a highly antifungal 56 fig. 7 structure of callipeltin a peptide isolated from the sea hare dolabella auricularia mentioned later was reported from the marine hydroid gymnangium regae, though no antifungal activity was described (milanowski et al., 2004). this peptide contains o-desmethyldolaproline (ddap), n-desmethyldolaisoleucine (ddil), l-threo-phenylserine (l-pser), and l-guanidinoserine (gser). perhaps this peptide was originated from symbiotic or sequestered cyanobacteria as the case of dolastatins. a 40-residue amp named aurelin isolated from the jellyfish aurelia aurita exhibited structural features of defensins and channel-blocking toxins of sea anemone origin, but no antifungal activity was reported (ovchinnikova et al., 2006). mollusca in molluscs, hemocytes are predominantly responsible for innate immune defense and release amps (bulet et al., 2004; tincu and taylor, 2004). amps have been reported from bivalves and opisthobrachs as shown in table 1; defensins were identified in hemocytes of the mussel mytilus galloprovincialis (mgd-1 and -2) (mitta et al., 1999b) and in the mantle tissue of the oyster crassostrea gigas (cg-def) (gueguen et al., 2006; gonzalez et al., 2007), respectively. mgds and cg-def inhibited growth of the fungus fusarium oxysporum with an mci value of 4.5-9 μm, respectively. the solution structure of mgp-1 obtained using 1h nmr spectroscopy consists of a helical part and two antiparallel β-strands. the cys-stabilized α-β motif is stabilized by 4 disulfide bridges (yang et al., 2000). similar antibacterial defensins a and b were isolated from hemocytes of m. edulis, but their antifungal activity has not been reported (charlet et al., 1996). defensin a and mgp-1 share some common properties in distribution of hydrophobic and hydrophilic side chains (yang et al, 2000). molluscan defensins are composed of a β-sheet and 3 disulfide bonds, which is similar to arthropod defensins. interestingly, an amp coined cg-prp (37-residue peptide) remarkably enhanced the antifungal activity of cg-def, although it is not antifungal (gueguen et al., 2009). fig. 8 structure of hymenamide a 57 fig. 9 structure of gymnangiamide fig. 10 structure of dolastatin 10 ap, a polyproline-type amp (47 redidues) isolated from the chilean scallop argopecten purpuratus, showed antifungal activity against f. oxisporum and saprolegnia parasitica with ic50 values of 2.1 and 0.85 μm, respectively (arenas et al., 2009). it is also highly antibacterial; perhaps ap enters in lipid bilayer to exhibit antifungal activity. a big defensin named aibd of the scallop a. irradians, similar to arthropod big defensins, has been cloned and expressed; the recombinant aibd (120 residues) was reportedly not only highly antibacterial, but also strongly fungicidal, though detailed fungicidal activity was not available (zhao et al., 2007). a novel argand cys-rich amp named myticin b (40 amino acids) (table 1) isolated from hemocytes of m. galloprovincialis showed antifungal activity against f. oxysporum with mic 5-10 μm (mitta et al., 1999a). interstingly, myticin a possessing a similar amino acid sequence to that of myticin b was not antifungal at 20 μm. mytilin b, a 34-residue amp containing 4 intramolecular disulfide bonds, purified from hemocytes of the same species exhibited antifungal activity against f. oxysporum with mic 0.7-1.4 μm (mitta et al., 2000). mytimycin, a novel antifungal cys-rich polypeptide of 6.2 kda that hindered the growth of fungi, was isolated and partially characterized from m. edulis (charlet et al., 1996). opisthobranch molluscs often sequester bioactive peptides from their prey organisms, especially cyanobacteria and seaweeds (cimino and ghiselin, 2001). dolastatins are highly cytotoxic linear peptides of nrps metabolites sequested from cyanobacteria of the genus lyngbya by the sea hare dolabella auricularia (garson, 2001), among which dolastatin 10, a highly unusual pentapeptide comprising of new amino acids, (2r,3r,4s)-dolaproline (dap), (3r,4s,5s)-dolaisoleucine (dil), l-dolapherine (doe), and l-dolavaline (dov), was shown to be highly antifungal against cryptococcus neoformans with mic 0.37 μg/ml, but not against other fungi (pettit et al., 1998). dolastatin 10 is a potent inhibitor of tubulin polymerization. similarly, kahalalide f (fig. 11), an unusual cyclic depsipeptide containing (z)-2-amino-2-dehydrobutyric acid (z-dhb) and l-ornithine (l-orn) accumulated by the hawaiian sacoglossan elysia rufescens from the green alga bryopsis pennata is highly cytotoxic as well as strongly antifungal against c. albicans, c. neoformans and aspergillus fumigatus with mic 5-10 μm (shilabin et al., 2007). kahalalide f is currently under phase ii clinical trials as anticancer drugs. dolabellin b2, a 33-residue amp (table 1) isolated from the body wall of the sea hare d. auricularia, is fungicidal against s. cerevisiae (ic50~25 μg/ml), while it is fungistatic agaist c. albicans (iijima et al., 2003). 58 fig. 11 structure of kahalalide f annelida and uchiura marine worms dwell in sediments, indicating the requirement of antimicrobial strategy for their survival. amps have been isolated from polychaete and echiuroid worms. arenicin-1 and -2 are 21-residue amps isolated from coelomoycytes of the polychaete arenicola marina (table 2) and show no structural similarity to any amps reported (ovchinnikova et al., 2004). arenicins are cationic peptides having two antiparallel β-strands and one disulfide bond between cys3 and cys20 (ovchinnikova et al., 2007). arenicin-1 showed antifungal activity against c. albicans, c. parasilosis, malasseria furfur, trichosporon beigelli and trichophyton rubrum with mics of 4.5-9 μm comparable to that of mellitin by disrupting fungal phospholipid membranes (park and lee, 2009). it is also antibacterial and hemolytic, which may be interpreted by its permeabilization of model membranes composed of phospholipids or lipopolysaccharides (andrä et al., 2009). perinerin, a 51 residue amp isolated from homogenates of the polychaete perinereis aibuhitensis, is a highly cationic, hydrophobic peptide that is not related to any known amps (pan et al., 2004). it was antifungal against paecilomyces heliothis with mics 12.5-25 μg/ml, in addition to bactericidal activity against gram-negative and –positive bacteria. some neuropeptides show potent antimicrobial activity, which is suggested to be involved in innate immunity (brogden et al., 2005). in fact, urechistachynins i and ii, five residue neuropeptides found in the echiuroid urechis unicinctus exhibit antibacterial and antifungal activities (mics 42 and 25 μm against c. albicans, respectively) (sung et al., 2008). it was suggested that the plasma membrane of fungi is structurally disrupted by urechistachynins. arthropoda amps play a major role in innate immunity of crustaceans and horseshoe crabs. penaeidins, a family of amps of 47-63 residues, were initially characterized from hemocytes of the shrimp litopenaeus vannamei (destoumieux et al., 1997) and later found to be expressed in all penaeid shrimps (bachère et al., 2000; destoumieux et al., 2000; cuthbertson et al., 2004: gueguen et al., 2006). they are composed of a pro-rich n-terminal domain, followed by a c-terminal domain stabilized by 3 intramolecular disulfide bonds, which is unique among amps (table 3). penaeidins exhibit not only antibacterial activity against gram-positive bacteria, but also antifungal activity against various filamentous fungi, but not against yeasts (e.g. mic 5-10 μm against f. oxysporum that is pathogenic to shrimps) (destoumieux et al., 1999; bachère et al., 2000). the solution structure of penaeidin 3 (litvan pen3-1) demonstrated that the surface of the cys-rich domain exhibits an amphipathic character required for antimicrobial properties (yang et al., 2003). a similar result was reported for penaeidin 4 (litset pen4-1) (cuthbertson et al., 2005). the penaeidin database, penbase (www.penbase.immunaqua.com), has been constructed; detailed information of 34 penaeidins is contained at moment (gueguen et al., 2006). they are classified into three subgroups based on amino acid sequences: penaeidin 2 (pen2), penaeidin 3 (pen3) and penaeidin 4 (pen4) (culthbertson et al., 2004). actually, penbase 59 http://www.penbase.immunaqua.com/ table 1 amino acid sequences of molluscan antifungal peptides mgd-1: gfgcpnnyqchrhcksipgrcggycggwhrlrctcyrcg mgd-2: gfgcpnnyachqhcksirgrcggycagwfrlrctcyrcg defensin a: gfgcpndypcksipgrxggycggxhrlrctcyr defensin b: gfgcpndypcksipgryggycggxhrlrctc cg-def: gfgcpgnqlkcnnhcksiscragycdaatlwlrctctdcngkk mytilin a: gcasrckakcagrrckgwasasfrgrcyckcfrc mytilin b: scasrckghcrarrcgyyvsvlyrgrcyckclrc myticinb: hphyctsyycskfcgtagcrrygcrnlhrgklcfclhcsrv ap: tympveegeyivnisyadqpkknspftakkqpgpkvdlsgvkaygpg dolabellanin b2: shqdcyealhkcmashskpfscsmkfhmclqqq each disulfide pair is highlighted in red, pink, green and light blue for mgds. cys residues are highlighted in red for the rest of peptides, while arg in violet for mytilins and myticin and pro in blue for ap. x in defensins is a not identified residue. contains more than 200 entries of penaeidins and the number is growing rapidly due to the active genomic and proteomic research. crustins are cationic, cys-rich antibacterial polypeptides of ca. 7-14 kd occurring in circulating hemocytes of crustaceans that contain a whey acidic protein (wap) domain in the c-terminus (smith et al., 2008). more than 50 crustin sequences have been reported from a variety of decapods and classified into 3 subgroups, type i to iii. no crustins had been reported to be antifungal until recently when crupc of 98 residues belonging to type ii and cruha1/2 of 90 residues (type i) were characterized from the red king crab paralithodes camtschaticus and the spider crab hyas araneus, respectively (sperstad et al., 2009). cruha1 inhibited the growth of s. cerevisiae with mic 12.5-25 μm. hemocytes of h. araneus also contain hyastatin, a gly-rich multi-domain polypeptide of 11.7 kd resembling type ii curstins (sperstad et al., 2009). it exhibited mics of 12.5 and 6.3-12.5 μm against s. cerevisiae and c. albicans, respectively. horseshoe crabs rely completely on innate immune system which is the first line of inducible host defense against bacterial, fungal and viral pathogens (iwanaga and lee, 2005). quite recently, an excellent review on the molecular basis for innate immune system of horseshoe crabs has been published in this journal (kawabata et al., 2009). the most well-studied species is tachypleaus tridentatus (japanese horseshoe crab); its hemolymph contains a variety of soluble defense molecules, e.g. hemocyanin, lectins/c-reactive proteins and α2-macroglobulin, in addition to granular hemocytes that comprise 99 % of total hemocytes. granular hemocytes consist of large and small granules which are sensitive to lipopolysaccharides (lps) of gram-negative bacteria and secret defense substances by stimulation of lps. small granules contain various amps, including tachyplesins, tachycitin, tachystatins and big defensin (table 4), while large granules release enzymes, lectins and proteins involved in hemolymph coagulation (iwanaga et al., 1998; 2005; kawabata et al., 2009). all amps isolated from horseshoe crabs showed binding activity to chitin, a primary target of the innate immune system. tachyplesins i and ii isolated from t. tridentatus are composed of 17 amino acids and contain 2 intramolecular disulfide bonds as shown in table 4 (nakamura et al., 1988; 60 table 2 amino acid sequences of antifungal peptides retrieved in worms arenicin 1: rwcvyayvrvrgvlvryrrcw arenicin 2: rwcvyayvrirgvlvryrrcw perinerin: fnklkqgsskrtcakcfrkimpsvhelderrrganrwaagfrkcvssicry urechistachykinin i: lrqsqfvgsr-nh2 urechistachykinin ii: aagmgffgar-nh2 table 3 amino acid sequences of penaeidins from the shrimp lytopenaeus vannamei litvan pen2-1 (pen2): eayrggytgpiprpppigrppfrpvcnacyrlsvsdarnccikfgscchlvkg litvan pen3-1 (pen3): qvykggytrpiprpppfvrplpggpigpyngcpvscrgisfsqrsccsrlgrcchvgkgysg litvan pen4-1 (pen4): hssgytrplpkpsrpifirpigcdvcygipsstarlccfrygdcchrg each disulfide pair is highlighted in red, green and light blue; pro residues are in pink. miyata et al., 1989). similar 18-residue amps named polyphemusins i and ii were isolated from the american horseshoe crab limulus polyphemus (miyata et al., 1989). these four amps showed similar antimicrobial activity against gram-negative and –positive bacteria as well as fungi (ic50 0.2 μg/ml against c. albicans)(osaki et al., 1999). a big defensin consisting of 79 amino acids was isolated from t. tridentatus hemocytes (saito et al., 1995). it is similar to rat defensins and showed potent antimicrobial activity against bacteria, but weak activity against fungi (ic50 20 μg/ml against c. albicans)(osaki et al., 1999). tachystatin a (44 residues), b (42) and c (41) isolated from t. tridentatus exhibited potent antifungal activity against c. albicans and p. pastoris with ic50 values of 0.9-3.0 and 0.1-0.3 μg/ml, respectively (osaki et al., 1999). tachystatins a and b showed sequence similarity to ω-agatoxin-iva of a funnel web venom, but tachystatin c, which is no significant sequence similarity to the former two, is similar to insecticidal spider neurotoxins. the solution structure of tachystatin a analyzed by nmr spectroscopy showed an amphiphilic folding found in membrane-interactive peptides (fujitani et al., 2002). tachystatins contain three disulfide bridges as shown in table 4. t. tridentatus hemocytes contain another amp named tachycitin which consists of 73 amino acids and five intramolecular disulfides linkages, but showed no significant sequence similarity to known amps (kawabata et al., 1996). it showed weak antimicrobial activity against antibacterial and fungi (ic50 52 μg/ml against c. albicans).. it should be noted that the apparent antimicrobial activity of horseshoe crab apms are significantly reduced under isotonic conditions (0.5 m nacl for horseshoe crabs) to those under hypotonic conditions (kawabata et al., 2009). echinodermata and urochordata starfishes and sea cucumbers are known to contain antifungal saponins named asterosaponins and holothurins, respectively (fusetani and kem, 2009). only a small number of works have been done on amps in echinoderms, although antibacterial activity of their coelomocytes were reported; cationic, defensin-like amps have been 61 table 4 amino acid sequences of antifungal peptides from horseshoe crabs tachyplesin i: kwcfrvcyrgicyrrcr-nh2 tachyplesin ii rwcfrvcyrgicyrkcr-nh2 polyphemusin i: rrwcfrvcyrgfcyrkcr-nh2 polyphemusin ii: rrwcfrvcykgfcyrkcr-nh2 tachystatin a: ysrcqlqgfncvvrsyglptipccrgltcrsyfpgstygrcqry tachystatinb1: yvsclfrgarcrvysgrsccfgyycrrdfpgsifgtcsrrnf tachystatin c: dydwslrgppkcatygqkcrtwsprnccwnlrckafrcrpr tachycitin: ylafrcgryspclddgpnvnlysccsfynchkclarlencpkglhynaylkvcdwpskagct each sulfide bond is highlighted in red, light blue and green table 5 amino acid sequences of antifungal peptides from tunicates clavanin a: vfqflgkiihhvgnfvhgfshvf-nh2 clavanin b: vfqflgriihhvgnfvhgfshvf-nh2 clavanin c: vfhllgkiihhvgnfvygfshvfnh2 clavanin d: afkllgriihhvgnfvhgfshvf-nh2 clavaspirin: flrfigsvihgighlvhhigval-nh2 recently reported from a sea urchin, but no information about its antifungal activity is available (li et al., 2008). tunicates (ascidians) often contain cytotoxic cyclic peptides, most of which are derived by nonribosaomal peptide synthases, but their antifungal activity have not been examined (blunt et al., 2009). an unusual diketopiperazine named etzionin (fig. 12) reported from a unidentified red sea tunicate inhibited the growth of c. albicans and aspergillus nidulans with mic of 3-12.5 μg/ml (hirsch et al., 1989). tunicates employ a prototype of vertebrate innate immune system for host defense; in fact, ciona intertinalis hemocytes are shown to express a number of host defense-related genes involved in innate immune systems (shida et al., 2003). halocyamines was the first ascidian-derived amps isolated from halocynthia roretzi which are post-translationally modified ribosomal peptides. halocyamine a (fig. 12) showed weak antifungal activity against cryptococcus neoformans with an mic of 100 μg/ml (azumi et al., 1990). styelin d also contains post-translationally modified residues but its antifungal activity is unknown (lehrer et al., 2003). clavanins a-d, α-helical amps of 23 residues identified in the solitary tunicate styela clava (table 5), showed antifungal activity against c. albicans with mic 5-20 μg/ml, in addition to antibacterial activity (lee et al., 1997). a his-rich, 23-residue apm named clavaspirin was identified in pharyngeal tissues of the same species (lee et al., 2001). a synthetic peptide of clavaspirin was antibacterial against gram-positive and –negative bacteria as well as antifungal against c. albicans 62 fig. 12 structure of etzionin and halocyamine a with mic ~5-10 μg/ml. it is highly hemolytic and cytotoxic; α-helical nature of the peptide may responsible for these activities. recently, search for amps using expressed sequence tag (est) database has been attempted for the tunicate ciona intestinalis on which the genome project was completed, which resulted in identification of gene families coding novel amps of α-helical types ( fedders and leippe, 2008; fedders et al., 2008). a synthetic peptide of c-terminal region consisting 24 amino acids of a putative amp coded ci-mam-a showed potent antifungal activity against c. albicans with mic 3.1 μm, in addition to potent antibacterial activity (fedders et al., 2008). importantly, its antibacterial activity was retained at high nacl concentrations (up to 450 mm). antimicrobial activity and mode of action of the intact peptide are interesting subjects. conclusion a large array of invertebrates live in marine environments harboring high concentrations of pathogenic microorganisms. amps are considered to be a major component of the innate immune defense on which marine invertebrates solely rely (tincu and taylor, 2004). therefore, it is surprising that amps have been explored for only limited phyla, mainly mollusca, arthropoda and urochordata. innate immune systems in marine invertebrates which are increasingly important, since emerging numbers of diseases reported for marine invertebrates partly due to seawater warming and water pollution. for better understanding of marine ecosystem, more knowledge should be accumulated for amps in a wide range of marine phyla. it is expected that more and more amps will be identified in a wide variety of marine invertebrates by genomic and proteomic approaches. these data will contribute to medicinal uses of amps (zasloff, 2002; jenssen et al., 2006). obviously, more research on modes of action of marine amps is required. nonribosomal peptides of various bioactivities, most of which are cytotoxic, but those described as antifungal are few, have been discovered from sponges, nudibranchs and tunicates. although these peptides are considered to be accumulated from symbiotic microbes or prey organisms, their roles in host organisms remain mostly unknown. ecological roles of nonribosomal peptides in marine invertebrates are interesting subjects. references andrä j, hammer mu, grötzinger j, jakovkin i, lindner b, vollmer e, et al. significance of the cyclic structure and of arginine residues for the antibacterial activity of arenicin-1 and its interaction with phospholipid and lipopolysaccharide model membranes. biol. chem. 390: 337-349, 2009. arenas g, guzmán f, cárdenas c, mercado l, marshall sh. a novel antifungal peptide designed from the primary structure of a natural antimicrobial peptide purified from argopecten purpuratus hemocyes. peptides 30: 1405-1411, 2009. azumi k, yokosawa h, ishii s. halocyamines: novel antimicrobial tetrapeptide-like substances isolated from the hemocytes of the solitary ascidian halocynthia roretzi. biochemistry 29: 159-165, 1990. bachère e, destoumieux d, bulet p. penaeidins, antimicrobial peptides of shrimp: a comparison with other effectors of innate immunity. aquaculture 191: 71-88, 2000. bachère e, gueguen y, gonzalez m, de lorgeril j, garnier j, romestand b. insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster crassostrea gigas. immunol. rev. 198: 149-168, 2004. bewley ca, debitus c, faulkner dj. microsclerodermins a and b. antifungal cyclic peptides from the lithistid sponge microscleroderma sp. j. am. chem. soc. 116: 7631-7636, 1994. bewley ca, faulkner dj. theonegramide, an antifungal glycopeptide from the philippine lithistid sponge theonella swinhoei. j. org. chem. 59: 4849-4852, 1994. 63 bewley ca, faulkner dj. lithisthid sponges: star performers or hosts to the stars. angew. chem. int. ed. 37: 2162-2178, 1998. blunt jw, copp br, hu w-p, munro mhg, northcote pt, prinsep mr. marine natural products. nat. prod. rep. 26:: 170-244, 2009 and the previous reviews of this series. brogden ka. antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? nat. rev. microbiol. 3: 238-250, 2005. bubb mr, senderowicz amj, sausville ea, duncan klk, korn ed. jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to f-actin. j. biol. chem. 269: 14869-14871, 1994. bulet, p, stöcklin r, menin l. anti-microbial peptides: from invertebrates to vertebrates. immunol. rev. 198: 169-184, 2004. charlet m, chernysh s, philippe h, hetru c, hoffmann ja, bulet p. innate immunity. isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, mytilus edulis. j. biol. chem. 271: 21808-21813, 1996. cimino g, ghiselin mt. marine natural products chemistry as an evolutionary narrative. in: mcclintock jb, baker bj (eds), marine chemical ecology, crc press, boca raton, usa, pp115-154, 2001. clark dp, carroll j, naylor s, crews p. an antifungal cyclodepsipeptide, cyclolithistide a, from the sponge theonella swinhoei. j. org. chem. 63: 8757-8764, 1998. cuthbertson bj, shepard ef, chapman rw, gross ps. diversity of the penaeidin antimicrobial peptides in two shrimp species. immunogenetics 54: 442-445, 2002. cuthbertson bj, yang y, bachère e, büllesbach ee, gross ps, aumelas a. solution structure of synthetic penaeidin-4 with structural and functional comparisons with penaeidin-3. j. biol. chem. 280: 16009-16018, 2005. destoumieux d, bulet p, loew d, van dorsselaer a, bachète e. panaeidins, a new family of antimicrobial peptides isolated from the shrimp panaeus vannamei (decapoda). j. biol. chem. 272: 28398-28406, 1997. destoumieux d, bulet p, strub j-m, van dorsselaer a, rodriguez j, bachète e. recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp. eur. j. biochem. 266: 335-346, 1999. destoumieux d, munoz m, bulet p, bachète e. penaeidins, a family of antimicrobial peptides from penaeid shrimp (crustacea, decapoda). cell. mol. life sci. 57: 1260-1271, 2000. fedders h, leippe m. a reverse search for antimicrobial peptides in ciona intestinalis: identification of a gene family expressed in hemocytes and evaluation of activity. dev. comp. immunol. 32: 286-298, 2008. fedders h, michalek m, grötzinger j, leippe m. an exceptional salt-tolerant antimicrobial peptide derived from a novel gene family of haemocytes of the marine invertebrate ciona intestinalis. biochem. j. 416: 65-75, 2008. fujitani n, kawabata s, osaki t, kumaki y, demura m, nitta k, et al. structure of the antimicrobial peptide tachystatin a. j. biol. chem. 277: 23651-23657, 2002. fusetani n. antifungal substances from marine invertebrates. ann. ny acad. sci. 544: 113-127, 1988. fusetani n, kem w. marine toxins: an overview. in: fusetani n, w. kem (eds) marine toxins as research tools, springer-verlag, berlin, germany, pp 1-44, 2009. fusetani n., matsunaga s. bioactive sponge peptides. chem. rev. 93: 1793-1806, 1993. garson mj. ecological perspectives on marine natural product biosynthesis. in: mcclintock jb, baker jb (eds), marine chemical ecology, crc press, boca raton, usa, pp71-114, 2001. gonzalez m, gueguen y, desserre g, de lorgeril j, romestand b, bachère e. molecular characterization of two isoforms of defensin from hemocytes of the oyster crassostrea gigas. dev. comp. immunol. 31: 332-339, 2007. gueguen y, garnier j, robert l, lefranc m-p, mougenot i, de lorgeril j, et al. penbase, the shrimp antimicrobial peptide penaeidin database: sequence-based classification and recommended nomenclature. dev. comp. immunol. 30: 283-288, 2006. gueguen y, herpin a, aumelas a, garnier j, fievet j, escoubas j-m, et al. characterization of a defensin from the oyster crassostrea gigas. recombinant production, folding, solution structure, antimicrobial activities, and gene expression. j. biol. chem. 281: 313-323, 2006. gueguen y, romestand b, fievet j, schmitt p, destoumieux-garzon d, vandenbulcke f, et al. oyster hemocytes express a proline-rich peptide displaying synergistic amtimicrobial activity with a defensin. mol. immunol. 46: 516-522, 2009. hancock rew, brown kl, mookherjee n. host defence peptides from invertebrates emerging antimicrobial strategies. immunobiology 211: 315-322, 2006. hirsch s, miroz a, mccarthy p, kashman y. etzionin, a new antifungal metabolite from a red sea tunicate. tetrahedron lett. 30: 4291-4294, 1989. iijima r, kisugi j, yamazaki m. a novel antimicrobial peptide from the sea hare dolabella auricularia. dev. comp. immunol. 27: 305-311, 2003. iwanaga s, kawabata s, muta t. new types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions. j. biochem. 123: 1-15, 1998. iwanaga, s, lee bl. recent advances in the innate immunity of invertebrates animals. j. biochem. mol. biol. 38: 128-150, 2005. jenssen h, hamill p, hancock rew. peptide antimicrobial agents. clin. microbiol rev. 19: 491-511, 2006. kawabata s, koshiba t, shibata t. the lipopolysaccharide-activated innate immune response network of the horseshoe crab. invert. surv. j. 6: 59-77, 2009. 64 kawabata, s, nagayama r, hirata m, shigenaga t, agarwala kl, saito t, et al. tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity. j. biochem. 120: 1253-1260, 1996. kobayashi, j, tsuda m, nakamura t, mikami y, shigemori h. hymenamides a and b, new proline-rich cyclic heptapeptides from the okinawan marine sponge hymeniacidon sp. tetrahedron 49: 2391-2402, 1993. lee ih, zhao c, cho y, harwig ssl, cooper el, lehrer ri. clavanins, α-helical antimicrobial peptides from tunicate hemocytes. febs lett. 400: 158-162, 1997. lee i-h, zhao c, nguyen t, menzel l, waring aj, sherman ma, lehrer ri. clavaspirin, an antibacterial and haemolytic peptide from styela clava. j. pept. res. 58: 445-456, 2001. lehrer ri, tincu ja, taylor sw, menzel lp, waring aj. natural peptide antibiotics from tunicates: structures, functions and potential uses. integr. comp. biol. 43: 313-322, 2003. li c, haug t, styrvold ob, jørgensen tø, stensvåg k. strongylocins, novel antimicrobial peptides from the green sea urchin, strongylocentrotus droebachiensis. dev. comp. immunol. 32: 1430-1440, 2008. li hy, matsunaga s, fusetani n. antifungal metabolites from marine sponges. curr. org. chem. 2: 649-682, 1998. matsunaga s, fusetani n. nonribosomal peptides from marine sponges. curr. org. chem. 7: 945-966, 2003. mcintosh ja, donia ms, schmidt ew. ribosomal peptide natural products: bridging the ribosomal and nonribosomal worlds. nat. prod. rep. 26: 537-559, 2009. milanowski dj, gustafson kr, rashid ma, pannell lk, mcmahon jb, boyd mr. gymnangiamide, a cytotoxic pentapeptide from the marine hydroid gymnangium regae. j. org. chem. 69: 3036-3042, 2004. mitta g, hubert f, noël t, roch p. myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel mytilus galloprovincialis. eur. j. biochem. 265: 71-78, 1999a. mitta g, vandenbulcke f, hubert f, roch p. mussel defensins are synthesized and processed in granulocytes then released into the plasma after bacterial challenge. j. cell sci. 112: 4233-4242, 1999b. mitta g, vandenbulcke f, hubert f, salzet m, roch p. involvement of mytilins in mussel antimicrobial defense. j. biol. chem. 275: 12954-12962, 2000. miyata t, tokunaga f, yoneya t, yoshikawa k, iwanaga, s, niwa m, et al. antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin ii, and polyphemusins i and ii: chemical structures and biological activity. j. biochem. 106: 663-668, 1989. molinski tf. antifungal compounds from marine organisms. anti-infect. agents med. chem. 3: 197-220, 2004. müller weg, blumbach b, müller im. evolution of the innate and adaptive immune systems: relationships between potential immune molecules in the lowest metazoan phylum (porifera) and those in vertebrates. transplantation 68: 1215-1227, 1999. mydlarz ld, jones le, harvell cd. innate immunity, environmental drivers, and disease ecology of marine and freshwater invertebrates. annu. rev. ecol. evol. syst. 37: 251-288, 2006. nakamura t, furunaka h, miyata t, tokunaga f, muta t, iwanaga s. tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (tachypleus tridentatus). isolation and chemical structure. j. biol. chem. 263: 16709-16713, 1988. osaki t, omotezako m, nagayama r, hirata m, iwanaga s, kasahara j, et al. horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins. j. biol. chem. 274: 26172-26178, 1999. ovchinnikova tv, aleshina gm, balandin sv, krasnosdembskaya ad, markelov ml, frolova ei, et al. purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta arenicola marina. febs lett. 577: 209-214, 2004. ovchinnikova tv, balandin sv, aleshina gm, tagaev aa, leonova yf, krasnodembsky eg, et al. aurelin, a novel antimicrobial peptide from jellyfish aurelia aurita with structural features of defensins and channel-blocking toxins. biochem. biophys. res. commun. 348: 514-523, 2006. ovchinnikova tv, shenkarev zo, nadezhdin kd, balandin sv, zhmak mn, kudelina ia, et al. recombinant expression, synthesis, purification, and solution structure of arenicin. biochem. biophys. res. commun. 360: 156-162, 2007. pan w, liu x, ge f, han j, zheng t. perinerin, a novel antimicrobial peptide purified from the clamworm perinereis aibuhitensis grube and its partial characterization. j. biochem. 135: 297-304, 2004. park c, lee dg. fungicidal effect of antimicrobial peptide arenicin-1. biochim. biophys. acta 1788: 1790-1796, 2009. pettit rk, pettit gr, hazen kc. specific activities of dolastatin 10 and peptide derivatives afainst cryptococcus neoformans. antimicrob. agents chemother. 42: 2961-2965, 1998. piel j. metabolites from symbiotic bacteria. nat. prod. rep. 21: 519-538, 2004. saito t, kawabata s, shigenaga t, takayenoki y, cho j, nakajima h, et al. a novel big defensin identified in horseshoe crab hemocyes: isolation, amino acid sequence, and antibacterial activity. j. biochem. 117: 1131-1137, 1995. scott vr, boehme r, matthews ts. new class of antifungal agents: jasplakinolide, a cyclodepsipeptide from the marine sponge, jaspis species. antimicrob. agents chemother. 32: 1154-1157, 1988. 65 shida k, terajima d, uchio r, ikawa s, ikeda m, asano k, et al. hemocytes of ciona intestinalis express multiple genes involved in innate immune host defense. biochem. biophys. res. commun. 302: 207-218, 2003. shilabin ag, kasanah n, wedge de, hamann mt. lysosome and her3 (erbb3) selective anticancer agent kahalalide f: semisynthetic modifications and antifungal lead-exploration studies. j. med. chem. 50: 4340-4350, 2007. smith vj, fernandes jmo, kemp gd, hauton c. crustins: enigmatic wap domain-containing antibacterial proteins from crustaceans. dev. comp. immunol. 32: 758-772, 2008. sperstad sv, haug t, paulsen v, rode tm, strandskog g, solem st, et al. characterization of crustins from the hemocytes of the spider crab, hyas araneus, and the red king crab, paralithodes camtschaticus. dev. comp. immunol. 33: 583-591, 2009. sperstad sv, haug t, vasskog t, stensvåg k. hyastatin, a glycine-rich multi-domain antimicrobial peptide isolated from the spinder crab (hya araneus) hemocytes. mol. immunol. 46: 2604-2612, 2009. sung ws, park sh, lee dg. antimicrobial effect and membrane-active mechanism of urechistachykinins, neuropeptides derived from urechis unicinctus. febs lett. 582: 2463-2466, 2008. thakur nl, hentschel u, krasko a, pabel ct, anil ac, müller weg. antibacterial activity of the sponge suberites domuncula and its primmorphs: potential basis for epibacterial chemical defense. aquat. microb. ecol. 31: 77-83, 2003. tincu ja, taylor sw. antimicrobial peptides from marine invertebrates. antimicrob. angents chemother. 48: 3645-3654, 2004. wang g, li x, wang z. apd2: the updated antimicrobial peptide database and its application in peptide design. nucleic acids res. 37: d933-d937, 2009. yang y-s, mitta g, chavanieu a, calas b, sanchez jf, roch p et al. solution structure and activity of the synthetic four-disulfide bond mediterranean mussel defensin (mgd-1). biochemistry 39: 14436-14447, 2000. yang ys, poncet j, garnier j, zatylny c, bachère e, aumelas a. solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide. j. biol. chem. 278: 36859-36867, 2003. yeaman mr, yount ny. mechanisms of antimicrobial peptide action and resistance. pharmacol. rev. 55: 27-55, 2003. zampella a, d’auria mv, paloma lg, casapullo a, minale l, debitus c, et al. callipeltin a, an anti-hiv cyclic depsipeptide from the new caledonian litisthida sponge callipelta sp. j. am. chem. soc. 118: 6202-6209, 1996. zasloff m. antimicrobial peptides of multicellular organisms. nature 415: 389-395, 2002. zhao j, song l, li c, ni d, wu l, zhu l, et al. molecular cloning, expression of a big defensin gene from bay scallop argopecten irradians and the antimicrobial activity of its recombinant protein. mol. immunol. 44: 360-368, 2007. 66 short communication isj 4: 82-85, 2007 issn 1824-307x short communication detection of hsp27-like molecules in the annelid enchytraeus japonensis after exposure to extremely low frequency magnetic fields (50 hz) d malagoli1, m marchetti2, f gobba2 1department of animal biology, university of modena and reggio emilia, modena, italy 2department of public heath sciences, university of modena and reggio emilia, modena, italy accepted july 9, 2007 abstract the immunocytochemical study performed on the annelid enchytraeus japonensis revealed the presence of immunoreactive heat shock protein (hsp)27 molecules in different areas of the body. positivity was observed in coelomocytes and in epithelial cells of the intestine wall. the exposition of the animals to 400 μt magnetic fields (50 hz) for 30 min provoked an increased immunoreactivity in some specimens, but after immunoblot experiments, no significant differences in the total content of hsp27-like molecules were found between exposed and non-exposed animals. key words: enchytraeus japonensis; hsp27; immunocytochemistry; extremely low frequency magnetic field introduction from the literature, it emerges that invertebrates are a suitable model to study the effects of extremely low frequency magnetic fields (elf-mf). after the pioneering work performed by goodman’s group (1976) on the slime mold physarum polycephalum, studies were subsequently carried out on the insects sciara coprophila and drosophila melanogaster, the molluscs cepea nemoralis and mytilus galloprovincialis and the nematode caenorhabditis elegans (kavaliers et al., 1991; goodman et al., 1995; junkersdorf et al., 2000; miyakawa et al., 2001; ottaviani et al., 2002; gobba et al., 2003; malagoli et al., 2003, 2004, 2006). mfs are able to influence a variety of biological systems (goodman et al, 1995; del re et al., 2006; malagoli et al., 2006; bernardini et al., 2007), and several investigations have focused on the alterations provoked by mfs on the membrane ion channels and on the expression of heat shock proteins (hsps). regarding ion channels in invertebrates, we have demonstrated that the exposure of the mussel m. galloprovincialis to 50 hz mfs ranging from 200 to 1000 μt disturbs the reactivity of circulating cells (immunocytes) towards ___________________________________________________________________________ corresponding author: davide malagoli department of animal biology via campi 213/d 41100 modena, italy e-mail: malagoli.davide@unimore.it n-formyl-meth-leu-phe (fmlp) by altering potassium and calcium channel permeability (ottaviani et al., 2002; gobba et al., 2003). also in the mollusc c. nemoralis, mfs provoke an alteration in calcium channel function (kavaliers et al., 1991). as other physiological stressors, mfs can provoke the expression of highly conserved genes coding for hsps (lindquist, 1986; goodman and blank, 1998). in the nematode c. elegans, exposure to mfs induces the expression of the hsp70 (goodman and blank, 1998) and the hsp16 genes (miyakawa et al., 2001), while an augmented expression of hsp70 and hsp90 was observed in the mussel m. galloprovincialis after repeated exposure to 400 and 600 μt mf (malagoli et al., 2004). in order to increase our knowledge of the possible effects of elf-mf on the small hsps in invertebrates, the present paper investigates the presence and expression of hsp27-like molecules in the annelid enchytraeus japonensis following exposure to a 400 μt intensity elf-mf. materials and methods animals prior to experimental procedures, adult samples of enchytraeus japonensis (annelida, oligochaeta, enchytraeidae) were grown on agar medium for 1 month, as previously described in detail for enchytraeus crypticus (franchini and marchetti, 2006). 82 mailto:malagoli.davide@unimore.it fig. 1 hsp27-like molecules in coelomocytes (arrows) (a) and in epithelial cells (arrows) of the intestine wall (b) of e. japonensis. nuclei are counterstained with hematoxylin. bar = 10 μm. exposure of animals to mfs each experiment was performed by placing 10 specimens for 2 h under a 50 hz mf generated by two pvc coated coils (10x10 cm, 1400 windings, 0.2 mm ∅ of copper wire) (igea, carpi, mo, italy) mounted horizontally 13 cm apart. the sinusoidal elf mf intensity was controlled by an electronic power source (california instruments, usa) connected to a computer and regulated by the “pgui32” ac source control program (california instruments, usa). elf mf intensity and the temperature between the coils were constantly checked using a “7010 gauss/teslameter” (f.w. bell, usa). mfs of 400 μt were generated. shamexposed animals were maintained under the coils for the same time as the treated specimens, but in the absence of any current. all experiments were performed at room temperature. after 4 h of recovery, some animals were immediately fixed in bouin’s mixture and embedded in agar/paraffin, following franchini and marchetti (2006), while others were sacrificed for western blot analysis. hematoxylin-eosin stain and immunocytochemical reactions were performed on 7 µm longitudinal sections. immunocytochemical procedure the immunocytochemical reaction was performed using avidin-biotin-peroxidase complex, as described in detail elsewhere (franchini and marchetti, 2006). anti-hsp27 polyclonal antibody (pab) (1:500) (santa cruz, ca, usa) was used as the primary antibody. negative control experiments were performed by either omitting the primary antibody or substituting it with non-immune serum. western blot analysis for hsp27 western blot analysis was performed on animals exposed to mfs and on sham-exposed animals (controls). immediately after the exposure to mfs, the animals were frozen at -80 °c for 30 min, then re-suspended in 95 μl of sample buffer (12.5 % 0.5 m tris-hcl ph 6.8, 10 % glycerol, 2 % sds, 0.5 % 2-mercaptoethanol, 0.025 % bromophenol blue) and boiled for 4 min at 1200 rpm in a thermomixer (eppendorf, germany). whole lysates were centrifuged at 13000xg at 4 °c for 30 min, the supernatant was collected, and the protein content quantified following bradford’s method (1976). protein extracts were separated by 12 % sds-polyacrylamide gel electrophoresis (laemmli, 1970) and electrophoretically transferred onto pvdf membranes (0.2-μm pore size). western blots were performed using anti-hsp27 pab (1:500) (santa cruz) and anti-β-tubulin monoclonal antibody (mab) (1:1000) (sigma, mo, usa) as primary antibodies. immunoreactive bands were visualized using a nbt/bcip detection system. densitometric analysis immunoblots were acquired using “gel doc xr” and digitally evaluated with the “quantity one” software (bio rad lab., milano, italy) and “imagej 1.32j” (wayne rasband, national institute of health, usa). statistical analysis statistical analysis of densitometric values was performed by anova. results and discussion the pot worm e. japonensis has normally been used as a model to study the mechanisms of annelid regeneration (myohara et al., 1999, 2006). in the present research, we sought to verify if the animal could also be used as an indicator for ecotoxicological stress, particularly in areas subjected to mf irradiation. previously, our and others laboratories have found interesting invertebrate models from different habitats that are very sensitive to mfs and hsp expression as a result of mf irradiation (lindquist, 1986; goodman and blank, 1998; miyakawa et al., 2001; malagoli et al., 2004). the majority of these investigations examined hsp70 and 90, which are typically related to stress-response (morimoto et al., 1997), but did not evaluate the effects of elf-mf on the small hsps, including hsp27. however, relationships between elf-mf and hsp27 could be of some interest in considering the role of these proteins in cytoskeletal dynamics (dalle donne et al., 2001; mounier and arrigo, 2002), as well as with regards to the suggested involvement of some cytoskeletal components in determining elf-mf effects (gartzke and lange, 2002). as far as the small hsps in 83 fig. 2 pot worm exposed for 2 h to 400 μt elf-mf shows an increased immunoreactivity to anti-hsp27 pab (b) with respect to control (a). nuclei are counterstained with hematoxylin. bar = 20 μm. invertebrates are concerned, miyakawa et al. (2001) demonstrated the presence of the hsp16 gene in c. elegans. working with d. melanogaster, tanguay’s group (2006) found that the family of small hsps is composed of 4 components: hsp22, 23, 26 and 27, localized in different cell compartments, i.e. hsp22 in the mitochondria, hsp23 and hsp26 in the cytosol and hsp27 in the nucleus. in e. japonensis, the hsp27-like molecules are distributed in the cytoplasm of coelomocytes and in epithelial cells of the intestine wall (fig. 1). even if in some specimens exposed to mfs, a higher immunoreactivity was detected compared to shamexposed animals (fig. 2), the hematoxylin-eosin staining did not reveal significant morphological modifications in the cells of treated animals. furthermore, western blot experiments performed on protein extracts of the whole animal failed to evidence any difference between exposed and control specimens (fig. 3). this findings are in agreement with previous data from the bivalve mollusc m. galloprovincialis. the exposure of mussels to elf-mfs in a range of 200-1000 μt induced intensity-correlated effects on immunocyte functionality. however, at 400 μt mfs only transient damage was observed, while the injury became permanent only after exposure to mf intensities ranging from 600 to 1000 t (ottaviani et al., 2002). moreover, hsp70 and 90 were not induced in mussel immunocytes at 400 μt after a single 30 min exposure (malagoli et al., 2004), a result that has also been confirmed by rt-pcr experiments (malagoli et al., 2006). as small hsps exert their cytoprotective role via antioxidant, antiapoptotic and actin-stabilizing properties during cell stress (ciocca et al., 1993; de franco et al., 2004; franklin et al., 2005), we can conclude that a single 2 h exposure to 400 μt elf-mf is not able to influence significantly cell integrity and hsp27 expression in the pot worm e. japonensis. this conclusion is based on western blot experiments performed on pooled animals. however, the immunocytochemical approach performed on single specimens revealed that some animals may be more sensitive to mf irradiation, fig. 3 western blot analysis of hsp-27 immunoreactivity in e. japonensis after 2 h exposure to 400 μt elf-mf (a). immunoreactivity towards βtubulin was adopted as loading control (b). mw = molecular weight standard; c = sham-exposed pot worms; t = exposed pot worms. displaying a high level of hsp27-like material after the elf-mf exposure. acknowledgements we thank prof. m myohara (developmental biology department, national institute of agrobiological sciences, tsukuba, ibaraki, japan) who kindly supplied the enchytraeus japonensis population and prof. e ottaviani (university of modena and reggio emilia, italy) for the critical 84 reading of the manuscript. this work was supported by an italian ministry of the university and the research (miur) grant to dm, and by the italian “istituto superiore per la prevenzione e la sicurezza del lavoro” (ispesl) and ministry of labour, grants cm10/dil/03 and res. project 906 to fg. references bernardini c, zannoni a, turba me, bacci ml, forni m, mesirca p, et al. effects of 50 hz sinusoidal magnetic fields on hsp27, hsp70, hsp90 expression in porcine aortic endothelial cells (paec). bioelectromagnetics 28: 231-237, 2007. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254, 1976. ciocca dr, oesterreich s, chamness gc, mcguire wl, fuqua sa. biological and clinical implications of heat shock protein 27,000 (hsp27): a review. j. natl. cancer inst. 85: 15581570, 1993. dalle-donne i, rossi r, milzani a, di simplicio p, colombo r. the actin cytoskeleton response to oxidants: from small heat shock protein phosphorylation to changes in the redox state of actin itself. free radic. biol. med. 31: 16241632, 2001. de franco db, ho l, falke e, callaway cw. small molecule activators of the heat shock response and neuroprotection from stroke. curr. atheroscler. rep. 6: 295-300, 2004. del re b, bersani f, mesirca p, giorgi g. synthesis of dnak and groel in escherichia coli cells exposed to different magnetic field signals. bioelectrochemistry 69: 99-103, 2006. franchini a, marchetti m. the effects of okadaic acid on enchytraeus crypticus (annelida: oligochaeta). inv. surv. j. 3: 111-117, 2006. franklin tb, krueger-naug am, clarke db, arrigo ap, currie rw. the role of heat shock proteins hsp70 and hsp27 in cellular protection of the central nervous system. int. j. hyperthermia 21: 379-392, 2005. gartzke j, lange k. cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli. am. j. physiol. cell physiol. 283: c1333-c1146, 2002. gobba f, malagoli d, ottaviani e. effects of 50 hz magnetic fields on fmlp-induced shape changes in invertebrate immunocytes: the role of calcium ion channels. bioelectromagnetics 24: 277-282, 2003. goodman em, greenebaum b, marron mt. effects of extremely low frequency electromagnetic fields on physarum polycephalum. radiat. res. 66: 531-540, 1976. goodman em, greenebaum b, marron mt. effects of electromagnetic fields on molecules and cells. int. rev. cytol. 158: 279-338, 1995. goodman r, blank m. magnetic field stress induces expression of hsp70. cell stress chaperones 3: 79-88, 1998. junkersdorf b, bauer h, gutzeit ho. electromagnetic fields enhance the stress response at elevated temperatures in the nematode caenorhabditis elegans. bioelectromagnetics 21: 100-106, 2000. kavaliers m, ossenkopp kp, tysdale dm. evidence for the involvement of protein kinase c in the modulation of morphine-induced 'analgesia' and the inhibitory effects of exposure to 60-hz magnetic fields in the snail, cepaea nemoralis. brain res. 554: 65-71, 1991. laemmli uk. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227: 680-685, 1970. lindquist s. the heat-shock response. annu. rev. biochem. 55: 1151-1191, 1986. malagoli d, gobba f, ottaviani e. 50 hz magnetic fields of constant or fluctuating intensity: effects on immunocyte hsp70 in the mussel mytilus galloprovincialis. bioelectromagnetics 27: 427429, 2006. malagoli d, gobba f, ottaviani e. effects of 50-hz magnetic fields on the signalling pathways of fmlp-induced shape changes in invertebrate immunocytes: the activation of an alternative "stress pathway". biochim. biophys. acta 1620: 185-190, 2003. malagoli d, lusvardi m, gobba f, ottaviani e. 50 hz magnetic fields activate mussel immunocyte p38 map kinase and induce hsp70 and 90. comp. biochem. physiol. 137c: 75-79, 2004. miyakawa t, yamada s, harada s, ishimori t, yamamoto h, hosono r. exposure of caenorhabditis elegans to extremely low frequency high magnetic fields induces stress responses. bioelectromagnetics 22: 333-339, 2001. morimoto ri, kline mp, bimston dn, cotto jj. the heat shock response: regulation and function of heat shock proteins and molecular cheperones. biochemistry 32: 17-29, 1997. morrow g, heikkila jj, tanguay rm. differences in the chaperone-like activities of the four main small heat shock proteins of drosophila melanogaster. cell stress chaperones 11: 5160, 2006. mounier n, arrigo ap. actin cytoskeleton and small heat shock proteins: how do they interact? cell stress chaperones 7: 167-176, 2002. myohara m, niva cc, lee jm. molecular approach to annelid regeneration: cdna subtraction cloning reveals various novel genes that are upregulated during the large-scale regeneration of the oligochaete, enchytraeus japonensis. dev. dyn. 235: 2051-2070, 2006. myohara m, yoshida-noro c, kobari f, tochinai s.fragmenting oligochaete enchytraeus japonensis: a new material for regeneration study. dev. growth differ. 41: 549-55, 1999. ottaviani e, malagoli d, ferrari a, tagliazucchi d, conte a, gobba f. 50 hz magnetic fields of varying flux intensity affect cell shape changes in invertebrate immunocytes: the role of potassium ion channels. 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 3 isj 6: 44-48, 2009 issn 1824-307x short communication seasonal changes in functional parameters of the hemolymph of mytilus galloprovincialis c ciaccia, r fabbrib, m bettia, p rochc, l canesib adisuan, dipartimento di scienze dell’uomo, dell’ambiente e della natura, urbino, italy bdipartimento di biologia, universita` di genova, italy cjru ecosystèmes lagunaires, cnrs-université de montpellier 2-ifremer, france accepted april 7, 2009 abstract in bivalves, many functional parameters show seasonal changes in relation to both abiotic (such as temperature and salinity) and biotic factors (such as gonad maturation, food availability). available data indicate that also immune parameters can show seasonal fluctuations in the marine mussel mytilus spp.. in this work we report data on hemocyte lysosomal membrane stability (lms) and phagocytic activity, as well as on soluble lysozyme activity, in the hemolymph of mussels (mytilus galloprovincialis) collected over a 24 month period in the adriatic sea (2006-2007). the results indicate that all the parameters measured show seasonal fluctuations over the year, with lysozyme activity showing the largest changes. lowest lms values were observed in early winter and early autumn, whereas maximal values of phagocytic activity were observed in winter and increasing serum lysozyme activities were recorded in autumn. the observed seasonal fluctuations are discussed in relation to both abiotic (temperature) and biotic (changes in endogenous modulators) factors. key words: mytilus; hemocytes; lysosomal membrane stability; phagocytosis; lysozyme; immune parameters; seasonal variation introduction bivalves (such are mussels, clams and oysters) possess both cellular and humoral defence mechanisms that co-operate to kill and eliminate invading bacteria (mitta et al., 2000; canesi et al., 2002). hemocytes are responsible for cell-mediated immunity through phagocytosis and various cytotoxic reactions, such as the release of lysosomal enzymes and antimicrobial peptides, and the production of oxygen metabolites (mitta et al., 2000, canesi et al., 2002). in the recent years, we have investigated the immune responses of mytilus galloprovincialis to different stimuli, from bacterial challenge to exposure to endogenous modulators and heterologous cytokines, in both in vitro and in vivo studies (canesi et al., 2001, 2003, 2004, 2005, 2006a, b; betti et al., 2006). although a number of assays can be utilized in order to evaluate the immune function in invertebrates (ballarin et al., 2008), the phagocytic activity is generally considered ______________________________________________________________________ corresponding author: laura canesi department of biology university of genoa corso europa 26, 16132, genoa, italy e-mail: laura.canesi@unige.it as one of the most important parameters, especially in the bivalve mytilus spp. where active phagocytes represent the main circulating cell type (carballal et al., 1998; ottaviani et al., 1998; wottoon et al., 2003). however, another hemocyte parameter, lysosomal membrane stability, evaluated in live hemocytes by the neutral red retention time (nrr) assay, has emerged as an extremely sensitive indicator not only of cellular stress due to environmental perturbations (lowe et al., 1995), but also of the functional status of immunocytes (hauton et al., 2001; pruzzo et al., 2005). in fact, the majority of mytilus hemocytes are endowed with an extremely developed lysosomal vacuolar system which is involved not only in digestion of engulfed foreign particles, but that also contains a number of hydrolases that are secreted for extracellular degradation of components from invading microorganisms, such as bacterial cell wall (canesi et al., 2002). in addition, lysosomal production of oxygen radicals has been demonstrated (winston et al., 1996). in mussels, lms response to a variety of extracellular stimuli has been long widely investigated in a number of studies so that recorded changes can be related to a different functional status of the cell; in particular, moderate lysosomal   44 mailto:laura.canesi@unige.it destabilisation indicates activation of lysosomal membrane fusion processes related to endo/exocytosis, whereas larger decreases in lms correspond to increasing cellular stress conditions that can lead to irreversible cellular damage and autophagy (moore et al., 2006). changes in both lms and phagocytosis are induced by immune stimuli, like bacterial challenge or exposure to cytokines (canesi et al., 2001, 2003; 2005; betti et al., 2006). moreover, we have identified the natural estrogen 17β-estradiol as an endogenous modulator of these parameters, as well as of lysosomal enzyme release and oxyradical production (canesi et al., 2004, 2006b). in bivalves, many functional parameters show seasonal changes in relation to both abiotic (such as temperature and salinity) and biotic factors (such as gonad maturation or food availability). these factors may affect also the immune function. in mytilus spp., only a few studies have been focused on seasonal variations in immune parameters (carballal et al., 1997, 1998; malagoli et al., 2006, 2007; novas et al., 2007). in this work, we report data on hemocyte lms and phagocytic activity, as well as on soluble lysozyme activity in mussels, mytilus galloprovincialis, collected over a 24 month period in the adriatic sea in 2006-2007. materials and methods chemicals all reagents were of analytical grade and were purchased by sigma (st. louis, mo). animals specimens of the bivalve mollusc mytilus galloprovincialis, collected in the cesenatico area (rn, italy) were purchased monthly from a local fishing company (sea, gabicce mare, pu) for two years (from january to december 2006 and 2007). analysis were carried out on individuals of 4-5 cm size. after collection, animals (30 mussels) were taken immediately to the laboratory where they were kept in an aquarium for 24 h in static tanks containing aerated artificial sea water (asw) (1 l/mussel), 36 % psu, at different temperatures (from 15 to 20 °c, depending on the sampling period to minimize the effect of laboratory conditions). hemolymph was sampled from the posterior adductor muscle using a sterile 1 ml syringe with an 18 g1/2” needle. with the needle removed, hemolymph was filtered through sterile gauze and pooled in 50 ml falcon tubes at 18°c. hemolymph samples from 8-10 mussels were pooled and utilised for subsequent analyses. hemolymph serum was obtained by centrifugation of whole hemolymph at 200xg and the supernatant was sterilised through a 0.22 µm pore size filter. all analyses were performed in quadruplicate. lysosomal membrane stability (lms) lms was evaluated by the nrr (neutral red retention time) assay as previously described (canesi et al., 2005) according to lowe et al. (1995). hemocyte monolayers on glass slides were incubated with 30 µl of a neutral red (nr) solution (final concentration 40 mg/ml from a stock solution of nr 40 µg/ml in dmso); after 15 min excess dye was washed out, 30 µl of asw was added, and slides were sealed with a coverslip. every 15 min, slides were examined under an optical microscope and the percentage of cells showing loss of the dye from lysosomes in each field was evaluated. for each time point 10 fields were randomly observed, each containing 8-10 cells. the end point of the assay was defined as the time at which 50 % of the cells showed sign of lysosomal leaking (the cytosol becoming red and the cells rounded). all incubations were carried out at 18 °c. phagocytosis assay phagocytosis of neutral red-stained zymosan was used to assess the phagocytic ability of hemocytes as previously described (canesi et al., 2006b) according to pipe et al. (1995). neutral redstained zymosan in 0.05m tris-hcl buffer (tbs), ph 7.8, containing 2 % nacl was added to each monolayer at a concentration of about 1:50 hemocytes:zymosan diluted in asw, and allowed to incubate for 30 and 60 min at 18 °c. monolayers were then washed three times with tbs, fixed with baker’s formol calcium (4 %, v/v, formaldehyde, 2 % nacl, 1 % calcium acetate) for 30 min and mounted in kaiser’s medium for microscopical examination with a vanox (olympus italy 1.2.1, mi) optical microscope. for each slide, the percentage of phagocytic hemocytes was calculated from a minimum of 200 cells. data are expressed as % of phagocytizing cells. serum lysozyme activity lysozyme activity in aliquots of serum was determined as previously described (pruzzo et al., 2005) following chu and la peyre (1989). briefly, lysozyme activity was determined as the ability to lyse a standard suspension of m. lysodeikticus (15 mg/100 ml in 66 mm phosphate buffer, ph 6.4) and measured as decrease in absorbance at 450 nm at room temperature. hen egg-white (hew) lysozyme was used to construct a standard curve and lysozyme activity was expressed as hew lysozyme equivalents/mg protein/ml. protein content was determined according to the lowry method using bovine serum albumin (bsa) as a standard. data analysis results are presented as the arithmetical mean ± sd of experiments carried out in quadruplicate. statistical analysis was performed by using the mann-whitney u-test with significance at p<0.05. results and discussion mussels were sampled for 24 months during 2006 and 2007. since no significant differences in the results obtained were recorded between the two years, only data from jan-dec 2007 are reported in fig. 1. figure 1a shows the results obtained for hemocyte lms: mean annual values of nr retention times were 117.75 ± 13.6 min. lowest lms (about 100 min) were recorded in winter (janfeb) and early autumn (sept-oct). with respect to these values, significantly higher lms were   45 recorded in spring, with a maximum in may (+39 %; p<0.05) and december + 31 %; p < 0.05). however, both minima and maxima did not significantly differ from mean annual values. data on hemocyte phagocytic activity are reported in fig. 1b as % of phagocytosing cells. mean values (%) were 53.87 ± 5.55. higher values were observed in winter, with a maximum in january (64 %), followed by a slow decrease in spring-early summer, that reached lowest values in june and september (-27 % and 26 %, respectively, with respect to january; p<0.05). however, as observed for lms data, also for phagocytosis neither minima nor maxima were significantly different from mean annual values. in figure 1c data on soluble lysozyme activity are reported. mean annual values were 147.25 ± 68 mu/mg protein. in this case, larger seasonal differences were observed, with lower values in late winter-early spring (less than 100 mu/mg protein from february to april, with a minimum in march of 84±15 mu/mg protein.). a large, progressive rise was observed from late summer to autumn (up to a +150 % increase in october and november with respect to july; p<0.05). maximal values recorded in october and november were also significantly different from mean annual values (+83 % and +100 %, respectively; p<0.05). taken together, the results indicate seasonal fluctuations in the parameters measured in the hemolymph of mussels from the adriatic sea. lowest lms values were observed in early winter and early autumn. on the other hand, maximal values of phagocytic activity were observed in winter and increasing serum lysozyme activities were recorded in autumn. these observations are in line with the fact that decreases in lms are associated with membrane fusion processes during both endo/phagocytosis and release of lysosomal enzymes by exocytosis. however, when data were analysed by the spearman rank correlation test, no significant correlation was observed among the different parameters measured (data not shown). the observed changes in hemolymph functional parameters can be related to both abiotic (such as temperature) and biotic factors (such as reproductive stage and also food availability). in the adriatic sea, fluctuations in hemolymph cytotoxicity of m. galloprovincialis were reported (malagoli et al., 2006, 2007), with two peaks at the end of spring and of summer. however, the temperature apparently was not the main parameter affecting cytotoxicity (malagoli et al., 2007). moreover, no significant correlation was found between changes in immune parameters recorded in the present work and environmental temperature (data not shown). although circulating hemocyte concentration did not show seasonal variations in m. galloprovincialis (carballal et al., 1998), the proportion of different hemocyte subpopulations endowed with different phagocytic activity due to different hemocyte maturation stages may change at different times of the year. since phagocytosis represents one of the main components of the innate immune response, lower phagocytic activities observed in this work during the summer may be fig. 1 seasonal trend of lysosomal membrane stability (lms) (a) and phagocytic activity (b) in hemocytes and of serum lysozyme activity (c) in hemolymph of mussels from adriatic sea. data, representing the mean ± sd of four replicates were analysed by the mann-whitney u test. a) * = p<0.05: may vs january and february; december vs september and october b) * = p<0.05: january vs june and september c) * = p<0.05: july vs october and november responsible for lower immune defence against invading micro-organisms during this period. moreover, the effect of seasonal changes in endogenous modulators (such as neuropeptides, cytokines and sex steroids) should be taken into account. in the hemocytes of m. galloprovincialis, physiological concentrations of the natural estrogen 17β-estradiol can stimulate immune parameters, including phagocytosis, both in vitro and in vivo (canesi et al., 2004, 2006b). in bivalves, 17βestradiol has a role in reproduction: seasonal changes in estrogen content have been observed in   46 different bivalve species in relation to gametogenesis, with increases during gonad maturation and decreases at spawning (osada et al., 2004; gauthier-clerc et al., 2006). however, in the present study, different extents of gonad ripening were observed in different individuals (males and females) throughout the year; although during acclimation in the laboratory a major gamete emission was observed in early spring, minor spawnings were also recorded at different times of the year. the absence of marked seasonal changes in gametogenesis may be partly related to a relatively constant food availability throughout the year in the adriatic sea. in m. galloprovincialis from the northern spanish coast, basal hemocyte no production showed lowest values in winter. however, only in mussels collected in winter il-2 greatly induced no production probably through an immunoreactive enos protein that was expressed only in this period by the hemocytes (novas et al., 2007). these data suggest that, in addition to fluctuations in basal values of immune parameters, changes in the inducibility of the immune response over the year should be considered. our data give a further insight on the seasonal inter-relationship among immune parameters in m. galloprovincialis. accumulation of this knowledge may greatly help understanding and predicting the immune response against potential pathogens, as well as the possible immunotoxic effects of exposure to environmental contaminants in an economically and ecologically relevant species of bivalves. acknowledgements this work was supported by the eu program imaquanim (food-ct-2005-007103) subcontract cnrs 009252. references ballarin l, cammarata m, cima f, grimaldi a, lorenzon s, malagoli d, et al. immuneneuroendocrine biology of invertebrates: a collection of methods. inv. surv. j. 5: 192-215, 2008. betti m, ciacci c, lorusso lc, canonico b, falcioni t, gallo g, et al. effects of tumor necrosis factor alpha (tnfalpha) on mytilus hemocytes: role of stress-activated map kinases. biol cell. 98: 233-244, 2006. canesi l, pruzzo c, tarsi r, gallo g. surface interactions between escherichia coli and the hemocytes of the mediterranean mussel mytilus galloprovincialis lam. leading to efficient bacterial clearance. appl. environ. microbiol. 67: 464-468, 2001. canesi l, gavioli m, pruzzo c, gallo g. bacteriahemocyte interactions and phagocytosis in marine bivalves. microsc. res. tech. 57: 469476, 2002. canesi l, betti m, ciacci c, citterio b, pruzzo c, gallo g. tyrosine kinase mediated cell signaling in the activation of mytilus hemocytes: possible role of stat proteins. biol. cell. 95: 608-618, 2003. canesi l, ciacci c, betti m, lorusso lc, marchi b, burattini s, et al. rapid effect of 17betaestradiol on cell signaling and function of mytilus hemocytes. gen. comp. endocrinol. 136: 58-71, 2004. canesi l, betti m, ciacci c, lorusso lc, gallo g, pruzzo c. interactions between mytilus hemocytes and different strains of escherichia coli and vibrio cholerae o1 el tor: role of kinase-mediated signalling. cell. microbiol. 7: 667-674, 2005. canesi l, betti m, ciacci c, lorusso lc, pruzzo c, gallo g. cell signaling in the immune response of mussel hemocytes. inv. surv. j. 3: 40-49, 2006a. canesi l, ciacci c, lorusso lc, betti m, guarnieri t, tavolari s, et al. immunomodulation by 17βestradiol in bivalve hemocytes. am. j. physiol. integr. resp. comp. physiol. 29: r664-r673, 2006b. carballal mj, lopez c, azevedo c, villalba a. enzymes involved in defense functions of hemocytes of mussel mytilus galloprovincialis. j. invertebr. pathol. 70: 96-105, 1997. carballal mj, villalba a, lopez c. seasonal variation and effects of age, food availability, size, gonadal development, and parasitism on the hemogram of mytilus galloprovincialis. j. invert. pathol. 72: 304-312, 1998. chu fle, la peyre jf. effect of environmental factors and parasitism on hemolymph lysozyme and protein of american oysters (crassostrea virginica). j. invert. pathol. 54: 224-232, 1989. gauthier-clerc s, pellerin j, amiard jc. estradiol17β and testosteorne concentrations in male and female mya arenaria (mollusca bivalvia) during the reproductive cycle. gen. comp. endocrinol. 145: 133-139, 2006. hauton c, hawkins le, hutchinson s. response of hemocyte lysosomes to bacterial inoculation in the oysters ostrea edulis l. and crassostrea gigas (thunberg) and the scallop pecten maximus (l.). fish shellfish immunol. 11: 143153, 2001. lowe dm, fossato vu, depledge mh. contaminantinduced lysosomal membrane damage in blood cells of mussels mytilus galloprovincialis from the venice lagoon: an in vitro study. mar. ecol. prog. ser. 129: 89-196, 1995. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005. inv. surv. j. 3: 1-3, 2006. malagoli d, casarini l, ottaviani e.. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2006: regular changes of cytotoxicity during the year. inv. surv. j. 4: 10-12, 2007. mitta g, vandenbulcke f, roch p. original involvement of antimicrobial peptides in mussel innate immunity. febs lett. 486: 185-190, 2000. moore mn, icarus allen j, mcveigh a. environmental prognostics: an integrated model supporting lysosomal stress responses as predictive biomarkers of animal health status. mar. environ. res. 61: 278-304, 2006.   47 http://www.isj.unimo.it/articoli/isj176.pdf http://www.isj.unimo.it/articoli/isj176.pdf http://www.isj.unimo.it/articoli/isj176.pdf http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids=16060858&query_hl=1 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids=16060858&query_hl=1 http://www.ncbi.nlm.nih.gov/pubmed/16343609?ordinalpos=10&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum novas a, barcia r, ramos-martinez ji. nitric oxide production by haemocytes from mytilus galloprovincialis shows seasonal variations. fish shellfish immunol., 23: 886-891, 2007. osada m, tawarayama h, morii k. estrogen synthesis in relation to gonadal development of japanese scallop patipecten yeasoensis: gonadal profile and immunolocalization of p450 aromatase and estrogen. comp. biochem. physiol. 139b: 123-128, 2004. ottaviani e, franchini a, barbieri d, kletsas d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two aging-related hemocyte stages. it. j. zool. 65: 349-354, 1998. pipe r, coles ja, farley sr. assays for measuring immune response in the mussel mytilus edulis. in: stolen j, fletcher tc, smith sa, zelikoff jt, kaattari sl, anderson, rs (eds), techniques in fish immunology: fish immunology technical communications, immunological and pathological techniques of aquatic invertebrates, vol. 4, sos publications, fair haven nj, pp 93-100, 1995. pruzzo c, gallo g, canesi l. persistence of vibrios in marine bivalves: the role of interactions with hemolymph components. environ. microbiol. 7: 761-772, 2005. winston gw, moore mn, kirchin ma, soverchia c. production of reactive oxygen species by hemocytes from the marine mussel mytilus edulis: lysosomal localization and effect of xenobiotics. comp. biochem. physiol. 113c: 221-229, 1996. wootton ec, dyrynda ea, ratcliffe n.a. bivalve immunity: comparison between the marine mussel (mytilus edulis), the edible cockle (cerastoderma edule) and the razor-shell (ensis siliqua). fish shellfish immunol. 15: 195210, 2003.   48 isj104.pdf 82 isj 2: 82-90, 2005 issn 1824-307x review mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides (hypothesis) o schmidt1, mm rahman1, g ma1, u theopold2, y sun3, m sarjan4, m fabbri5, h roberts1 1insect molecular biology, faculty of sciences (waite campus), university of adelaide, glen osmond, sa 5064, australia 2 department of molecular biology and functional genomics, stockholm university, s-10691 stockholm, sweden 3 the biotechnology research center, shanxi academy of agricultural sciences, 64 n.nongke, taiyuan, shanxi 030031, china 4 faculty of agriculture, university of mataram, lombok, indonesia 5 department of experimental oncology, european institute of oncology, i-20141 milan, italy accepted july 1, 2005 abstract antimicrobial peptides and pore-forming toxins are important effectors in innate immune defence reactions. but their mode of action, comprising the insertion into cholesterol-containing membranes is not known. here we explore the mechanical implications of pore-formation by extracellular protein assemblies that drive cellular uptake reactions by leverage-mediated (lm) processes, where oligomeric adhesion molecules bent membrane-receptors around ‘hinge’-like lipophorin particles. the interactions of antimicrobial peptides, pore-forming toxins and biologically active proteins with lmassemblies provide a new paradigm for the configurational specificity and sterical selectivity of biologically active peptides. key words: antimicrobial peptides; pore-forming toxins; peptide hormones; endocytosis; lipophorin; cholesterol; lipid rafts; channel formation; lectins introduction antimicrobial proteins are effectors of innate immunity with unique structural properties mediating mainly pore-forming activities in membranes (boman, 2000). since its discovery more than thirty years ago (boman et al., 1974; faye et al., 1975), peptides with antibacterial activities have been divided into a number of different categories (boman, 2003), including alpha-helical peptides, peptides with cysteine bonds and peptides enriched in one or more amino acids. a unique feature of anti-microbial peptides is their ability to permeate and disrupt target membranes (shai, 2002). corresponding author: otto schmidt insect molecular biology, faculty of sciences (waite campus), university of adelaide, glen osmond, sa 5064, australia e-mail: otto.schmidt@adelaide.edu.au antimicrobial peptides are believed to kill microorganisms via non-receptor-mediated mechanisms, although some peptides, such a nisin z bind to bacterial cell wall components (breuking et al., 1999). according to (shai, 2002) monomeric peptides that have random structures gain amphipathic structures and form oligomers in solution such that the hydrophobic regions are buried in the lumen of the oligomer and the hydrophilic regions are exposed to the solution. upon reaching the membrane the organization is reversed. the hydrophobic regions are exposed to the lipid constituents of the membrane, and the hydrophilic regions are either segregated in the lumen of the oligomer (if the peptide oligomerizes and inserts into the membrane via the ‘barrel’ mechanism (ehrenstein and lecar, 1977), or exposed to the solution (if the peptides lay on the surface of the membrane and insert via the ‘carpet’ mechanism (pouny and shai, 1992). two different mechanisms of peptide insertion have been implicated in related antimicrobial peptides (chen et al., 2003), where cecropin b molecules, (with one amphipathic and one hydrophilic á-helix) may be inserted into the 83 membrane in a concentration-dependent ‘barrel-stave’ mechanism, whereas synthetic cecropin b3 molecules (with two hydrophobic á-helices) may disrupt the membrane by a ‘carpet-like’ lysis mechanism. although many peptide properties have been described within the framework of the two models, a fundamental question remains: why are these peptides specific to bacterial membranes, but not damaging to most eukaryotic membranes? one argument is that the positive net charge of antibacterial peptides enables binding and permeation of negatively charged phospholipid membranes of bacteria but not to zwitterionic membranes, which are the major constituents of the outer leaflet of erythrocytes and other eukaryotic membranes (shai, 2002). but there are important exceptions, which suggest that it is not the charge alone, but that the secondary and tertiary structure of the peptide is also crucial for insertion into the membrane. this is apparent in some antimicrobial peptides that are active in bacteria and in mammalian cells, such as melittin, its hybrid cecropin a permutations and its diastereomeric analogs (merrifield et al., 1995b). it appears that neither the direction of the peptide bond, nor the turn of the helix in d-enantiomers (merrifield et al., 1995a) interferes with the membrane-lytic mechanism of the peptides (boman, 2003). this and other examples (staubitz et al., 2001) suggest that chiral and sequence-specific determinants are not required for membrane-disrupting activity, while target specificity is strongly influenced by the overall physico-chemical nature of the analogues (merrifield et al., 1995b; staubitz et al., 2001). apart from the observation that changes in a peptide’s sequence are more likely to destroy its activity to eukaryotic than to prokaryotic cells, there are no identifiable amino acid sequences that are responsible for the specificity (hancock and rozek, 2002). another argument put forward to explain peptide-specificities against bacterial membranes is that cholesterol, which is present in eukaryotic membranes, protects against the action of antibacterial peptides (boman, 2003). although this has been confirmed in artificial membrane systems, it raises the question of why a large portion of antibacterial peptides, such as defensins (hoffmann and reichart, 2002), dermaseptins (shai, 2002), cathelicidines (zanetti et al., 1997), pardaxin analogs (shai, 2002), and tachystatin (osaki et al., 1999) are also active against fungal membranes, which contain ergosterol. also difficult to understand in the context of current models are the non-lytic modes of action, such as the transfer of some peptides through the membrane into the underlying cytoplasm, where active peptides interfere with a diverse range of metabolic processes (hancock and rozek, 2002). in fact, some argue that the lytic action may not be the primary cause of death for bacteria (boman, 2003), since antibacterial peptides may have already irreversibly impaired the viability of bacteria before the apparent disruption of the membrane. membrane trafficking – an achilles’ heel? although most experiments are performed with artificial membrane systems comprising regularshaped vesicles, it is very likely that in vivo membrane disruptions by peptides are instigated during lipidbilayer disturbances, such as cellular processes involving extreme membrane curvature and membrane fusion. this is relevant for membrane trafficking in higher organisms, but also applies to prokaryotes during cell division. the in vitro studies mentioned above suggest that membrane lipid composition is crucial for susceptibility to peptide insertion. artificial membranes lacking cholesterol are more susceptible to peptide insertions than cholesterol-containing membranes (boman, 2003). furthermore, insertion of amphiphilic molecules forming nanotubes into cholesterol-free lipid bilayers has been modelled around hydrophobic-hydrophilic matching using molecular dynamics simulations (lopez et al., 2004). thus the presence of cholesterol and sphingolipids appear to be a major barrier for the insertion of peptides into the membranes. this may explain why bacteria are susceptible to most peptides, while animals and plants are not. in this context it is interesting to note that higher organisms with intensive membrane trafficking perform non-clathrin mediated uptake reactions in special membrane domains, called ‘lipid rafts’, which represent accumulations of cholesterol and sphingolipids (brown and london, 1998; ikonen, 2001). the fact that uptake reactions involve reorganisation of the membrane bilayer makes membrane trafficking vulnerable to peptide attacks. but if these membrane domains are perceived to protect against pore-forming toxins, why do many toxins interact with receptors in lipid rafts (kurzchalia, 2003; zhuang et al., 2002)? is it possible that poreforming toxins and channel-forming peptides exploit the protein machinery of cellular uptake reactions to achieve insertion into the membrane? pore-forming toxins an alternative mechanism of channel formation involves the membrane insertion of pore-forming toxins with â-barrel peptides (lesieur et al., 1997) by putative leverage-mediated (lm) uptake reactions (schmidt and theopold, 2004). in line with the model, receptors are assembled around ring-shaped lipoproteins, such as lipophorin or hexamerin. oligomeric adhesion molecules, such as lectins, cross-link receptors using membrane-distal mucindomains thereby bending receptors around hinge-like lipoproteins. in this model the configuration of the complex provides the leverage to curve the membrane and internalise receptors in a cluster of lm-complexes affecting cell shape changes (schmidt and theopold, 2004) and adhesive properties of the cell (schmidt and schreiber, in press). since many pore-forming toxins, such as crystal toxins from bacillus thuringiensis (bt-toxins), are lectins, the insertion into the membrane may be mediated by an lm-uptake reaction (schmidt and theopold, 2004). in the lm-scenario, the ring-shaped pore complex is formed before or during the assembly of receptors (fig. 1), which are bend around the hingelike lipoprotein by the oligomeric pore-forming complex (fig. 2). this is in contrast to the current assumption that bt-toxin molecules are inserted into the membrane as monomers by a receptor-mediated 84 fig. 1 schematic illustration of receptor-assemblies with the potential to provide configurational energy by leverage-mediated mechanisms. a) lipoproteins, such as lipophorin, contain ring-shaped proteins filled with lipids (canavoso et al., 2001), including phospholipids (green), cholesterol (blue) and diacylglycerols (yellow). lipophorin particles can bind to membrane-bound receptors to form lipophorin complexes (theopold and schmidt, 1997). alternatively, lectins may bind to glycolipids or glycoproteins on lipid particles to form lectinlipophorin complexes, before interacting with receptors. (glycodeterminants are indicated by black dots) b) formation of a receptor complex with the potential to create configurational energy by a leverage-mediated (lm) process. 1b 1a 85 fig. 2 schematic illustration of leverage-mediated uptake reactions. lm-uptake reactions are mediated by oligomeric adhesion molecules, such as lectins. a) assemblies, consisting of lipoproteins and multimeric lectins, interact with membrane-anchored molecules, such as membrane receptors, lipid anchored glycoproteins or glycolipids molecules. as a result of the lm-process the lipid particle is pushed into the underlying membrane thereby ‘unloading’ the lipophorin without the need to internalize the complex. note that multiple lm-complexes predict membrane domains enriched in particle-derived lipids, such as cholesterol that may alter the local composition and protect the membrane against antibacterial peptide attack. b) putative ‘shuttle’ mechanism: if the lipophorin particle changes shape in the process of lipid unloading, the resulting complex may not support a leverage-mediated uptake reaction and the complex unravels before internalization occurs. the unloaded lipophorin particles may then be released to become loaded on the gut membrane by an unknown process. 2a 2b 86 fig. 3 insertion of pore-forming toxins into the membrane by a putative lm-mechanism. a) lm-complex comprising oligomeric lectins containing amphipathic alpha-helices (red), which can engage in uptake reactions like other lectins. b) leverage-mediated uptake reactions may push the amphipathic peptide into the lipid layer opening a membrane gap to the cytoplasm. this allows ions and water to pass from the endosome into the cytoplasm, causing osmofragility in some lectins (pande et al., 1998) and pores in endotoxins. note that the poreforming peptides can be covalently attached to the oligomeric adhesion molecule (e.g. pore-forming toxin, such as bt-toxin). alternatively, the active peptide may be assembled with the lm-complex without being covalently attached to any components by its space-filling properties. in this case the active peptide may be toxic and form a pore, such as mellittin, or interfere with lm-functions by altering cellular processes and signalling functions as observed with biologically active peptides. 3a 3b 87 reaction and only assembled into pore-forming oligomeric complexes once inside the membrane bilayer (de maagd et al., 2001). however, it has been shown that some mature bt-toxins form tetrameric complexes when processed in vitro (ma et al., 2005), which would enable multiple interactions with receptors and lipoproteins before membrane insertion. another intriguing observation is that some plant lectins increase osmofragility (pande et al., 1998), which in itself is not damaging to membranes, but may provide clues for our understanding of how poreforming toxins are inserted into the membrane (schmidt and theopold, 2004). since many poreforming toxins are lectins, which recognise mucin-like glycoprotein-receptors (armstrong et al., 1996; kuwahara et al., 2000) and glycolipids (griffitts et al., 2005), such ring-shaped adhesion complexes could potentially be internalised by an lm-mechanism (schmidt and theopold, 2004), where the structural features of the complex predict a disruption of the lipid bilayer (fig. 2). in this context the structure of pore-forming toxins can be viewed as oligomeric lectins that have amphipathic peptides with antibacterial properties covalently attached. indeed, secondary structure predictions, helical wheel/net diagrams and molecular mechanics calculations of membrane-inserting peptides from the bt-toxin, form a strongly amphiphilic alpha-helix and show haemolytic activity in vitro comparable to that of bee venom peptide melittin (szabo et al., 1993). similar results were obtained with the isolated á4-loop-á5 hairpin from the bt-toxin, which showed that this peptide is extremely active compared to the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes (gerber and shai, 2000). the concept that pore-forming toxins are oligomeric adhesion molecules with covalently attached antibacterial peptides is compatible with the idea that antibacterial peptides exploit lm-uptake reactions and are inserted into the membrane together with oligomeric adhesion molecules. a prediction of this model is that some antibacterial peptides can potentially overcome the cholesterol barrier of the membrane by assembling into the lm-machinery and become inserted into the membrane during lm-reactions. the fact that antibacterial peptides targeting eukaryotic cells are attached to oligomeric adhesion molecules, whereas peptides targeting prokaryotes are not, could indicate that peptides are effective in prokaryotes without the help of lm-mechanisms. alternatively, prokaryotic uptake mechanisms may be different (e.g. in the absence of cholesterol) and less selective against active peptides. lipid exchange in higher organisms cholesterol is transported between cells by a ring-shaped protein complex of apolipophorins that stabilize a mix of other hydrocarbons, such a phospholipids and diacylglycerols (dags). these lipid particles are in general internalised by cells via endocytosis reactions, except in insects, where lipophorin particles can act as a reusable shuttle by taking up lipid and delivering it to target tissues without internalization and degradation of the particle. for example, a single lipophorin particle, synthesized in the fat body, can take up dietary lipid at the midgut in the form of dag, diffuse through the hemolymph to the fat body, and deliver the dag for storage in the fat body without being internalised and degraded. this same particle can then return to the midgut surface and repeat the process (canavoso et al., 2001). how the lipophorin complex is able to release cholesterol and other lipids into the underlying cell membrane is not known. one of the possible implications of the lm-model is that an lm-uptake mechanism may provide the extracellular energy that is required to merge zwitterionic lipid layers. in this scenario, the lipid-loaded lipophorin complex is pushed into the membrane as receptors form linkages with an oligomeric adhesion molecule (fig. 2). while the nature and identity of receptors and adhesion molecules involved in lipophorin unloading is not known at this stage, recent observation suggest that lipophorin particles carry glycolipids and lipidanchored glycoproteins that can interact with lectins and membrane receptors. for example, when lectins were mixed with cell-free hemolymph from lepidopteran insects and lipophorin separated on density gradients, lectins were enriched in the lipophorin fractions (sarjan, 2002). conversely when lipophorin fractions where analysed on western blots with lectins a number of glycoproteins were found to be co-purified with lipophorin particles. one of these proteins, a ca 50 kda protein was identified (fabbri, 2003) and shown to belong to a group of chitinase-like molecules known as imaginal disc growth factors (idgfs) (asgari and schmidt, 2004) with possible lectin-like properties (homma et al., 1996; kawamura et al., 1999; li and aksoy, 2000) that are conserved in other invertebrates (akalal and nagle, 2001) and vertebrates (riazi et al., 2000). more recently, lipidanchored morphogens, such as wingless and hedgehog, have been found in association with drosophila lipophorin particles and shown to be morphogen carriers in the extracellular space of imaginal discs (panakova et al., 2005). it is therefore conceivable that lipophorin particles are pushed onto the membrane by functional lmcomplexes, and in the process may allow the lipid moieties to mix, releasing the lipids into the underlying membrane (fig. 2a). in this context a reversible shuttle function of lipophorin particles is possible if apolipophorin changes its conformation as a result of lipid depletion of the particle. in this case the hinge-like properties of lipophorin may be lost, disrupting the internalisation process and releasing the unloaded lipophorin (fig. 2b). while this is hypothetical, the importance of a lipophorin shuttle based on lm-mechanisms is that it makes specific predictions that can be experimentally tested. for example, mature crystal toxin from b. thuringiensis was co-purified with lipophorin particles on density gradients (fig. 4). since apolipophorin is not recognised by the toxin on western blots this implies that the toxin binds to other glycodeterminants, such as lipid-anchored glycoproteins (luo et al., 1999; panakova et al., 2005) and glycolipids (griffitts et al., 2005; hatakeyama et al., 1999; inoue et al., 2001; nedelkoska and benjamins, 1998; sandvig et al., 88 fig. 4 low density gradient of cell-free hemolymph (plasma) from galleria mellonella larvae mixed with mature bt-toxin (cry1ac). aliquots of fractions were analysed on western blots using anti-cry1ac antibodies. a 69 kda monomer was found predominantly in high density fractions (arrowhead) together with a minor 60 kda protein resulting from over-digestion of the pro-toxin. in addition, monomers were also found in low-density regions of the gradient between fractions 11 and 14, where apolipophorin i and ii subunits peak in addition to a minor peak between fractions 16 and 20. apart from monomers, oligomeric forms of the toxin, such as trimers and tetramers, are enriched in the lipophorin fractions (sarjan, 2002). it is not clear whether pre-existing oligomers preferentially bind to lipophorin particles or whether monomers are bound to particles and form oligomers on the particle as observed in other systems (park et al., 2005). 1989). although the identity of lectin-binding proteins from lipophorin particles in the gut lumen remains to be determined, this observation could suggest toxicity mechanisms at the gut lining involving lipophorinmediated lipid exchange or uptake. conversely, the fact that lipophorin (li et al., 2002) and other lipid carrying storage proteins (ma et al., 2005) are known to be involved in coagulation reactions could potentially be responsible for the observed aggregation reactions in the gut lumen (ma et al., 2005). the sequestration of toxin molecules in the gut lumen by immune-related coagulation reactions can also explain the observed tolerance to the toxin by immune induction (rahman et al., 2004; gunning et al., 2005; ma et al., 2005). configurational specificity if lm-uptake reactions are the driving force for the insertion of pore-forming complexes into membranes, the functioning of such multi-protein complexes will depend on the correct configurational compilation of the protein assembly. for example, only oligomeric adhesion molecules that are able to generate leverage by interacting with membrane receptors across a hinge-like protein will be able to curve the membrane (fig. 2a), a prerequisite for the insertion of amphipathic peptides into the membrane (fig. 3). in this context the role and specificity of antibacterial peptides that are inserted into cholesterol-containing membranes may depend on structural requirements, which allow the peptide to intercalate into gaps provided by the lm-assemblies comprising oligomeric adhesion molecules, membrane-receptors and hingelike proteins (fig. 3a), without damaging the functionality of the complex. this implies that the observed peptide-specificity may be based on spacefilling rather than protein-binding properties and that biologically active peptides are able to specifically interact with lm-assemblies without the need to bind to any individual proteins or receptors. the outcome of this interaction may be the formation of a damaging pore, but also non-toxic ion flux or alteration of the lmuptake process, which can modify cell behavior and signaling. in fact the evolution of eukaryotic protein assemblies with lm-uptake properties may have been under selection pressure to protect against peptides with lytic properties, which makes cholesterolcontaining lipoproteins attractive components in the process. if the lipid exchange during uptake reduce the toxicity of some antibacterial peptides and instead reduce the effect to modulate lm-uptake and signaling reactions, this may explain, why some antibacterial peptides function as biologically active peptide in vertebrates, such as vaso-active intestinal peptide hormones (vip), pr-39 and other non-lytic peptides (boman, 2003). in summary, we propose that toxin-producing pathogens exploit a cellular uptake mechanism involving receptor rearrangements by a leveragemediated process. the mechanical and configurational specifications of these lm-assemblies predict new types of protein specificity based on space-filling properties rather than protein-binding interactions. references akalal dbg, nagle gt. mollusk-derived growth factor: cloning and developmental expression in the central nervous system and reproductive tract of aplysia. mol. brain res. 91: 163-168, 2001. armstrong pb, swarnakar s, srimal s, misquith s, hahn ea, aimes rt, quigley jp. a cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins. j. biol. chem. 271: 14717-14721, 1996. asgari s, schmidt o. isolation of an imaginal disc growth factor homologue from pieris rapae and its expression following parasitization by cotesia rubecula. j. insect physiol. 50: 687-694, 2004. boman hg. innate immunity and the normal microflora. immunol. rev. 173: 5-16, 2000. boman hg. antibacterial peptides: basic facts and emerging concepts. j. intern. med. 254: 197-215, 2003. 1 2 3 4 5 6 7 8 9 bt 10 11 12 13 14 15 16 17 18 19 1.1 1.3 1 2 3 4 5 6 7 8 9 bt 10 11 12 13 14 15 16 17 18 19 89 boman hg, nilsson-faye ipk, rasmuson t. insect immunity. i characteristics of an inducible cell-free antibacterial reaction in haemolymph of samia cynthia pupae. infect. immun. 10: 136-145, 1974. breuking e, wiedemann i, van kraaij c, kuipers op, sahl h, de kruijff b. use of the cell wall precursor lipid ii by a pore-forming peptide antibiotic. science 286: 23612364, 1999. brown da, london e. functions of lipid rafts in biological membranes. annu. rev. cell dev. biol. 14: 111-136, 1998. canavoso le, jouni ze, karnas kj, pennington je, wells ma. fat metabolism in insects. annu. rev. nutr. 21: 2346, 2001. chen hm, leung kw, thakur nn, tan a, jack rw. distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin b, b1 and b3. eur. j. biochem. 270: 911-920, 2003. de maagd r, bravo a, crickmore, n. how bacillus thuringiensis has evolved specific toxins to colonize the insect world. trends genet. 17: 193-199, 2001. ehrenstein g, lecar h. electrically gated ionic channels in lipid bilayers. q. rev. biophys. 10: 1-34, 1977. fabbri m. evolutionary and functional relationships of insect immune proteins. university of adelaide, adelaide, 2003. faye i, pye a, rasmuson t, boman hg, boman ia. insect immunity. ii simultaneous induction of antibacterial activity and selective synthesis of some hemolymph proteins in diapausing pupae of hyalophora cecropia and samia cynthia. infect. immun. 12: 1426-1438, 1975. gerber d, shai y. insertion and organization within membranes of the delta-endotoxin pore-forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide. j. biol. chem. 275: 2360223607, 2000. griffitts js, haslam sm, yang t, garczynski sf, mulloy b, morris h, cremer ps, dell a, adang mj, aroian rv. glycolipids as receptors for bacillus thuringiensis crystal toxin. science 307: 922-925, 2005. gunning rv, dang ht, kemp fc, nicholson ic, moores gd. new resistance mechanism in helicoverpa armigera threatens transgenic crops expressing bacillus thuringiensis cry1ac toxin. appl. environ. microbiol. 71: 2558-2563, 2005. hancock rew, rozek a. role of membranes in the activities of antimicrobial cationic peptides. fems microbiol. lett. 206: 143-149, 2002. hatakeyama t, sato t, taira e, kuwahara h, niidome t, aoyagi h. characterization of the interaction of hemolytic lectin cel-iii from the marine invertebrate, cucumaria echinata, with artificial lipid membranes: involvement of neutral sphingoglycolipids in the poreforming process. j. biochem. 125: 277-284, 1999. hoffmann ja, reichart jm. drosophila innate immunity: an evolutionary perspective., nat. immunol. 3: 121-126, 2002. homma k, matsushita t, natori s. purification, characterization, and cdna cloning of a novel growth factor from the conditioned medium of nih-sape-4, an embryonic cell line of sarcophaga peregrina (flesh fly). j. biol. chem. 271: 13770-13775, 1996. ikonen e. roles of lipid rafts in membrane transport. curr. opin. cell biol. 13: 470-477, 2001. inoue k, fujinaga y, honke k, arimitsu h, mahmut n, sakaguchi y, ohyama t, watanabe t, oguma k. clostridium botulinum type a haemagglutinin-positive progenitor toxin (ha(+)-ptx) binds to oligosaccharides containing gal beta 1-4glcnac through one subcomponent of haemagglutinin (ha1). microbiology 147: 811-819, 2001. kawamura k, shibata t, saget o, peel d, bryant pj. a new family of growth factors produced by the fat body and active on drosophila imaginal disc cells. development 126: 211-219, 1999. kurzchalia t. anthrax toxin rafts into cells. j. cell biol. 160: 295-296, 2003. kuwahara h, funada t, hatakeyama t, aoyagi h. effects of chemical modification of carboxyl groups in the hemolytic lectin cel-iii on its hemolytic and carbohydrate-binding activities. biosci. biotechnol. biochem. 64: 1278-1281, 2000. lesieur c, vecseysemjen b, abrami l, fivaz m, vandergoot fg. membrane insertion the strategies of toxins. mol. membr. biol. 14, 45-64, 1997. li d, scherfer c, korayem am, zhao z, schmidt o, theopold u. insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade. insect biochem. mol. biol. 32: 919-928, 2002. li s, aksoy s. a family of genes with growth factor and adenosine deaminase similarity are preferentially expressed in the salivary glands of glossina m. morsitans. gene 252: 83-93, 2000. lopez cf, nielsen so, moore pb, klein ml. understanding nature's design for a nanosyringe. proc. natl. acad. sci. usa 101: 4431-4434, 2004. luo k, mclachlin jr, brown mr, adang mj. expression of a glycosyl phosphatidylinositol-linked manduca sexta aminopeptidase n in insect cells. protein expr. purif. 17: 113-122, 1999. ma g, roberts h, sarjan m, featherstone n, lahnstein j, akhurst r, schmidt o. is the mature endotoxin cry1ac from bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant helicoverpa armigera larvae? insect biochem. mol. biol. 35: 729739, 2005. merrifield el, mitchell s, ubach j, boman h, andreu d, merrifield r. d-enantiomers of 15 residue cecropin amellitin hybrids. int. j. pept. protein res. 46: 214-220, 1995a. merrifield rb, juvvadi p, andreu d, ubach j, boman a, boman h. retro and retroenantio analogs of cecropinmelittin hybrids. proc. natl. acad. sci. usa 92: 34493453, 1995b. nedelkoska l, benjamins ja. binding of cholera toxin b subunit a surface marker for murine microglia but not oligodendrocytes or astrocytes. j. neurosci. res. 53: 605-612, 1998. osaki t, omotezako m, nagayama r, hirata m, iwanaga s, kasahara j, hattori j, ito i, sugiyama h, kawabata s. horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins. j. biol. chem. 274: 26172-26178, 1999. panakova d, sprong h, marois e, thiele c, eaton s. lipoprotein particles are required for hedgehog and wingless signalling. nature 435: 58-65, 2005. pande ah, sumati n, hajela n, hajela k. carbohydrate induced modulation of cell membrane vii binding of exogenous lectin increases osmofragility of erythrocytes. febs lett. 427: 21-24, 1998. park k-h, yang h-b, kim h-g, lee y-r, hur h, kim j-s, koo b-s, han m-k, kim j-h, jeong y-j, kim j-s. low density lipoprotein inactivates vibrio vulnificus cytolysin through the oligomerization of toxin monomer. med. microbiol. immunol. 194: 137-141, 2005. pouny y, shai y. interaction of d-amino acid incorporated analogs of pardaxin with membranes. biochemistry 31, 9482-9490, 1992. rahman mm, roberts hls, sarjan m, asgari s, schmidt o. induction and transmission of bacillus thuringiensis tolerance in the flour moth ephestia kuehniella. proc. natl. acad. sci. usa 101: 2696-2699, 2004. riazi ma, brinkman-mills p, nguyen t, pan hq, phan s, ying f, roe ba, tochigi j, shimizu y, minoshima s et al. the human homolog of insect-derived growth factor, 90 cecr1, is a candidate gene for features of cat eye syndrome. genomics 64: 277-285, 2000. sandvig k, olsnes s, brown je, petersen ow, van deurs b. endocytosis from coated pits of shiga toxin: a glycolipid-binding protein from shigella dysenteriae 1. j. cell biol. 108: 1331-1343, 1989. sarjan m. resistance against endotixin from bacillus thuringiensis endotoxins in lipidopteran insects. university of adelaide, adelaide, 2002. schmidt o, schreiber a. intregration of cell adhesion reactions a balance of forces? j. theor. biol. (in press). schmidt o, theopold u. an extracellular driving force of endocytosis and cell-shape changes (hypothesis). bioessays 26: 1344-1350, 2004. shai y. mode of action of membrane active antimicrobial peptides. biopolymers 66: 236-248, 2002. staubitz p, peschel a, nieuwenhuizen wf, otto m, götz f, jung g, jack rw. structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin. j. pept. sci. 7: 552-564, 2001. szabo e, murvai j, fabian p, fabian f, hollosi m, kajtar j, buzas z, sajgo m, pongor s, asboth b. is an amphiphilic region responsible for the haemolytic activity of bacillus-thuringiensis toxin? int. j. pept. protein res. 42: 527-532, 1993. theopold u, schmidt o. helix pomatia lectin and annexin v, molecular markers for hemolymph coagulation. j. insect physiol. 43: 667-674, 1997. zanetti m, gennaro r, romeo d. the cathelicidin family of antimicrobial peptide precursors: a component of the oxygen-independent defense mechanisms of neutrophils. ann. ny acad. sci. 832: 147-162, 1997. zhuang m, oltean di, gomez i, pullikuth ak, soberon m, bravo a, gill ss. heliothis virescens and manduca sexta lipid rafts are involved in cry1a toxin binding to the midgut epithelium and subsequent pore formation. j. biol. chem. 277: 13863-13872, 2002. may 4, 2010 isj 7: 165-180, 2010 issn 1824-307x review role of α2-macroglobulin in the immune responses of invertebrates pb armstrong marine biological laboratory, 7 mbl street, woods hole, ma 02543, and department of molecular and cellular biology, university of california, davis, ca 95616, usa accepted june 30, 2010 abstract although proteases play essential roles in the lives of all organisms, they are also important agents of disease and pathogenesis in metazoans. most notably, proteases are essential virulence factors for a broad array of prokaryote and eukaryote parasites. one strategy used by the immune system of metazoans to defend against parasitic attack is to neutralize the toxins and essential virulence factors that allow the parasite to gain entry to the host and to survive and proliferate in the internal environment of the metazoan host. the particular defense system of interest to the present review is the system of endogenous protease inhibitors that operate to inactivate the secreted proteases utilized by invading parasites during the infection cycle within the host. protease inhibitors are of two broad classes, active-site inhibitors that bind to and inactivate the active sites of target proteases and the α2-macroglobulin class of inhibitors that operate as opsonins to bind and mark proteases in a manner that allows the subsequent endocytosis and intracellular proteolytic degradation of the α2-macroglobulin-protease complex. members of the α2-macroglobulin class of inhibitors interact with target proteases by the novel process of enfolding the protease into a pocket within the interior of the α2-macroglobulin molecule, which is followed by binding of the complex to the α2-macroglobulin receptor at the surfaces of macrophages and other endocytotic cells and its endocytosis and degradation. in contrast to the active-site protease inhibitors, each of which is specialized to interact with a small subset of all endopeptidases, the α2-macroglobulin inhibitors are remarkably promiscuous, binding proteases of all enzymatic classes and origins. this characteristic allows α2-macroglobulin to play an important role in immune defense because this one protein is capable of binding and neutralizing the diverse array of proteases that function as virulence factors of the diverse array of parasites out there in the environment of metazoa. key words: α2-macroglobulin; thiol ester protein family; protease inhibitor; innate immunity introduction an important barrier to the successful reproduction of metazoans is the disease and premature death that attends the attack by parasites. barriers to parasitic attack are the integuments, which limit the range of parasites that can gain entry to the internal milieu, and the immune system, which defends against parasites and their toxic products both at the surface and in the internal spaces. parasites may be unicellular or multicellular, prokaryote, eukaryote, or virus. in coelomate animals, the most important organ of the ___________________________________________________________________________ corresponding author: peter b armstrong department of molecular and cellular biology university of california, davis, ca 95616-8535, usa e-mail: pbarmstrong@ucdavis.edu 165 immune system is the blood, presumably because the blood has ready access to all parts of the body and is best prepared to concentrate defense effectors at sites of pathogen invasion. this review will be concerned with one of the important immune effector proteins found in the plasma of all major taxa of metazoans, α2-macroglobulin. α2-macroglobulin is an evolutionarily conserved element of the innate immune system whose bestcharacterized function is the clearance of active proteases from the tissue fluids. proteases have a diverse set of essential roles in the lives of eukaryotes, including protein digestion, the activation of peptide hormones and other effector proteins that are secreted as zymogens, the physiological turnover of intracellular proteins, the removal of effete extracellular proteins, and mailto:pbarmstrong@ucdavis.edu 166 remodeling of the extracellular matrix (lopez-otin and bond, 2008). however, in addition, proteases, whether of endogenous or exogenous origin, are capable of considerable mischief when free in the blood and tissue spaces. they are important agents in a variety of connective tissue diseases (perlmutter and pierce, 1989), contribute to neoplastic invasion and metastasis (deryugina and quigley, 2006; kessenbrock et al., 2010), and are important virulence factors contributing to the pathogenicity of prokaryotic and eucaryotic parasites (armstrong, 2006; mckerrow et al., 2006). in response to protease challenge, animals have evolved a diverse array of protease inhibitors (travis and salvesen, 1983). in mammals, approximately 3-5 % of the plasma proteins are protease inhibitors (laskowski and kato, 1980) and in the american horseshoe crab, limulus polyphemus, the hemolymph protease inhibitor, α2macroglobulin, is the third most abundant protein in the hemolymph (enghild et al., 1990). α2macroglobulin is one of the most abundant proteins of human plasma, at a concentration of 2-4 mg/ml (sottrup-jensen, 1989) and is the second-most abundant protein of the hemolymph of the cephalopod, sepia (vanhoorelbeke et al., 1993). α2macroglobulin, the subject of this review, is present in representatives of several metazoan phyla, including coelenterates (fujito et al., 2010) and representative species of the deuterostomia (echinodermata, tunicata, vertebrata), ecdysozoa (arthropoda, nematoda), and lophotrochozoa (mollusca) [for reviews see (armstrong and quigley, 1999; armstrong, 2006)]. a diverse variety of gramnegative eubacteria have acquired a gene encoding α2-macroglobulin from eukaryote associates (budd et al., 2004; doan and gettins, 2008). mechanisms for attack on proteases the protease inhibitors are of two fundamental classes, the active-site inhibitors, which bind to and inactivate the activate site of the targeted endopeptidase (huntington et al., 2000), and the α2macroglobulins, which react by a unique mechanism that involves the physical entrapment of the target protease within the folds of a molecule of α2macroglobulin (table 1). by binding to the active site of the protease, the active-site inhibitors destroy both its proteolytic activity against proteins and its ability to cleave the ester and amide bonds of low molecular mass artificial substrates. dedication for a particular active site means that the active-site inhibitors are restricted in their activity to one particular class, or even sub-class, of protease. by contrast, α2-macroglobulin shows a unique mechanism for interaction with target proteases which is initiated by its proteolytic cleavage at a defined motif that is constructed as an exposed and highly flexible stretch of 30 40 amino acids that presents a suite of peptide bonds that are inviting targets for most extant proteases (sottrup-jensen et al., 1989). this means that, whatever the protease, it will likely find a target for proteolytic attack at this “bait” region of the α2-macroglobulin molecule. this is the basis for the broadly reactive ability of α2-macroglobulin to bind proteases of diverse enzymatic reactivity and source. the forms of α2macroglobulin found in the tick (saravanan et al., 2003; buresova et al., 2009), horseshoe crab (husted et al., 2002), and carp (mutsuro et al., 2000) show multiple forms of the protein that differ by bait region sequence. presumably this functions to expand the list of proteases recognized by the α2macroglobulins of these species. proteolytic cleavage of the bait region is followed by the rapid re-folding of the α2-macroglobulin molecule so as to produce an enclosed interior pocket that now contains the target protease, entrapped within the folds of the α2-macroglobulin protein (starkey and barrett, 1973). protease entrapment disables the enzyme’s ability to proteolyze macromolecular substrates too large to penetrate the α2macroglobulin cage, but leaves intact the ability of the entrapped enzyme to hydrolyze low molecular mass substrates small enough to enter the α2macroglobulin cage and interact with the active site of the protease (starkey and barrett, 1973). one diagnostic feature to show that α2-macroglobulin is responsible for a protease inhibitory activity of an uncharacterized sample is the demonstration that the sample inhibits proteolytic but not amidolytic activity of an exogenously added protease. i am not aware of any other enzyme inhibitor that operates by this unique “trap” mechanism. the conformational change in the proteaseactivated α2-macroglobulin molecule exposes a domain at its carboxy terminus, the receptor-binding domain, that now targets the molecule, with its entrapped cargo of protease, for receptor-mediated endocytosis and proteolytic degradation by phagocytes and other cells that display the α2macroglobulin receptor at the cell surface (van leuven, 1984). in this fashion, α2-macroglobulin operates as an opsonin that delivers proteases of every enzymatic class to endocytotic cells of the innate immune system for internalization and destruction. several strands of evidence support this unusual scheme for the removal of potentially destructive proteolytic enzymes. active site protease inhibitors block both the proteolytic activity and the amidase and esterase activities against low molecular mass substrates. in contrast, proteases bound to α2-macroglobulin remain active against low molecular mass substrates while losing the ability to cleave peptide bonds of proteins (barrett and starkey, 1973; quigley and armstrong, 1983). this was interpreted to mean that the active site of α2-macroglobulin-bound proteases remained intact but was sterically blocked from reacting with macromolecular substrates. protein sequencing of α2-macroglobulins from mammals (sottrup-jensen et al., 1989) and a variety of invertebrates (iwaki et al., 1996; husted et al., 2002; saravanan et al., 2003) has identified the bait region as a defined stretch of approximately 30 amino acids that contains the protease-sensitive peptide bonds whose cleavage initiates the refolding of α2macroglobulin around the target protease. protein 167 table 1 comparison of α2-macroglobulin with active site protease inhibitors α2-macroglobulin active site inhibitors inhibits the proteolytic activity of proteases without inhibiting the hydrolysis of low molecular mass amide or ester substrates inhibits activity of target proteases against polypeptide and low molecular mass substrates reacts with endopeptidases of diverse catalytic mechanisms and substrate specificities reacts with a narrow spectrum of related proteases shields bound proteases from antibodies and high molecular mass active site inhibitors bound proteases remain reactive with antibodies presence of a unique internal reactive thiol ester group internal thiol ester is found only in proteins of the α2macroglobulin family and c3 families (the tep superfamily) sequencing has facilitated the molecular characterization of the receptor-recognition domain that is exposed by proteolytic cleavage (sottrupjensen et al., 1986; van leuven et al., 1986; enghild et al., 1989b; holtet et al., 1994). the molecular compaction responsible for the physical entrapment of a protease molecule was initially shown by the retarded elution of the proteasereacted form during gel filtration chromatography and by the increased electrophoretic mobility of the reacted form during non-denaturing polyacrylamide gel electrophoresis. this latter observation generated the term for the reacted and more compact form of the molecule as the “fast form” (barrett et al., 1979). the molecular compaction has also been demonstrated by direct electron microscopic comparison of α2-macroglobulin molecules before and after reaction with a protease (armstrong et al., 1991). the spectrum of proteases that interact with α2macroglobulin is unusually broad and includes proteases of all major enzymatic classes. although active-site protease inhibitors have been shown to function in immune defense (kanost, 1999), the promiscuous reactivity of α2-macroglobulin has adapted this single molecule to function as a particularly effective scavenger of the novel proteases introduced by parasites and pathogens. indeed, the α2-macroglobulins of vertebrates and invertebrates have been shown to react against the exoproteases of important parasites (freedman, 1991; araujo-jorge et al., 1992; fryer et al., 1996; srimal and armstrong, 1996; scharfstein, 2006). the broad spectrum of reactivity of α2macroglobulin contrasts with the much more restricted reactivity of the naturally-occurring activesite protease inhibitors and is another feature that can be used to demonstrate that an uncharacterized protease inhibitor is, indeed, α2-macroglobulin. interestingly, some microbial proteases are immune to certain versions of α2-macroglobulin. in some instances, the protease is too large to fit into the interior hydrophilic pocket of the reacted form of α2-macroglobulin [the collagenase of chlostridium perfringens has a molecular mass greater than 100 kda (abe et al., 1989)] but other non-reactive enzymes cleave peptide bonds not found in the bait region of certain versions of α2-macroglobulin. human α2-macroglobulin lacks the lysine residues in the bait region targeted by the lysyl endopeptidase of acromobacter lyticus and fails to react with that enzyme (ikai et al., 1999). a related example of this restriction is the relative insensitivity of the proteases of the limulus clotting system to limulus α2-macroglobulin, but with a sensitivity of those same proteases to human α2-macroglobulin (armstrong et al., 1984; iwaki et al., 1994). the limulus clotting protease specifically cleaves the clottable protein, coagulogen, at the carboxyl sides of two arg residues with the sequences leu-gly-arg and ser-gly-arg, which are conserved in coagulogens isolated from the four extant species of horseshoe crabs (iwanaga et al., 1992). limulus α2macroglobulin lacks the target sequence in the bait domain, but met-gly-arg is present in human α2macroglobulin, which may account for the differences in reactivity of these two forms of α2macroglobulin to limulus clotting enzyme. presumably, this is adaptive because blood clotting might be seriously compromised if the proteases necessary for the clotting reaction were rapidly inhibited by the principal protease inhibitor in the hemolymph. a similar situation may obtain in the hemolymph of the crayfish, pacifastacus, in which the proteases involved in activation of the prophenol oxidase system, a component of the systems involved in humoral immunity in crustaceans, are unaffected by the α2-macroglobulin found in the hemolymph of that organism (hergenhain et al., 1987). entrapment in the α2-macroglobulin cage both prevents the entrapped enzyme from accessing external proteins as substrates for proteolytic attack and protects the entrapped enzyme molecule from inactivation by macromolecular active site protease inhibitors. in other words, the protease molecule cannot get out of the α2-macroglobulin cage, but neither can macromolecular protein inhibitors get in. this has allowed the development of a specific and semi-quantitative assay for α2-macroglobulin, in which an uncharacterized sample is reacted first with trypsin, next with excess soybean trypsin inhibitor (sti, mr 21.5 kda), and then the residual trypsin activity is assayed with the low molecular mass amide substrate, bapna (na-benzoyl-dlarginine p-nitroanilide) (ganrot, 1966; armstrong et al., 1985). any trypsin not sequestered by α2macroglobulin is inactivated by sti and the only enzyme still able to hydrolyze bapna is that which is protected within the α2-macroglobulin cage. in the absence of α2-macroglobulin in the sample, degradation of bapna is zero. in the presence of increasing amounts of α2-macroglobulin, the hydrolysis of bapna is increased in a dosedependent fashion, indicative of increasing amounts of α2-macroglobulin-sequestered, and thus protected, trypsin. a positive reaction in a properly controlled sti-protection assay is a sure indicator for the presence of α2-macroglobulin in the sample, but the assay is subject to false negative responses that can occur if the sample also contains low molecular mass trypsin inhibitors small enough that they can enter the α2-macroglobulin cage and inactivate bound enzyme or if the α2-macroglobulin in the sample contacts proteases during sample collection and storage. because α2-macroglobulin is large, low molecular mass trypsin inhibitors can be removed by dialysis and premature reaction of α2macroglobulin can often be prevented by the inclusion of appropriate cocktails of low molecular mass protease inhibitors that can subsequently be removed by dialysis prior to assay with the stiprotection assay. we have seen now three diagnostic features of the α2-macroglobulin family of protease inhibitors: a broad reactive capacity against proteases of all classes, a unique molecular trap mechanism for interaction with target proteases, and a failure to inactivate the active site of the entrapped protease. a fourth diagnostic feature of the members of the α2-macroglobulin family of proteins is the presence of a reactive internal thiol ester bond linking cysteinyl and glutamyl residues, ⋅⋅⋅⋅g-c-g-e-q-n-m⋅⋅⋅⋅ * s____ c=o 168 which in limulus α2-macroglobulin are cys-999 and glx-1002 (iwaki et al., 1996). the thiol ester bond is cleaved when α2-macroglobulin experiences proteolytic attack on the bait domain, thereby exposing the cysteinyl thiol and the carbonyl of glutamic acid. this was seen by the exposure of a new titratable thiol group, the thiol of cys-999 in limulus α2-macroglobulin (armstrong and quigley, 1987). the newly-exposed carbonyl group of the glutamyl residue is highly reactive and can engage in isopeptide bonding with ε-amino groups of lysines of protein targets in the reaction environment (fig. 1). in human α2-macroglobulin, crosslinking of the thiol esterified glutamic acid is preferentially with the entrapped protease (sottrup-jensen et al., 1990b), whereas with α2-macroglobulin from the american horseshoe crab, limulus, the isopeptide bonds crosslink chains of α2-macroglobulin itself (dolmer et al., 1996). although the internal thiol ester of α2macroglobulin is stable in the intact protein, it will react with small primary amines such as ammonium and methylamine in the absence of proteolysis (fig. 1). an important diagnostic feature of α2macroglobulin is its sensitivity to methylamine. the contribution of α2-macroglobulin to the protease inhibitory activities of a biological sample is inactivated following methylamine treatment. protease clearance although α2-macroglobulin is usually thought of as a protease inhibitor, it might better be considered a protease-binding molecule whose principal function is to deliver its protease cargo to an endocytotic protease clearance pathway. in this context, unreacted α2-macroglobulin serves the recognition function and the protease-reacted fastform α2-macroglobulin serves the delivery function. the role of α2-macroglobulin in protease clearance is especially well illustrated in limulus because α2macroglobulin is the only protease inhibitor in the hemolymph (quigley and armstrong, 1983). clearance was studied by injecting fluoresceinlabeled proteins into the lumen of the heart and using a fluorometer to assay their concentration in blood drawn from the peripheral circulation at various times after injection. native proteins such as hemocyanin or native α2-macroglobulin (fig. 2b) require approximately 10 min to exit the heart and mix with the blood of the peripheral circulation, and then remain at constant concentration. in contrast, trypsin (fig. 2a) or the α2-macroglobulin-trypsin complex (fig. 2b) show rapid clearance from the hemolymph with a half-clearance time of 10 15 min. and maximal clearance at 20 25 min after dispersal from the injection site. clearance of trypsin depends on its catalytic activity because trypsin that has been inactivated by treatment with phenylmethylsulfonylfluoride (pmsf) is not cleared (fig. 2a). during the clearance stage, fluorescent trypsin is associated with a protein of high molecular mass. later, fluorescence reappears in the circulation as a form with mr < 10 kda (melchior et al., 1995). our interpretation of these observations is that the clearance of trypsin is mediated by binding to α2-macroglobulin and the protease-α2macroglobulin complex is then degraded to low molecular mass components that later reappear in the hemolymph. the blood cell appears to be involved in clearance and degradation because fig. 1 activation and cleavage of the internal thiol ester of α2-macroglobulin exposes a new thiol group on the cysteine and a reactive (-carbonyl on the glutamyl residue, which in limulus α2-macroglobulin are cys 999 and glx1002 . the reactive internal thiol ester of members of the α2-macroglobulin protein family is cleaved following proteolysis at the distantly-located protease bait region of the protein. thiol ester cleavage generates an activated (-carbonyl at the glutamyl residue and a free thiol at the cystenyl residue (top line of the diagram). the reactive glutamyl can form amide linkages with proteins (right arm of the diagram). the thiol ester can also react slowly with small primary amines, such as methylamine (left arm of diagram), even in the absence of proteolytic cleavage at the bait region. methylamine treatment eliminates many of the functional activities of α2-macroglobulin in parallel with its inactivation of the thiol ester. in general, sensitivity of a molecular function such as protease inhibition to treatment with methylamine is a useful test for the possibility that that function is dependent on the activity of a protein of the thiol ester protein family. fluoresceinated trypsin and fast-form α2macroglobulin associate with the blood cells at the very stages that these proteins are being cleared from the hemolymph (fig. 2b). the conformational change of protease-reacted α2-macroglobulin serves both to entrap the protease and to expose a previously cryptic domain at the carboxyl terminus, the receptor-binding domain (fig. 3) (sottrup-jensen et al., 1986; van leuven et al., 1986; enghild et al., 1989b; holtet et al., 1994), that is recognized by cell surface receptors, leading to the binding and endocytosis of the protease-α2macroglobulin complex (van leuven, 1984). this removes the protease from the circulation, thereby neutralizing its potentially damaging actions. the three major steps in protease recognition, capture, and delivery are mediated in part by three identifiable domains of the α2-macroglobulin molecule, the bait region, the thiol ester domain, and the receptor-recognition domain (fig. 3). presumably, there are additional domains that are under molecular strain in the unreacted form of α2macroglobulin and that are most actively involved in the active change in molecular shape following protease reaction and hydrolysis of the thiol ester, putative “hinge-domains”. in the absence of high resolution information of the structure of the native and fast-forms of the molecule, these have not been characterized. the best-characterized mammalian receptor for protease-reacted α2-macroglobulin (jensen et al., 1989; moestrup and gliemann, 1989; ashcom et al., 1990) has been identified as low density lipoprotein receptor-related protein-1 (lrp/α2m-r, a.k.a. lrp1, cd91) (kristensen et al., 1990; strickland et al., 1990). cd91 is synthesized as a 600 kda protein with a single transmembrane domain and is a member of the low density lipoprotein receptor family (for review see lillis et al., 2008). cd91, in addition to binding the α2-macroglobulin-protease complex, is also active as a receptor for a diverse variety of other exogenous ligands and plays an important regulatory role in a diverse array of important cellular processes (gonias et al., 2004; 169 fig. 2 clearance of proteases from the hemolymph of limulus is mediated by α2-macroglobulin. fluoresceinated proteins were injected into the lumen of the heart and their concentration in the peripheral hemolymph drawn from the leg joints was measured with a fluorometer. fluoresceinated protein reached the periphery by 5 10 min after injection. enzymatically active trypsin, but not trypsin inactivated by treatment with pmsf, was substantially cleared from the hemolymph by 30 min (fig. 1a). the fluorescence that appeared at later times was of low molecular mass, and presumably consisted of peptide digest products of the injected protein. fast-form, but not slow-form limulus α2-macroglobulin showed a similar pattern of clearance from the hemolymph. during the period of clearance of fast-form α2-macroglobulin from the hemolymph, fluorescence accumulated in the blood cells. this is interpreted to indicate that clearance of the introduced protease is mediated by its binding to α2-macroglobulin and that the blood cells participate in the uptake and clearance of the α2-macroglobulin-protease complex. may et al., 2007). of practical interest is its ability to bind two or more mols/mol of a 39 kda endogenous ligand, lrp receptor-associated protein (rap) (ashcom et al., 1990; herz et al., 1991). rap functions as a dedicated chaperone and regulator of members of the ldl receptor family of proteins (bu, 1998) and its high affinity binding makes rapconjugated sepharose an appropriate affinity matrix for purification of these proteins. to date, a receptor for protease-reacted α2macroglobulin has not been identified in an invertebrate. the family of proteins that includes vertebrate cd91 is, like α2-macroglobulin itself, an ancient family that was present prior to the great evolutionary radiation that established the divergent deuterostome and protostome invertebrate superphyla. representatives of the cd91 family have been found in modern representatives of lineages that diverged at the time of the precambrian radiation, notably in vertebrates, the nematode, caenorhabditis elegans (yochem and greenwald, 1993), and the arthropod, drosophila melanogaster (locus tag dmel cg33087). whether invertebrate orthologs of vertebrate cd91 operate as α2-macroglobulin receptors has not been decided. the limulus blood cell does contain a protein of very high molecular mass that, like cd91, binds rap (aimes et al., 1995) and human cd91 binds to protease-reacted limulus α2-macroglobulin (iwaki et al., 1996). although these observations hint at a possibility that the clearance pathway for protease-reacted limulus α2-macroglobulin involves an ortholog of cd91, they fall well short of any convincing demonstration of that prospect. phylogenetic distribution of the α2macroglobulins application of the criteria cited above has permitted the protein-based demonstration of α2macroglobulin in the plasma of lower vertebrates (starkey and barrett, 1982), arthropods, and molluscs (armstrong and quigley, 1999). the initial demonstration of α2-macroglobulin in an invertebrate occurred when james quigley and i stumbled upon the molecule while looking for fibrinolytic proteases active in the dissolution of blood clots in the american horseshoe crab, limulus polyphemus. we failed to find a fibrinolytic system, but we did find a hemolymph protease inhibitor that 170 fig. 3 domain structure of limulus α2-macroglobulin. based on the amino acid sequence of limulus α2macroglobulin (iwaki et al., 1996), it is possible to identify important functional domains by comparison with the already established domain structure of human α2-macroglobulin. the amino terminal end of limulus α2macroglobulin has a unique stretch identified as the c8 (domain, based on modest sequence identity with the human defense protein c8 (of the mammalian complement pathway. the bait region contains a sequence of amino acids that present target sites for nearly all proteases. proteolysis of the bait region initiates the compaction of α2-macroglobulin that is responsible for entrapment of the molecule of reacting protease. the thiol ester domain contains the unique thiol ester bond linking cysteine-999 and glutamic acid-1002. the receptor-binding domain at the carboxyl terminus of the polypeptide chain is exposed following molecular compaction and makes the protease-reacted form of α2-macroglobulin a target for binding to the cell surface α2-macroglobulin receptor. suppressed the action of serine, thiol, and metalloproteases against [14c]-casein, a protein substrate, but that failed to suppress hydrolysis of the amide substate bapna by trypsin. the antiprotease activity of hemolymph was eliminated if limulus hemolymph was treated with methylamine. this suggested the presence of α2-macroglobulin in the hemolymph of this invertebrate (quigley et al., 1982). our early analysis was facilitated by the absence of other trypsin inhibitors in limulus hemolymph (quigley and armstrong, 1983). subsequently, we showed that limulus hemolymph protected trypsin from inactivation by the macromolecular active-site inhibitor, soybean trypsin inhibitor (armstrong et al., 1985), that purified limulus α2-macroglobulin did indeed contain an internal thiol ester domain (armstrong and quigley, 1987), and that the protein from limulus shows significant sequence identity in key functional domains with human α2-macroglobulin (sottrupjensen et al., 1990a; iwaki et al., 1996). although overall sequence identity between human and limulus α2-macroglobulin is relatively low, the thiol ester domain shows significant sequence conservation, the receptor binding domains shows conservation of key basic amino acids, and the bait regions shows no sequence similarity. the strict conservation of the thiol ester domain of the various α2-macroglobulins has facilitated the molecular cloning of the genes for a variety of invertebrates from c-dna libraries. with the advent of complete genomic sequences for an everincreasing variety of species, data mining has revealed additional examples of genes encoding α2macroglobulin from these species. for example, the presence of genes for α2-macroglobulin in gramnegative bacteria was first demonstrated using the data-mining strategy (budd et al., 2004). these methods have identified both representatives of α2macroglobulin that, upon further investigation, have shown the unique suites of functional and structural characteristics of canonical α2-macroglobulin, and have also revealed novel related proteins, several of which await functional characterization. the α2-macroglobulin family includes the canonical α2-macroglobulins, a variety of similar protease-binding proteins, including pregnancyzone protein of humans, α1i3 of the rat, ovomacroglobulin of avian and reptile eggs, and proteins to be discussed below that were initially identified from the vertebrate complement system (sottrup-jensen, 1987). additionally, humans have two newly identified and incompletely characterized members of the family, cd109, which is linked to the cell surface by gpi-bonding (lin et al., 2002), and cpamd8, an acute-phase protein expressed by kidney, brain, and testis (li et al., 2004), and the settlement-inducing protein of the barnacle balanus amphitrite is an α2-macroglobulin (dreanno et al., 2006). the different α2-macroglobulins show differing orders of multimerization. human (jones et al., 1972; swenson and howard, 1979) and snail (bender and bayne, 1996) α2-macroglobulins are homotetramers, whereas many forms of the α2macroglobulins from a variety of lower vertebrates and arthropods are homodimers (starkey and barrett, 1982; feldman and pizzo, 1984; sand et al., 1985; feldman and pizzo, 1986; spycher et al., 1987; enghild et al., 1990; armstrong et al., 1991), and a few monomeric forms of α2-macroglobulin have been identified (enghild et al., 1989a). as indicated above, the molecular homologues of the canonical α2-macroglobulins have a wide phylogenetic distribution and share a defined suite of structural and functional characters (table 2). thiol ester protein family α2-macroglobulin is one of the founding members of the larger protein family, the thiol ester protein (tep) super-family (dodds and law, 1998). the thiol ester proteins share a similar domain 171 172 table 2 molecular and functional conservation of the α2-macroglobulins character specific attribute ref. 1. genetic identity overall peptide sequence identity 28-29 % identity of limulus and mammalian α2macroglobulins (sottrup-jensen et al., 1984; kan et al., 1985; gehring et al., 1987; van leuven et al., 1992; iwaki et al., 1996) bait region bounded by pxxc and exxr consensus sequences in limulus and mammalian α2macroglobulin (sottrup-jensen et al., 1989; iwaki et al., 1996) thiol ester domain >50 % identity for α2-macroglobulins of mammals, crustaceans, chelicerates, cephalopods, and gastropods (sottrup-jensen et al., 1984; spycher et al., 1987; hall et al., 1989; enghild et al., 1990; sottrup-jensen et al., 1990a; stocker et al., 1991; thogersen et al., 1992; bender and bayne, 1996; iwaki et al., 1996) receptor recognition domain conservation of two of the key lys residues of human α2-macroglobulin in limulus α2macroglobulin; of 73 residues conserved amongst the mammals, 45 are conserved in limulus α2macroglobulin (nielsen et al., 1996; iwaki et al., 1996) 2. biochemical homology susceptibility of bait region to proteolysis by proteases of all catalytic mechanisms reactivity of purified α2-macroglobulins of mammals, arthropods and molluscs to serine, cysteine, and metalloproteases (starkey and barrett, 1977; quigley and armstrong, 1985; hergenhahn and soderhall, 1985; enghild et al., 1990; thogersen et al., 1992; bender and bayne, 1996) molecular compaction; noncovalent trapping of proteases molecular compaction of protease-reacted α2macroglobulin of mammals and limulus (barrett and starkey, 1973; barrett et al., 1979; quigley et al., 1991; armstrong et al., 1991) covalent isopeptide bonding of thiol esterified glutamyl residue covalent bonding to trapped protease in mammalian α2-macroglobulin; covalent crosslinking to lys-254 of partner chain of α2macroglobulin dimer in limulus (sottrup-jensen et al., 1980; sottrup-jensen et al., 1990b; jacobsen and sottrup-jensen, 1993; dolmer et al., 1996) protease treatment exposes the recognition stretch for receptor-binding thiol ester-reacted limulus α2-macroglobulin binds the human α2-macroglobulin receptor (sottrup-jensen et al., 1986; van leuven et al., 1986; enghild et al., 1989b; iwaki et al., 1996) 3. functional similarity protease clearance mammals and limulus utilize α2-macroglobulin for the clearance of proteases from the internal milieu (feldman et al., 1985; davidsen et al., 1985; melchior et al., 1995) 173 architecture (janssen et al., 2005; doan and gettins, 2007), sequence similarity in key functional domains, and most, but not all, contain a stable internal thiol ester bond linking the cysteinyl and glutamyl residues of the thiol ester motif. members of this family that lack the internal thiol ester include complement component c5 (tack, 1983) and ovostatin from the albumin of the chicken egg (nagase et al., 1983) and drosophila mcr (ncbi accession number y11116). in all three proteins, the cys has has been substituted with another residue (nielsen and sottrup-jensen, 1993). but these proteins retain the other signature features that validate their assignment to the thiol ester protein family and ovostatin retains the ability to capture proteases by the molecular trap mechanism (nagase and harris, 1983). the taxonomy of the tep family is still a work in progress. one classification identifies two subfamilies, c3 and a2m (sottrup-jensen et al., 1985; fujito et al., 2010) named for their founding members, human complement component c3 and human α2-macroglobulin, respectively. a second scheme posits three distinct subfamilies, c3, a2m, and insect tep. members of the insect teps show sequence relatedness intermediate between the c3 and a2m subfamilies and functional similarity to members of the c3 subfamily. they also, like c3, the canonical member of the c3 subfamily, have a histidine residue (his 951 in drosophila tep1r; his 1,106 in human c3) in position to convert the reactive glx of the activated thiol ester to an intermediate that favors its subsequent covalent linkage to hydroxyl groups rather than showing the default reactivity with amines (dodds et al. 1966; law and dodds 1997). this residue is asp, asn, or ala in thiol ester proteins where the covalent reactivity of the thiol ester is with amines. subfamily membership is based on amino acid sequence and functional domain relatedness and is reflective of important functional differences between the members of the subfamilies. the best-characterized function of the a2m family members is protease binding, whereas the best-characterized function of the members of the c3 and insect tep proteins is covalent binding to the surfaces of foreign cells to mark them for immune destruction (dodds and sim, 1997; blandin et al., 2008). as will be described below, some members of the a2m subfamily, with well established protease-binding characters also, like members of the c3 and insect tep subfamilies, bind to the surfaces of foreign cells and facilitate their phagocytotic uptake. i am unaware of any reports of members of the c3 or insect tep proteins showing the unique protease capture abilities of the members of the a2m subfamily. the common ancestor of the tep family is presumably ancient because representatives of both the c3 and a2m subfamilies are found in cnidarians, which are basal metazoans. the sea anemone, haliplanella has two members of the c3 subfamily and two of the a2m subfamily (fujito et al., 2010). representatives of both families have been identified in vertebrates (sottrup-jensen et al., 1985), non-vertebrate chordates (nonaka et al., 1999), echinoderms (alsharif et al., 1998), and representatives of the ecdysozoans, the horseshoe crab (zhu et al., 2005; ariki et al., 2008), and the lophotrochozoans, a squid (castillo et al., 2009) and a clam (pradoalvarez et al., 2009). the insect tep subfamily has been reported only from insects and possibly nematodes (blandin and levashina, 2004), but not from other arthropods. it is important to note that the subdivision of the thiol ester proteins into just two or just three major subfamilies is not yet definitive. there are several tep proteins that may not fit within a twoor three-subfamily classification schemes (lin et al., 2002; li et al., 2004; dreanno et al., 2006). although the best-characterized function of the proteins of the a2m subfamily, both from vertebrates and invertebrates, is protease binding, biochemical pathways other than protease clearance have been identified. the α2macroglobulins also modulate cell proliferation and cell survival pathways (lamarre et al., 1991; shi et al., 2008), protease-independent pathways of the innate immune system (swarnakar et al., 2000; arnold et al., 2006; craig-barnes et al., 2010), antigen delivery in the operation of the adaptive immune system (bowers et al., 2009), and can operate as molecular chaparones (yerbury et al., 2009). as mentioned above, cd91, the canonical cell-surface receptor for protease-reacted α2-macroglobulin recognizes and binds a diverse suite of ligands and is an essential agent for a diverse array of important biological processes. the best-characterized function for members of the c3 subfamily is their covalent binding to the surfaces of foreign cells to target them for immune destruction, a function shared with members of the insect tep subfamily. an appealing example of this is the protein tep1, the mediator of an important pathway for the immune defense of the mosquito vector against the protozoan parasite, plasmodium falciparum, the agent of human malaria (reviewed in blandin et al., 2008). tep1 is a member of the thiol ester protein family from the anopheles mosquito. tep1 binds to bacteria and plasmodium cells and targets the cells for phagocytosis. the experimental elevation of the concentration of tep1 in the hemolymph of the mosquito reduces infection rates and experimental reduction increases susceptibility of the mosquito to infection (reviewed in volohonsky et al., 2010). different members of the insect teps show specialization of their targeting to the surfaces of different classes of microbes, for example, with drosophila mcr targeting candida albicans cells, drosophila tepiii targeting s. aureus, and drosophila tepii targeting e. coli (stroscheinstevenson et al., 2006). prospects for future research it is always presumptuous to predict the direction of research in any field of biology; the natural world holds many unexpected surprises and the ingenuity of biologists to identify and investigate those surprises seems without limit. that being said, some prospects for the future study of α2macroglobulin and of other members of the thiol ester protein superfamily are appropriate for a 174 review of this topic. a full characterization of the function(s) of the α2-macroglobulins in a diverse array of species will involve both the functional characterization of the molecule in vitro, which has been the principal topic of this review, and the elucidation of its function in vivo. what functions in the animal require α2-macroglobulin? the gene knock-out mouse has been available for a decade and a half and shows a surprisingly mild phenotype (umans et al., 1995; umans et al., 1999). interpretation is complicated by the artificially sanitary conditions of the life of the laboratory mouse. this mouse model will be particularly interesting when its sensitivity to an extended suite of proteasewielding pathogens is investigated (coutinho et al., 1999). in appropriate model invertebrates, rnai knock-down trials are possible and reduction of expression of various teps have been shown to significantly diminish host resistance to infection by some, but not all pathogens (buresova et al., 2009; volohonsky et al., 2010). one imagines refinements on this strategy where modified versions of α2macroglobulin are used to reconstitute function in α2-macroglobulin-depleted animals. in this context, the modified α2-macroglobulin constructs might feature bait regions with enhanced or restricted sensitivity to the various proteases of an appropriate pathogen (ikai et al., 1999). these could provide information on the roles of different proteases of the pathogen in the infection cycle and on the precise roles for α2-macroglobulin in immune defense. for example, might forms of α2-macroglobulin modified to include the appropriate novel cleavage sites for previously unrecognized microbial proteases now offer protection to the host from those bacteria? a related challenge to the functional characterization of the diverse members of the thiol ester protein family is the high resolution structural characterization of representative thiol ester protein family members for a detailed understanding of the relation of protein structure to the several functions of different members of this protein family. the goal of establishing a detailed understanding of the relations between protein structure and function is best exemplified by the characterization of human c3 (janssen et al., 2005) and an insect tep (baxter et al., 2007). the establishment of a high resolution structural characterization of α2-macroglobulin has proven elusive (jenner et al.,1998) but the insights provided by the characterization of the domain structure of c3 have been used to develop a model for human α2-macroglobulin (doan and gettins, 2007). one additional bonus of this line of research will be the provision of information for the refinement of the still-unsettled molecular taxonomy of the thiol ester protein superfamily. it will be interesting and illuminating to identify and characterize the clearance pathways for protease-reacted α2-macroglobulin in taxa other than mammals. to date, the sole experimental study is the investigation of the protease clearance pathway for the horseshoe crab (melchior et al., 1995) and this has implicated a cell-based clearance pathway that is selective for fast-form α2macroglobulin. initial evidence described above hints at a role for a role for a cell surface receptor with properties similar to mammalian cd91 in this pathway. although cd91 is the best-characterized cell surface receptor for mammalian α2macroglobulin, a second cell surface protein, grp78, has been shown to bind protease-reacted α2-macroglobulin with nm affinity (quinones et al., 2008; gonzalez-gronow et al., 2009). grp78 is a protein found in diverse eukaryotes and it will be interesting to discover if this or other undiscovered receptors operate to bind α2-macroglobulin in different metazoans. in mammals, both α2-macroglobulin and its receptor, cd91, have been shown to contribute to the operation of a number of physiological processes that are independent of protease clearance. it will be interesting to identify and characterize the possible diversity of functions of α2macroglobulin in invertebrates. a scattering of examples have already been identified. in invertebrates, α2-macroglobulin also regulates the activities of other immune effector proteins. in the shrimp, penaeusmonodon, α2-macroblobulin binds syntenin (tonganunt et al., 2005). syntenin is an acute-phase protein that is dramatically upregulated in shrimp infected with the white spot syndrome virus. in the horseshoe crab, limulus, protease-activated, but not native α2-macroglobulin binds and inactivates the cytolytic actions of limulin (armstrong et al., 1998; swarnakar et al., 2000). cytolytic destruction of foreign cells is a ubiquitous strategy of metazoan immune systems (canicatti, 1990; gabay, 1994). in mammals, cytolytic destruction of foreign cells is mediated in part by the multi-protein complement system (law and reid, 1995), whereas the hemolymph cytolytic pathway of limulus is mediated by the single protein limulin, a sialic acid-binding member of the pentraxin gene family (armstrong et al., 1996; harrington et al., 2008). the adaptive significance of the α2macroglobulin-limulin binding reaction has not been identified, but the pentraxins of limulus are multifunctional mediators of immunity, with potentially important roles in the inactivation of bacteral lipopolysaccharide (ng et al., 2004) and in the formation of hydrophilic pores across the lipopolysaccharide-rich outer membrane of gramnegative bacteria (harrington et al., 2009). it will be interesting to discover if the binding to proteasereacted α2-macroglobulin affects other functions of the limulus pentraxins or the pentraxins of other invertebrates. perhaps the reduced cytolytic activity of limulin complexed with α2-macroglobulin is correlated with augmentation of other immune functions of limulin. the recent identification of members of the c3 subfamily of tep proteins in diverse invertebrate taxa (zhu et al., 2005; fujito et al., 2010) and the insect tep subfamily in insects (blandin and levashina, 2004; blandin et al., 2008) have opened fertile fields for investigation. functional characterization of proteins of this class indicates that they operate much as c3 of the vertebrate complement system by their covalent binding to the surfaces of foreign cells to mark them for 175 subsequent immune destruction. it will be interesting to determine if this functional characterization is universal amongst metazoans or whether there is functional diversity for members of the c3 family from different taxa. for example are there instances where proteins of the c3 or insect tep sub-families function in a manner similar to the a2ms as protease binding proteins in addition to their canonical cell-binding functions or are there members of the a2m family that, in addition to the display of an ability to bind proteases, also function as opsonins that promote the phagocytosis of foreign cells that have become decorated with surface-associated α2-macroglobulin? for example, murine α2-macroglobulin binds to the surfaces of the pathogen, trypanosome cruzi (coutinho et al., 1997), and a form of α2-macroglobulin from a hard tick binds target bacteria and promotes their ingestion by phagocytes (buresova et al., 2009). conclusion it is the solemn duty of every animal to live to adulthood and to reproduce the species. survival requires efficient means to thwart the myriad invading pathogens that would compromise that survival. since many invertebrates regularly live to a considerable age, at least 20 years for limulus, 80 years for lobster, and 375 years for the ocean quahog, a mollusc (finch, 1990; philipp and abeke, 2010), while inhabiting highly septic environments, they have to possess efficient immune processes. these are largely of the innate class of immune systems, because invertebrates lack lymphocytes and the traditional rag-mediated adaptive immune system, which are restricted to the vertebrate lineage (marchalonis and schluter, 1990; agrawal et al., 1998). as we and others have shown, certain of these innate immune systems arose early in evolution and have been faithfully preserved in species of diverse lineage, and inhabiting very different environments and displaying very different life styles. for example both vertebrates and arthropods utilize the pentraxin (a.k.a., the c-reactive protein) and the tep families of proteins to protect from invading parasites. interest in immunity in invertebrates is driven by a basic curiosity in how species other than our own survives pathogenic attack and may have application to the development ___________________________________________________________________________ list of abbreviations: a2m, the α2-macroglobulin family of the tep protein superfamily; bapna, na-benzoyl-dl-arginine pnitroanilide; c3, the complement component 3 family of the tep protein superfamily; cd91, the canonical cell-surface receptor for fast-form α2macroglobulin and also known as lrp1, ldlreceptor-related protein/α2-macroglobulin-receptor; ma, methylamine, a small primary amine that reacts with the internal thiol ester of the teps; pmsf, phenylmethylsulfonylfluoride; rap, cd91associated protein, a natural ligand of cd91; sti, soybean trypsin inhibitor; tep, the superfamily of proteins characterized by the presence of a stable internal thiol ester motif and other conserved motifs. of rational veterinary care for aquacultured invertebrates of commercial importance and the development of improvements in the application of disease organisms for the biological control of invertebrates that are agricultural pests or disease vectors. acknowledgments supported by grant 0344360 from the national science foundation. references abe k, yamamoto k, sinohara h. proteinase inhibitory spectrum of mouse murinoglobulin and α-macroglobulin. j. biochem. 106: 564568, 1989. agrawal a, eastman qm, schatz dg. transposition mediated by rag1 and rag2 and its implications for the evolution of the immune system. nature 394: 744-751, 1998. aimes rt, quigley jp, swarnakar s, strickland d, armstrong pb. preliminary investigations on the scavenger receptors of the amebocyte of the american horseshoe crab, limulus polyphemus. biol. bull. (woods hole) 189: 225-226, 1995. al-sharif wz, sunyer jo, lambris jd, smith lc. sea urchin coelomocytes spedifically express a homologue of the complement component c3. j. immunol. 160: 2983-2997, 1998. araujo-jorge tc, lage mjf, rivera mt, carlier z, van leuven f. trypanozoma cruzi-enhanced α-macroglobulin levels correlate with the resistance of balb/cj mice to acute infection. parasitol. res. 78: 215-221,1992. ariki s, takahara s, shibata t, fukuoka t, ozaki a, endo y, et al. factor c acts as a lipopolysaccharide-responsive c3 convertase in horseshoe crab complement activation. j. immunol. 181: 7994-8001, 2008. armstrong pb. proteases and protease inhibitors: a balance of activities in host-pathogen interaction. immunobiology 211: 263-281, 2006. armstrong pb, levin j, quigley jp. role of endogenous proteinase inhibitors in the regulation of blood clotting system of the horseshoe crab, limulus polyphemus. thrombos. haemost. 52: 2631-2634, 1984. armstrong pb, mangel wf, wall js, hainfield jf, van holde ke, ikai a, et al. structure of α2macroglobulin from the arthropod limulus polyphemus. j. biol. chem. 266: 2526-2530, 1991. armstrong pb, melchior r, swarnakar s, quigley jp. α2-macroglobulin does not function as a c3 homologue in the hemolymph hemolytic system of the american horseshoe crab, limulus. mol. immunol. 35: 47-53, 1998. armstrong pb, quigley jp. limulus α2macroglobulin. first evidence in an invertebrate for a protein containing an internal thiol ester bond. biochem. j. 248: 703-707, 1987. armstrong pb, quigley jp. α2-macroglobulin: an evolutionarily conserved arm of the innate immune system. dev. comp. immunol. 23: 375 176 390, 1999. armstrong pb, rossner mt, quigley jp. an α2macroglobulin-like activity in the blood of chelicerate and mandibulate arthropods. j. exp. zool. 236: 1-9, 1985. armstrong pb, swarnakar s, srimal s, misquith s, hahn ea, aimes rt, et al. a cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins. j. biol. chem. 271: 14717-14721, 1996. arnold jn, wallis r, willis ac, harvey dj, royle l, dwek ra, et al. interaction of mannan binding lectin with α2-macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family? j. biol. chem. 281: 6955-6963, 2006. ashcom jd, tiller se, dickerson k, cravens jl, argraves ws, strickland dk. the human α2macroglobulin receptor: identification of a 420 kd cell surface glycoprotein specific for the activated conformation of α2-macroglobulin. j. cell biol. 110: 1041-1048, 1990. barrett aj, brown ma, sayers ca. the electrophoretically 'slow' and 'fast' forms of the α2-macroglobulin molecule. biochem. j. 181: 401-418, 1979. barrett aj, starkey pm. the interaction of α2macroglobulin with proteinases. characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. biochem. j. 133: 709-724, 1973. baxter rhg, chang c-i, chelliah y, blandin s, levashina ea, deisenhofer j. structural basis for conserved complement factor-like function in the antimalarial protein tep1. proc. natl. acad. sci. usa 104: 11615-11620. 2007. bender rc, bayne cj. purification and characterization of a tetrameric α2-macroglobulin protease inhibitor from the gastropod mollusc, biomphalaria glabrata. biochem. j. 316: 893900, 1996. blandin s, levashina ea. thioester-containing proteins and insect immunity. mol. immunol. 40: 903-908, 2004. blandin sa, marois, e, levashina ea. antimalarial response in anopheles gambiae: from a complement-like protein to a complement-like pathway. cell host & microbe 3: 364-374, 2008. bowers ev, horvath jj, bond je, cianciolo gj, pizzo sv. antigen delivery by α2-macroglobulin enhances the cytotoxic t lymphocyte response. j. leukoc. biol. 86: 1259-1268, 2009. bu g. receptor-associated protein: a specialized chaperone and antogonist for members of the ldl receptor gene family. curr. opin. lipidol. 9: 149-155, 1998. budd a, blandin s, levashina ea, gibson tj. bacterial α2-macroglobulin: colonization factors acquired by horizontal gene transfer from the metazoan genome? genome biol. 5: r38, 2004. buresova v, hajdusek o, franta z, sojka d, kopacek p. iram-an α2-macroglobulin from the hard tick ixodes ricinus: characterization and function in phagocytosis of a potential pathogen chryseobacterium indologenes. dev. comp. immunol. 33: 489-498, 2009. canicatti c. hemolysins: pore-forming proteins in invertebrates. experientia (basel) 46: 239-244, 1990. castillo mg, goodson ms, mcfall-ngai m. identification and molecular characterization of a complement c3 molecule in a lophotrochozoan, the hawaiian bobtail squid euprymna scolopes. dev. comp. immunol. 33: 69-76, 2009. coutinho cm, cavalcanti gh, van leuven f, araujo-jorge tc. α2-macroglobulin binds to the surface of trypanosoma cruzi. parasitol. res. 83: 144-150, 1997. coutinho cm, van leuven f, araujo-jorge tc. detection of α2-macroglobulin in the heart of mice infected with trypanosoma cruzi. parasitol. res. 85: 249-255, 1999. craig-barnes ha, doumouras bs, palaniyar n. surfactant protein d interacts with α2macroglobulin and increases its innate immune potential. j. biol. chem. 285: 13461-13470, 2010. davidsen o, christensen ei, gliemann j. the hemolymph clearance of human α2macroglobulin-trypsin in the rat is mainly accounted for by uptake into hepatocytes. biochim. biophys. acta 846: 85-92, 1985. deryugina e, quigley jp. matrix metalloproteinases and tumor metastasis. cancer metastasis rev. 25: 9-34, 2006. doan n, gettins pg. α-macroglobulins are present in some gram-negative bacteria: characterization of the α2-macroglobulin from escherichia coli. j. biol. chem. 283: 28747-28756, 2008. dodds aw, law sk. the phylogeny and evolution of the thioester bond-containing proteins c3, c4 and α2-macroglobulin. immunol. rev. 166: 15-26, 1998. dodds aw, sim rb. complement: a practical approach. oxford university press, oxford, 1997. dodds aw, xiang-dong r, willis ac, law ska. the reaction mechanism of the internal thioester in the human complement component c4. nature 379: 177-179 1996. dolmer k, husted lb, armstrong pb, sottrupjensen l. localisation of the major reactive lysine residue involved in the self-crosslinking of proteinase-activated limulus α2macroglobulin. febs lett. 393: 37-40, 1996. dreanno c, matsumura k, dohmae n, takio k, hirota h, kirby rr, et al. an α2-macroglobulinlike protein is the cue to gregarious settlement of the barnacle balanus amphitrite. proc. natl. acad. sci. usa 103: 14396-14401, 2006. enghild jj, salvesen g, thogersen ib, pizzo sv. proteinase-binding and inhibition by the monomeric α2-macroglobulin rat α1-inhibitor-3. j. biol. chem. 264: 11428-11435, 1989a. enghild jj, thogersen ib, roche pa, pizzo sv. a conserved region in α-macroglobulins participates in binding to the mammalian αmacroglobulin receptor. biochemistry 28: 1406 177 1412, 1989b. enghild jj, thogersen ib, salvesen g, fey gh, figler nl, gonias sl, et al. α-macroglobulin from limulus polyphemus exhibits proteinase inhibitory activity and participates in a hemolytic system. biochemistry 29: 10070-10080, 1990. feldman sr, pizzo sv. comparison of the binding of chicken α-macroglobulin and ovomacroglobulin to the mammalian α2macroglobulin receptor. arch. biochem. biophys. 235: 267-275, 1984. feldman sr, pizzo sv. purification and characterization of "half-molecule" α2 macroglobulin from the southern grass frog: absence of binding to the mammalian α2macroglobulin receptor. biochemistry 25: 721727, 1986. feldman sr, rosenberg mr, ney ka, michalopoulos g, pizzo sv. binding of α2macroglobulin to hepatocytes: mechanism of in vivo clearance. biochem. biophys. res. commun. 128: 795-802, 1985. finch ce. longevity, senescence and the genome. university of chicago press, chicago, 1990. freedman sj. the role of α2-macroglogulin in furunculosis: a comparison of rainbow trout and brook trout. comp. biochem. physiol. 98b: 549553, 1991. fryer se, bender rc, bayne cj. inhibition of cysteine proteinase from schsitosoma mansoni larvae by α-macroglobulin from the hemolymph of biomphalaria glabrata. j. parasitol. 82: 343347, 1996. fujito nt, sugimoto s, nonaka m. evolution of thioester-containing proteins revealed by cloning and characterization of their genes from a cnidarian sea anemone, haliplanella lineate. dev. comp. immunol. 34: 775-784, 2010. gabay je. ubiquitous natural antibiotics. science 264: 373-374, 1994. ganrot po. determination of α2macroglobulin as trypsin-protein esterase. clin. chim. acta 14: 493-501, 1966. gehring mr, shiels br, northemann w, de bruijn mh, kan cc, chain ac, et al. sequence of rat liver α2-macroglobulin and acute phase control of its messenger rna. j. biol. chem. 262: 446454, 1987. gonias sl, wu l, salicioni am. low density lipoprotein receptor-related protein: regulation of the hemolymph membrane proteome. thromb. haemost. 91: 1056-1064, 2004. gonzalez-gronow m, selim ma, papalas j, pizzo sv. grp78: a multifunctional receptor on the cell surface. antioxid. redox. signal. 11: 22992306, 2009. hall m, soderhall k, sottrup-jensen l. amino acid sequence around the thiolester of α2macroglobulin from hemolymph of the crayfish, pacifastacus leniusculas. febs lett. 254: 111114, 1989. harrington, jm, chou ht, gutsmann t, gelhaus c, stahlberg h, leippe m, et al. membrane pore formation by pentraxin proteins from limulus, the american horseshoe crab. biochem. j. 413: 305-313, 2008. harrington jm, chou ht, gutsmann t, gelhaus c, stahlberg h, leippe m, et al. membrane activity of a c-reactive protein. febs lett. 583: 10011005, 2009. hergenhahn h-g, soderhall k. α2-macroglobulinlike activity in hemolymph of the crayfish, pacifastacus leniusculus. comp. biochem. physiol. 81b: 833-835, 1985. hergenhahn h-g, aspan a, söderhäll k. purification and characterization of a high-mr proteinase inhibitor of pro-phenol oxidase activation from crayfish hemolymph, biochem. j. 248: 223-228, 1987. herz j, goldstein jl, strickland dk, ho yk, brown ms. 39-kda protein modulates binding of ligands to low density lipoprotein receptorrelated protein/α2-macroglobulin receptor. j. biol. chem. 266: 21232-21238, 1991. holtet tl, nielsen kl, etzerodt m, moestrup sk, gliemann j, sottrup-jensen l, et al. recombinant α2m receptor binding domain binds to the α2m receptor with high affinity. ann. ny acad sci. 737: 480-482, 1994. huntington ja, read rj, carrell rw. structure of a serpin-protease complex shows inhibition by deformation. nature 407: 923-926, 2000. husted lb, sorensen es, armstrong pb, quigley jp, kristensen l, sottrup-jensen l. localization of carbohydrate attachment sites and disulfide bridges in limulus α2macroglobulin. evidence for two forms differing primarily in their bait region sequences. j. biol. chem. 277: 43698-43706, 2002. ikai a, ookata k, shimizu m, nakamichi n, ito m, matsumura t. a recombinant bait region mutant of human α2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum. cytotechnology 31: 53-60, 1999. iwaki d, kawabata s-i, miura y, kato a, armstrong pb, quigley jp, et al. molecular cloning of limulus α2-macroglobulin. eur. j. biochem. 242: 822-831, 1996. iwanaga s, miyata t, tokunaga f, muta t. molecular mechanisms of hemolymph clotting system in limulus. thromb. res. 68: 1-32, 1992. jacobsen l, sottrup-jensen l. localization of εlysyl-γ-glutamyl cross-links in α2-macroglobulinplasmin complex. biochemistry 32: 120-126, 1993. janssen bjc, huizinga eg, raaijmakers hca, roos a, daha mr, nilsson-ekdahl k, et al. strictures of complement component c3 provide insights into the function and evolution of immunity. nature 437: 505-511 2005. jenner l, husted l, thirup s, sottrup-jensen l. crystal sgtrucure of the receptor-binding domain of α2-macroglobulin. structure 15: 595604 1998. jensen ph, moestrup sk, gliemann j. purification of the human placental α2-macroglobulin receptor. febs lett. 255: 275-280, 1989. jones jm, creeth jm, kekwick ra. thiol reduction of human α2-macroglobulin. the subunit 178 structure. biochem. j. 127: 187-197, 1972. kan cc, solomon e, belt kt, chain ac, hiorns lr, fey g. nucleotide sequence of cdna encoding human α2-macroglobulin and assignment of the chromosomal locus. proc. natl. acad. sci. usa 82: 2282-2286, 1985. kanost mr. serine proteinase inhibitors in arthropod immunity. dev. comp. immunol. 23: 291-301, 1999. kessenbrock k, plaks v, werb z. matrix metalloproteinases: regulators of the tumor microenvironment. cell 141: 52-67, 2010. kristensen t, moestrup sk, gliemann j, bendtsen l, sand o, sottrup-jensen l. evidence that the newly cloned low-density-lipoprotein receptor related protein (lrp) is the α2-macroglobulin receptor. febs lett. 276: 151-155, 1990. lamarre j, wollenberg gk, gonias sl, hayes ma. cytokine binding and clearance properties of proteinaseactivated α2-macroglobulins. lab. invest. 65: 3-14, 1991. laskowski mj, kato i. protein inhibitors of proteinases. annu. rev. biochem. 49, 593-626 1980. law ska, dodds aw. the internal thioester and the covalent binding properties of the complement proteins c3 amd c4. protein sci. 6: 263-274, 1997. law sk, reid kbm. complement, 2nd edition. irl press, oxford, 1995. li zf, wu xh, engvall e. identification and characterization of cpamd8, a novel member of the complement 3/α2-macroglobulin family with a c-terminal kazal domain. genomics 83: 1083-1093, 2004. lillis ap, van duyn lb, murphy-ullrich je, strickland dk. ldl receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies. physiol. rev. 88: 887-918, 2008. lin m, sutherland dr, horsfall w, totty n, yeo e, nayar r, et al. cell surface antigen cd109 is a novel member of the a2 macroglobulin/c3, c4, c5 family of thioester-containing proteins. blood 99: 1683-1691, 2002. lopez-otin c, bond js. proteases: multifunctional enzymes in life and disease. j. biol. chem. 283: 30433-30437, 2008. marchalonis jj, schluter sf. origin of immunoglobulins and immune recognition molecules. bioscience 40: 758-768, 1990. may p, woldt e, matz rl, boucher p. the ldl receptor-related protein (lrp) family: an old family of proteins with new physiological functions. ann. med. 39: 219-228, 2007. mckerrow jh, caffrey c, kelly b, loke p, sajid m. proteases in parasitic diseases. annu. rev. pathol. 1: 497-536, 2006. melchior r, quigley jp, armstrong pb. α2macroglobulin-mediated clearance of proteases from the hemolymph of the american horseshoe crab, limulus polyphemus. j. biol. chem. 270: 13496-13502, 1995. moestrup sk, gliemann j. purification of the rat hepatic α2-macroglobulin receptor as an approximately 440-kda single chain protein. j. biol. chem. 264: 15574-15577, 1989. mutsuro j, nakao m, fujiki k, yano t. multiple forms of α2-macroglobulin from a bony fish, the common carp (cyprinus carpio): striking sequence diversity in functional sites. immunogenetics 51: 847-855, 2000. nagase h, harris ed. ovostatin: a novel proteinase inhibitor from chicken egg white ii. mechanism of inhibition studied with collagenase and thermolysin. j. biol. chem. 258: 7490-7498, 1983. nagase h, harris edj, woessner jfj, brew k. ovostatin: a novel proteinase inhibitor from chicken egg white. i. purification, physicochemical properties, and tissue distribution of ovostatin. j. biol. chem. 258: 7481-7489, 1983. ng pm, jin z, tan ss, ho b, ding jl. c-reactive protein. a predominant lps-binding acute phase protein responsive to pseudomonas infection. j. endotoxin res. 10: 163-174, 2004. nielsen kl, holtet tl, etzerodt m, moestrup sk, gliemann j, sottrup-jensen l, et al. identification of residues in α-macroglobulins important for binding to the α2-macroglobulin receptor/low density lipoprotein receptorrelated protein. j. biol. chem. 271: 1290912912, 1996. nielsen kl, sottrup-jensen l. evidence from sequence analysis that hen egg-white ovomacroglobulin (ovostatin) is devoid of an internal β-cys-γ-glu thiol ester. biochim. biophys. acta 1162: 230-232, 1993. nonaka m, azumi k, ji x, namikawa-yamada c, sasaki m, saiga h, et al. opsonic complement component c3 in the solitary ascidian, halocynthia roretzi. j. immunol. 162: 387-391, 1999. perlmutter dh, pierce ja. the α1-antitrypsin gene and emphysema. amer. j. physiol. 257: l147l162, 1989. philipp ee, abeke d. masters of longevity: lessons from long-leved bivalves a mini-review. gerentology 56: 55-65. prado-alvarez m, rotllant j, gestal c, novoa b, figueras a. characterization of a c3 and a factor b-like in the carpet-shell clam, ruditapes decussatus. fish shellfish immunol. 26: 305315, 2009. quigley jp, armstrong pb. an endopeptidase inhibitor, similar to mammalian α2macroglobulin, detected in the hemolymph of an invertebrate, limulus polyphemus. j. biol. chem. 258: 7903-7906, 1983. quigley jp, armstrong pb. a homologue of α2macroglobulin purified from the hemolymph of the horseshoe crab limulus polyphemus. j. biol. chem. 260: 12715-12719, 1985. quigley jp, armstrong pb, gallant p, rickles fr, troll w. an endopeptidase inhibitor, similar to vertebrate α2macroglobulin, present in the hemolymph of limulus polyphemus. biol. bull. (woods hole) 163: 402, 1982. quigley jp, ikai a, arakawa h, osada t, armstrong 179 pb. reaction of proteinases with α2macroglobulin from the american horseshoe crab, limulus. j. biol. chem. 266: 1942619431, 1991. quinones qj, de ridder gg, pizzo sv. grp78: a chaperone with diverse roles beyond the endoplasmic reticulum. histol. histopathol. 23: 1409-1416, 2008. sand o, folkersen j, westergaard jg, sottrupjensen l. characterization of human pregnancy zone protein. comparison with human α2-macroglobulin. j. biol. chem. 260: 15723-15735, 1985. saravanan t, weise c, sojka d, kopacek p. molecular cloning, structure and bait region splice variants of α2-macroglobulin from the soft tick ornithodoros moubata. insect biochem. mol. biol. 33: 841-851, 2003. scharfstein j. parasite cysteine proteinase interactions with α2-macroglobulin or kininogens: differential pathways modulating inflammation and innate immunity in infection by pathogenic trypanosomatids. immunobiology 211: 117-125, 2006. shi z, rudzinski m, meerovitch k, lebrun-julien f, birman e, di polo a, et al. α2-macroglobulin is a mediator of retinal ganglion cell death in glaucoma. j. biol. chem. 283: 29156-29165, 2008. sottrup-jensen l. α2-macroglobulin and related thiol ester hemolymph proteins. in: putnam fw (ed), the hemolymph proteins: structure, function and genetic control, academic press, orlando, fl, pp 191-291, 1987. sottrup-jensen l. α-macroglobulins: structure, shape, and mechanism of proteinase complex formation. j. biol. chem. 264: 11539-11542, 1989. sottrup-jensen l, borth w, hall m, quigley jp, armstrong pb. sequence similarity between α2macroglobulin from the horseshoe crab, limulus polyphemus, and the proteins of the α2-macroglobulin family from mammals. comp. biochem. physiol 96b: 621-625, 1990. sottrup-jensen l, gliemann j, van leuven f. domain structure of human α2-macroglobulin. characterization of a receptor-binding domain obtained by digestion with papain. febs lett. 205: 20-24, 1986. sottrup-jensen l, hansen hf, pedersen hs, kristensen l. localization of ε-lysyl-γ-glutamyl cross-links in 5 human α2-macroglobulinproteinase complexes nature of the high molecular weight cross-linked products. j. biol. chem. 265: 17727-17737, 1990. sottrup-jensen l, petersen te, magnusson s. a thiol-ester in α2-macroglobulin cleaved during proteinase complex formation. febs lett. 121: 275-279, 1980. sottrup-jensen l, sand o, kristensen l, fey gh. the α-macroglobulin bait region. sequence diversity and localization of cleavage sites for proteinases in five mammalian αmacroglobulins. j. biol. chem. 264: 1578115789, 1989. sottrup-jensen l, stepanik tm, kristensen t, lonblad pb, jones cm, wierzbicki dm. common evolutionary origin of α2macroglobulin and complement components c3 and c4. proc. natl. acad. sci. usa 82: 9-13, 1985. sottrup-jensen l, stepanik tm, kristensen t, wierzbicki dm, jones cm, lonblad pb, et al. primary structure of human α2-macroglobulin. v. the complete structure. j. biol. chem. 259: 8318-8327, 1984. spycher se, arya s, isenman de, painter rh. a functional, thioester-containing α2macroglobulin homologue isolated from the hemolymph of the american lobster (homarus americanus). j. biol. chem. 262: 14606-14611, 1987. srimal s, armstrong pb. interaction of immune defense systems of limulus polyphemus with an extracorporeal protease secreted by the principal ectoparasite of limulus, the triclad turbellarid worm, bdelloura candida. indian j. biol. 34: 1081-1084, 1996. starkey pm, barrett aj. inhibition by αmacroglobulin and other serum proteins. biochem. j. 131: 823-831, 1973. starkey pm, barrett aj. α2-macroglobulin, a physiological regulator of proteinase activity. in: barrett aj (ed), proteinases in mammalian cells and tissues, elsevier/north holland biomedical press, amsterdam, pp 663-696, 1977. starkey pm, barrett aj. evolution of α2macroglobulin. the demonstration in a variety of vertebrate species of a protein resembling human α2macroglobulin. biochem. j. 205: 9195, 1982. stocker w, breit s, sottrup-jensen l, zwilling r. α2macroglobulin from hemolymph of the freshwater crayfish astacus astacus. comp. biochem. physiol. 98b: 501-509, 1991. strickland dk, ashcom jd, williams s, burgess wh, migliorini m, argraves ws. sequence identity between the α2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. j. biol. chem. 265: 17401-17404, 1990. stroschein-stevenson sl, foley e, o’farrell ph, johnson ad. identification of drosophila gene products required for phagocytosis of candida albicans. plos biol. 4: 87-99, 2006. swarnakar s, asokan r, quigley jp, armstrong pb. binding of α2-macroglobulin and limulin: regulation of the hemolymph hemolytic system of the american horseshoe crab, limulus. biochem. j. 347: 679-685, 2000. swensonrp, howard jb. characterization of alkylamine-sensitive site in α2macroglobulin. proc. natl. acad. sci. usa 76: 4313-4316, 1979. tack bf. the β-cys-γ-glu thiolester bond in human c3, c4, and α2macroglobulin. springer semin. immunopathol. 6: 259-282, 1983. thogersen ib, salvesen g, brucato fh, pizzo sv, 180 enghild jj. purification and characterization of an α-macroglobulin proteinase inhibitor from the mollusc octopus vulgaris. biochem. j. 285: 521-527, 1992. tonganunt,m., phongdara,a., chotigeat,w., fujise,k. identification and characterization of syntenin binding protein in the black tiger shrimp penaeusmonodon. j. biotechnol. 120: 135-145, 2005. travis j, salvesen gs. human hemolymph proteinase inhibitors. annu. rev. biochem. 52: 655-709, 1983. umans l, serneels l, overbergh l, lorent k, van leuven f, van den berghe h. targeted inactivation of the mouse α2-macroglobulin gene. j. biol. chem. 270: 19778-19785, 1995. umans l, serneels l, overbergh l, stas l, van leuven f. α2-macroglobulinand murinoglobulin-1deficient mice. a mouse model for acute pancreatitis. am. j. pathol. 155: 983-993, 1999. van leuven f. human α2 macroglobulin. mol. cell biochem. 58: 121-128, 1984. van leuven f, marynen p, sottrup-jensen l, cassiman jj, van den berghe h. the receptorbinding domain of human α2-macroglobulin. isolation after limited proteolysis with a bacterial proteinase. j. biol. chem. 261: 11369-11373, 1986. van leuven f, torrekens s, overbergh l, lorent k, de strooper b, et al. the primary sequence and the subunit structure of mouse α2 macroglobulin, deduced from protein sequencingof the isolated subunits and from molecular cloning of the cdna. eur. j. biochem. 210: 319-327, 1992. vanhoorelbeke k, goossens a, gielens c, preaux,g. an α2-macroglobulin-like proteinase inhibitor inthe haemolymph of the decabrachia cephalopod sepia officinalis. arch. inter. physiol. biochim. biophys. 102: b25, 1993. volohonsky g, steinert s, levashina ea. focusing on complement in the antiparasitic defense of mosquitoes. trends parasitol. 26: 1-3, 2010. yerbury jj, kumita jr, meehan s, dobson cm, wilson mr. α2-macroglobulin and haptoglobin suppress amyloid formation by interacting with prefibrillar protein species. j. biol. chem. 284: 4246-4254, 2009. yochem j, greenwald i. a gene for a low density lipoprotein receptor-related protein in the nematode caenorhabditis elegans. proc. natl. acad. sci. usa 90: 4572-4576, 1993. zhu y, thangamani s, ho b, ding jl. the ancient origin of the complement system. embo j. 24: 382-394, 2005. baxter rhg, chang c-i, chelliah y, blandin s, levashina ea, deisenhofer j. structural basis for conserved complement factor-like function in the antimalarial protein tep1. proc. natl. acad. sci. usa 104: 11615-11620. 2007. review isj 7: 11-21, 2010 issn 1824-307x review signaling molecules involved in immune responses in mussels s koutsogiannaki, m kaloyianni laboratory of animal physiology, zoology department, school of biology, faculty of science, aristotle university of thessaloniki, 54124 thessaloniki, greece accepted december 22, 2009 abstract immune system of molluscs is constituted by hemocytes and humoral factors that cooperate for the protection of the organism, triggering a wide range of immune responses. in molluscs, immune responses include phagocytosis, encapsulation, respiratory burst leading to reactive oxygen species (ros) production and nitric oxide (no) synthesis, release of antimicrobial molecules and the activation of phenoloxidase system. these responses are mediated firstly by a variety of hemocyte receptors binding to ligands that results to a cascade of signaling events. the processes of hemocytes adhesion to and migration through extracellular matrix (ecm) proteins play a crucial role in cell immunity. results suggest that cadmium and oxidants induce adhesion to and migration through ecm proteins in mytilus gallorovincialis hemocytes with the involvement of na+/h+ exchanger (nhe), phosphatidylinositol-3 kinase (pi-3k), protein kinase c (pkc), nadph oxidase, ros and no as well as with α2 integrin subunit. furthermore, the data so far suggests the involvement of additional signaling molecules such as mitogen-activated protein kinases (mapks), signal transducers and activators of transcription (stats), c-jun n-terminal kinase (jnk), extracellular signal-regulated kinase (erk), cyclic adenosine monophosphate (camp), responsive element binding protein (creb) and nuclear factor kappa b (nf-kb) in molluscs immunity. further research in mollusc immune system may lead to a more sufficient protection and to a better control of these economically important organisms. key words: mytilus galloprovincialis; immune response; adhesion; migration; integrin   introduction immune responses are highly complex including a variety of different cellular and molecular processes. the study of the immune processes in invertebrates is of great importance from ecological, economic and public health points of view (peteiro et al., 2007). furthermore, the study of the immune mechanisms in molluscs is also significant due to their susceptibility to infection by bacteria, viruses, and parasites that makes them transmitters of many diseases affecting different vertebrate species (barcia and ramos-martinez, 2008). however, the available data on the immune responses in invertebrates should be implemented (humphries and yoshino, 2003; tiscar and mosca, 2004; canesi et al., 2006; mydlarz et al., 2006; ottaviani, 2006; barcia and ramos-martinez, 2008). in this review we will refer to the phagocytic behavior of m.  galloprovincialis hemocytes. ___________________________________________________________________________ corresponding author: martha kaloyianni zoology department, aristotle university of thessaloniki 54124 thessaloniki, greece e-mail: kaloyian@bio.auth.gr immune system of molluscs among invertebrates, molluscs represent the widest phylum after arthropods. they are considered as excellent bio-indicators and they have been used intensively in research studies. the defense mechanisms of molluscs consist firstly of chemicophysical barriers (external skeletons, cockles, cuticles, mucus) that prevent host invasion and secondly of the circulating hemocytes and humoral factors that operate in co-ordination triggering a wide range of immune responses (renwrantz, 1990; rinkevich and muller, 1996; hine, 1999). according to mydlarz et al. (2006) the three essential components of innate immunity in invertebrates are: 1) phagocytosis which represents the cell-mediated immunity, 2) activation of humoral responses that result to opsonization, coagulation and melanization and 3) the production of humoral antimicrobial components. m. galloprovincialis immune system consists in at least four subtypes of hemocytes charged with different tasks in host defense: large granular (r1), large semigranular (r2), small semigranular   11 fig. 1 adhesion of mytilus galloprovincialis hemocytes to laminin, collagen iv and oxidized collagen iv after treatment with cadmium and inhibitors of nhe, pkc, pi-3k, nadph oxidase and no synthase. hemocytes were pre-incubated with the inhibitors cariporide (20 nm), go6976 (500 nm) and gf109203χ (10 μμ), wortmannin (50 nm), dpi (10 μm) and l-name (10 μμ) for 15 min at 20 0c, cdcl2 (5 μμ) was then added and the samples were incubated for 30 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the control value. values that share a are significant different between each other. b,c,d indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) (r3), and small hyaline (r4) hemocytes (garciagarcia et al., 2008). while large granular (r1), large semigranular (r2), and small semigranular (r3) cells are thought to be phagocytic, and capable of activating the respiratory burst, small hyalinocytes (r4) lack these two capabilities. nevertheless, all hemocyte subpopulations seem to be capable of nitric oxide (no) production (garcia-garcia et al., 2008). the presence of many hydrolytic enzymes in the large granules indicates their connection with lysosomes (pipe, 1990). ottaviani et al. (1998a) proposed just one type of immunocytes in different stages (young and old) in m. galloprovincialis supporting mix’s (1976) suggestion that hyalinocytes (agranular type) are a proliferative condition that after various stages mature into granulocytes. two of these four subtypes, large granular and large semigranular cells share common features with the mammalian professional phagocytes (garcia-garcia et al., 2008). furthermore, hemocytes secrete humoral factors that play a fundamental role in the innate immune responses in molluscs including lysosomal enzymes, agglutinins or lectins, cytokine-like molecules, bioactive peptides, no, and antimicrobial peptides (ottaviani, 2006). in addition to these, the defense mechanisms of mussels include the activation of phenoloxidase system (little et al., 2005). hemocytes are also involved in detoxification through accumulation of metallic and organic xenobiotics in their well developed lysosomal system (cajaraville et al., 1995). phagocytosis represents the main cellmediated immune response and is mediated by the hemocytes. it is comprised by different phases involving recognition, chemotactic migration, adhesion, ingestion, destruction and elimination of foreign cells (tiscar and mosca, 2004). we will focus on properties such as cell adhesion and cell migration through extracellular matrix proteins collagen iv, laminin-1 and on the signaling molecules that mediate these processes. cell adhesion, cell migration and extracellular matrix proteins among the immune responses in mussels, the processes of hemocyte adhesion to and migration through extracellular matrix play a crucial role in cell immunity. hemocyte adhesion is an initial step in phagocytosis of foreign particles (hynes and lander, 1992). cell-cell adhesion and cellsubstratum adhesion (e.g., to extracellular matrix) are critical for the development, maintenance and 12 fig. 2 migration of mytilus galloprovincialis hemocytes through collagen iv and oxidized collagen iv after treatment with cadmium and inhibitors of nhe, pkc, pi-3k, nadph oxidase and no synthase. hemocytes were pre-incubated with the inhibitors cariporide (20 nm), go6976 (500 nm) and gf109203χ (10 μμ) for 15 min at 20 0c, cdcl2 (5 μμ) was then added and the samples were incubated for 30 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the control value. a,b indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) function of multicellular organisms. moreover, hemocyte adhesion to different surfaces can result in important cellular behaviors such as parasitic encapsulation and hemocyte-mediated clotting responses (yoshino, 1998). hemocyte migration depends on directed cytoskeletal reorganization, ion transport membrane recycling by endocytosis and formation of focal adhesion sites with extracellular matrix. m. galloprovincialis hemocytes migration through extracellular matrix (ecm) proteins was reported after heavy metals (koutsogiannaki, 2008) and interleukin (il)8 affect (ottaviani, 2000). chemotaxis was also detected in immunocytes of the hard clam mercenaria mercenaria as a result of bacteria stimuli (fawcett and tripp, 1994). ecm plays a central role in the structure and maintenance of tissue architecture (adams and watt, 1993). it is now evident that ecm turnover is a critical step in tissue remodelling that accompanies many physiological as well as pathological processes in vertebrates, invertebrates and plants (massova et al., 1998). the macromolecules that are present in all extracellular matrices include collagen, proteoglycans and glycoproteins (mainly laminins). the collagens are a family of extracellular matrix proteins involved in cell adhesion, chemotaxis and migration, and the dynamic interplay between cells and collagens regulates tissue remodelling during growth, differentiation, morphogenesis and wound healing. cells encounter collagen in a number of different ways. cells may stably adhere to collagen in tissues and thus receive survival signals (e.g., dermal fibroblasts), migrate through the collagen-rich stroma as part of a normal morphogenic process (e.g., mammary gland branching) or in disease (e.g., tumour metastasis), or interact with collagen as a result of injury (e.g., homeostasis). interestingly, molluscan hemocytes have been reported to be involved in collagen synthesis and extracellular matrix deposition (serpentini et al., 2000). in accordance with these, studies in sections of integument from bivalve species suggest that molluscan integumental ecm contains collagens similar to type i, iv, v and vi collagens (corbetta et al., 2002). in addition, molluscan motoneurons adhere to laminin and type iv collagen (wildering et al, 1998). furthermore, results suggest that hemocytes after treatment with either cadmium or oxidants adhere to and migrate through collagen iv and oxidized collagen iv at a higher degree compared to control cells (koutsogiannaki, 2008) (figs 1-4). it is also suggested that m. galloprovincialis hemocytes adhere to collagen with the involvement of α2 integrin subunit (koutsogiannaki, 2008) (fig. 5) apart from collagens, laminins are also components of the extracellular matrix that determine the histoarchitecture and provide cells with biological information. the laminins are the major family of non collagenous heterodimeric glycoproteins that provide an integral part of the structural scaffolding in almost every tissue of an organism. it has been demonstrated that laminins are mainly involved in the organization of the basal membrane network and are also present in cellassociated extracellular matrices. they are involved in multiple physiological processes including cell proliferation, differentiation, migration, adhesion and survival. the laminins are found as trimeric proteins 13 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_coverdate=03%2f31%2f2001&_rdoc=1&_fmt=full&_orig=search&_cdi=4925&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=fc7fb64f38f647acc255c8becabaecbf#bib1 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_coverdate=03%2f31%2f2001&_rdoc=1&_fmt=full&_orig=search&_cdi=4925&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=fc7fb64f38f647acc255c8becabaecbf#bib1 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_coverdate=03%2f31%2f2001&_rdoc=1&_fmt=full&_orig=search&_cdi=4925&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=fc7fb64f38f647acc255c8becabaecbf#bib16 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=1c26a6c00a2ccf01e53e8c6ec9f5fcc8#bbib29 fig. 3 adhesion of mytilus galloprovincialis hemocytes to laminin, collagen iv and oxidized collagen iv after treatment with cadmium, oxidants and antioxidants. hemocytes were incubated with the oxidant rotenone (25 μμ) for 60 min at 20 0c or with the antioxidant nac for 15 hr at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the control value. a,b,c indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) which form a cross, giving a structure that can bind to other cell membrane and extracellular matrix molecules (timpl and brown, 1994; yurchenco and cheng, 1994; yurchenco and o'rear, 1994). it is reported that m. galloprovincialis hemocytes after treatment with either cadmium or oxidants adhere to the ecm protein laminin at a higher degree compared to control cells (koutsogiannaki, 2008) (figs1, 3). hemocyte receptors involved in immune responses the processes of adhesion and migration are mediated through hemocytes receptors interactions with binding groups. hemocyte receptors are grouped into several broad groups including lectins (or lectin-like receptors), integrins (or integrinrelated receptors) and growth factor/hormone/cytokine-like receptors (humphries and yoshimo, 2003). lectins, are glycoproteins which serve as recognition molecules by binding to non-self material through carbohydrate recognition sites (ottaviani, 2006). in addition, a unique family of proteins with cho-activity, referred to as fibrinogenrelated proteins or freps has been found to be induced in snails in response to infection (adema et al., 1997; leonard et al., 2001). moreover, selectinlike proteins has been referred to exist in molluscs. selectins constitute a family of cho-reactive membrane proteins that are present in endothelial cells, leukocytes and platelets in mammals. they are adhesion receptors involved in many processes such as leucocyte extravascular trafficking and inflammation (patel et al., 2002). it has also been demonstrated that receptors for platelet-derived growth factor (pdgf-alpha/β) and transforming growth factor β (tgf-β) are present in m. gallorovincialis hemocytes involved in many cellular functions such as phagocytosis and cell motility (ottaviani et al., 1997a; kletsas et al., 1998). moreover, receptors for bioactive peptides such as proopiomelanocortin (pomc) including β-endorphin, adrenocorticotrophic hormone (acth) and alpha-melanotropin receptors as well as insulin-like receptors have been found in molluscan hemocytes (stefano et al., 1989; duvax-miret et al., 1992; ottaviani et al., 1998b; sassi et al., 1998; lardans et al., 2001). furthermore, cytokine-like receptors have been found to be present in molluscan hemocytes as well. it has been shown that bioactive peptides and cytokines in invertebrates are related to cell shape changes and cell migration (hughes et al., 1990; ottaviani et al., 1995), induce no synthase (ottaviani et al., 1995) and increase phagocytic activity by activating the classical signal transduction pathways, i.e., protein kinase a, c and b (ottaviani et al., 1997b). among cytokines, interleukins which belong to chemotactic cytokines also referred as chemokines, are involved in acute inflammation. il-8 has been demonstrated to induce increased phagocytic activity and chemotactic response in m. galloprovincialis hemocytes (ottaviani et al., 2000). barcia et al. (1999) also detected the il-2 receptor to be present in m. galloprovincialis hemocytes. 14 http://www3.interscience.wiley.com/cgi-bin/fulltext/72503858/main.html,ftx_abs#bib269 http://www3.interscience.wiley.com/cgi-bin/fulltext/72503858/main.html,ftx_abs#bib269 fig. 4 migration of mytilus galloprovincialis hemocytes through collagen iv and oxidized collagen iv after treatment with cadmium, oxidants and antioxidants. hemocytes were incubated with the oxidant rotenone (25 μμ) for 60 min at 20 0c or with the antioxidant nac for 15 hr at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the respective control value. a,b indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) integrins comprehend a large family of cell surface receptors. in mammals there are integrins binding to laminin (α1β1, α2β1, α3β1, α6β1, α7,β1 and α6β1), integrins binding to collagen (α1β1, α2β1, α3β1, α10β1 and α11β1), integrins of leukocytes (αlβ2, αmβ2, αxβ2 and αdβ2) and integrins recognizing the rgd motif (α5β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8 and αiibβ3) (heino et al., 2009) integrins function mainly as cell-matrix adhesion molecules and transducers of the signals between them (li et al., 2003). ecmintegrin interactions function in a bidirectional manner across cell membranes. as the extracellular domain of integrin receptors becomes occupied by ligand and cluster, the integrins set off a cascade of events termed “outside-in” signaling. in this regard integrins orchestrate multiple functions including proliferation, differentiation, gene expression, changes in intracellular ph and death (ross and borg, 2001). moreover, integrins interact with cytoskeleton regulating cell shape and cell migration. these interactions are mediated through binding of the cytoplasmatic domain of integrins to actin network and actin-binding proteins (ezrin, randixin, moesin) (hynes, 1992). this short cytoplasmatic domain serves also as a host of molecules such as kinases and small gtpases (ross and borg, 2001). integrins are presumed to be present in all the metazoan cells. in invertebrates the structures of integrins are well conserved and functions are correlated with adhesive processes and immune responses (tanzer, 2006). studies in molluscan neurons indicate that cells can attach to various substrates using both rgd-dependent and rgd-independent adhesion mechanisms suggesting that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in molluscan neurons (wildering et al., 1998). results have shown that α2 integrin subunit mediates the increased adhesion of m. gallorovincialis hemocytes to collagen and oxidized collagen induced by cadmium (koutsogiannaki, 2008) (fig. 5). in addition increased expression of α2 integrin subunit was observed after cadmium treatment in m. gallorovincialis hemocytes, which was due to na+/h+ exchanger (nhe), phosphatidylinositol-3 kinase (pi-3k), protein kinase c (pkc), nadph oxidase, reactive oxygen species (ros) and no involvement (koutsogiannaki, 2008) (fig. 6). among the adhesion receptors that have been also found in invertebrates are caderins and immunoglobulins (n-cam) as well as peroxinectin and psp1 peptide (plasmatocyte spreading peptide) (johansson, 1999). signaling molecules involved in immune responses the first step to initiate an immune response is the detection by hemocytes of foreign invaders and/or non-self cells, presumably through receptors associated with the surface membrane. signals generated by ligand binding are then transduced across the membrane resulting in a cascade of downstream chemical reactions, ultimately directing these signals to target organelles (e.g., nucleus, cytoskeleton) mediating the induction of appropriate cellular responses (heldin and purton, 1996). cells 15 fig. 5 adhesion of mytilus galloprovincialis hemocytes to collagen iv and oxidized collagen iv after treatment with the anti-alpha2 integrin subunit. hemocytes were incubated with the anti-alpha2 integrin subunit (cd49b) for 30 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the respective control value. a indicates significant difference between each other (koutsogiannaki, 2008) mediating immunity are able to communicate with both their internal and external environments through well developed signaling pathways (hynes and zhao, 2000). receptor-ligand interactions result in the modulation of many cellular processes mediated by complex intracellular signal transduction pathways. in invertebrates little is known about these signaling pathways although the cumulative data implies that there is high homology with those of mammals (humphries and yoshino, 2003). studies concerning the induction of the immune system of m. galloprovincialis by various stimuli (bacteria, cytokines, hormones, environmental chemicals) suggest the involvement of p38 [(stress-activated p38 mitogen-activated protein kinase (mapk)], c-jun n-terminal kinase (jnk), extracellular signal-regulated kinase (erk), signal transducer and activator of transcription (stat)5, stat 3, nuclear factor kappa b (nf-kb), pkc, cyclic adenosine monophosphate (camp) dependent pka (camp/pka), pi-3 k, ros and no (ottaviani et al., 2000; canesi et al., 2006; cao et al., 2007; novas et al., 2007; barcia and ramosmartinez, 2008; garcia-garcia et al., 2008; malagoli et al., 2008). in addition, malagoli et al. (2007) reported that stressfull conditions in mussel hemocytes trigger increased phagocytic activity and/or modulation of their signal transduction pathways, mainly erk and map kinases. this flexibility suggests the possibility that accumulated substances exert different effects in diverse situations. on the other hand, garcia-garcia et al. (2008) suggest that the role of erk and pkc in phagocytosis regulation is less generalized due to differential stimulation of phagocytic receptors. in addition it has been suggested that different bacteria and bacterial strains can differently affect the host signaling pathways (zampini et al., 2003; canesi et al., 2005, 2006). ottaviani et al. (2000) suggested that il-8 triggers conformational changes, induces chemotaxis and increased phagocytic activity in m. galloprovincialis hemocytes through pka and pkc pathway followed by reorganization of the actin microfilaments. this study also suggests that pka signaling pathway could be more important than the pkc in mediating cell shape changes induced by il8. on the other hand, il-2 mediated biogenin amines (ba) synthesis involves preferably pkc whereas the camp dependent pka plays secondary role (cao et al., 2004, 2007). it has been found that camp activates nucleotide depended protein kinases in molluscs (macdonald and storey, 1999) and modulates phagocytic behavior of hemocyte (chen and bayne, 1995). results from our laboratory showed that treatment with 3-isobutyl-1methylxanthin (ibmx), that results in high camp cell content, didn’t significantly affect the processes of adhesion and migration of m. galloprovincialis hemocytes to extracellular matrix proteins laminin and collagen (koutsogiannaki, 2008). the role of camp in these processes warrants further investigation. no, different forms of nitric oxide synthase (nos) and ros represent some of the main immune mechanisms in invertebrates (pipe, 1992; anderson et al., 1992; gourdon et al., 2001; ottaviani, 2006; barcia and ramos-martinez, 2008). ros are produced through respiratory burst, which is a series of biochemical reactions leading to ros generation such as superoxide (o2-), hydrogen peroxide (h2o2) and hydroxyl radical (oh .) (cross 16 fig. 6 integrins expession of mytilus galloprovincialis hemocytes. hemocytes were incubated with the anti-alpha 2 integrin subunit for 10 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the respective control value. a indicates significant difference between each sample value with cadmium value (koutsogiannaki, 2008) and segal, 2004). the activation of respiratory burst has been detected in hemocytes of many mollusc species including m. galloprovincialis (garcia-garcia et al., 2008). no is a highly cytotoxic and microbicidal molecule, that is responsible for the defense mechanisms mediated by macrophages in mammals. it is also capable of activating other leukocytes (armstrong, 2001). no synthesis has been demonstrated in many molluscs as well (ottaviani et al., 1993; arumugam et al., 2000; novas et al., 2004; stefano et al., 2004). it has been shown that no, o2and h2o2 are involved in the signaling pathway induced by cadmium leading to m. galloprovincialis hemocytes adhesion and migration through ecm proteins (koutsogiannaki, 2008). in addition, the use of oxidants caused increase in adhesion and migration of hemocytes through ecm proteins that was reversed in the presence of the antioxidant nac (koutsogiannaki, 2008) (figs 3, 4). the later observations confirm the fact that ros are implicated in immune responses of m. galloprovincialis hemocytes (koutsogiannaki, 2008). furthermore, the use of nos inhibitors resulted in elimination of the bacteria clumping induced by lipopolysaccharides (lps) in the molluscan hemocytes of m. edulis and v. alter (ottaviani et al., 1993). it has been demonstrated that metals can increase ros production in m. galloprovincialis hemocytes with the implication of pkc (kaloyianni et al., 2006). moreover, in mussel leukocytes no production seems to be mainly regulated by pi3-k, pkc and erk families (garciagarcia et al., 2008). according to the latter, erk and pkc regulate no production only in large semigranular hemocytes as a result of differential membrane phagocytic receptor stimulation. in addition, studies on lymnaea stagnalis relate pkc and erk to the signaling pathway that regulates no activity (wright et al., 2006). moreover, barcia and ramos-martinez (2008) showed that il-2 induces the synthesis of no in m. galloprovincialis hemocytes via activation mainly of the camp dependent pka and secondary of pkc. it has been also shown that pi-3k activation plays critical role in the immune responses of m. galloprovincialis against pathogens and environmental pollutants (canesi et al., 2002a-c). pi3k has central role in coordinating phagocytosis and is found to mediate production of ros, no synthesis and pkc activation in m. galloprovincialis hemocytes (chou et al. 1998; chen et al., 2003; garcia-garcia et al., 2008). in addition, there are studies that point out the role of pi3k in the signaling pathways involved in the interactions of cells with the extracellular matrix in invertebrates and in mammals (guan and chen, 1996; parson, 1996; wei et al., 1997; howe et al., 1998; koutsogiannaki, 2008; konstantinidis et al., 2009). it has been also reported that treatment with wortmannin (pi3-k inhibitor) caused inhibition of cell adhesion, migration, phagocytosis and reorganization of cytoskeleton in the colonial ascidian botryllus schlosseri (ballarin et al., 2002). similarly, it has been found that wortmannin effect 17 caused inhibition of hemocytes adhesion to and migration through ecm proteins (koutsogiannaki, 2008). finally, another signaling molecule that seems to be involved in immune processes is nhe. nhe plays a central role in intracellular ph regulation and homeostasis of cell volume and is also involved in many intracellular signaling pathways (dailianis and kaloyianni, 2004; dailianis et al., 2005; kaloyianni et al., 2005; koutsogiannaki et al., 2006). nhe activation is implicated in many other cell functions as cell survival and apoptosis (koliakos et al., 2008). it has been shown that treatment of m. galloprovincialis hemocytes with cadmium resulted in increased degree of hemocytes adhesion to and migration through laminin-1, collagen type iv and oxidized collagen type iv in relation to control cells, with the involvement of nhe and pkc (kaloyianni et al., 2006; koutsogiannaki, 2008). in addition, nhe’s implication in cell adhesion and cell migration is probably related to the fact that nhe is involved in focal anchoring sites together with focal adhesion kinase (fak) and proteins of the actin network (teline, vincoulin, paxiciline and others) through indirect connection with integrins (beningo et al., 2001; koliakos et al., 2001; webb et al., 2002; stock et al., 2005; kostidou et al., 2007) or cd44 (verfaillie et al., 1994). in conclusion, mussels are able to perform sophisticated responses regarding immune functions. the cumulative data implies the existence of numerous different signaling pathways that may participate in immune responses or the existence of a network of all these suggested pathways, that involve a number of molecules as nhe, pi3-k, pkc, no, ros, nadph oxidase, mapks, stats, jnk, erk, creb and nf-kb. most of the molecules involved in immune processes are well conserved from invertebrates to vertebrates. in the higher forms of life their function remains basically similar. further research is necessary in order to elucidate the signaling molecules that are involved in these processes and that may lead to a more clear understanding of the immune mechanisms operating in molluscs. references adams jc, watt fm. regulation of development and differentiation by the extracellular matrix. development 117: 1183-1198, 1993. adema cm, hertel la, miller rd, loker es. a family of fibrinogen-related proteins that precipitate parasite-derived molecules is produced by an invertebrate after infection. proc. natl. acad. sci. usa 94: 8691-8696, 1997. anderson d, paynter k, burreson e. increased reactive oxygen intermediate production by hemocytes withdrawn from crassostrea virginica infected with perkinsus marinus. biol. bull. 183: 476-481,1992. armstrong r. the physiological role and pharmacological potential of nitric oxide in neutrophil activation. int. immunopharmacol. 1: 1501-1512, 2001. arumugan m, romestand b, torreilles j, roch p. in vitro production of superoxide and nitric oxide (as nitrite and nitrate) by mytilus galloprovincialis haemocytes upon incubation with pma or laminarin or during yeast phagocytosis. eur. j. cell biol. 79: 513-519, 2000. ballarin l, scanferla m, cima f, sabbatin a. phagocyte spreading and phagocytosis in the compound ascidian botryllis schlosseri: evidence for an integrin-like, rdg-dependent recognition mechanism. develop. comp. immunol. 26: 345-354, 2002. barcia r, cao a, arbeteta j, ramos-martínez ji. the il-2 receptor in hemocytes of the sea mussel mytilus galloprovincialis lmk. iubmb life 48: 419-423, 1999. barcia r, ramos-martinez ji. effects of interleukin2 on nitric oxide production in molluscan innate immunity. inv. surv. j. 5: 43-49, 2008. beningo k, dembo m, kaverina i, small j, wang y. nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. j. cell biol. 153: 881-887, 2001. cajaraville mp, pal sg, robledo y. light and electron microscopical localization of lysosomal acid hydrolases in bivalve hemocytes by enzyme cytochemistry. acta histochem. cytochem. 28: 409-416, 1995. canesi l, gallo g, gavioli m, pruzzo c. bacteriahemocyte interactions and phagocytosis in marine bivalves. microsc. res. tech. 57: 469476, 2002a. canesi l, betti m, ciacci c, scarpato a, citterio b, pruzzo c, et al. signalling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 map kinases. dev. comp. immunol. 26: 325-334, 2002b. canesi l, scarpato a, betti m, ciacci c, pruzzo c, gallo g. bacterial killing by mytilus hemocyte monolayers as a model for investigating the signaling pathways involved in mussel immune defence. mar. environ. res. 54: 547-551, 2002c. canesi l, betti m, ciacci c, lorusso lc, gallo g, pruzzo c. interactions between mytilus hemocytes and different strains of escherichia coli and vibrio cholerae o1 el tor: role of kinase-mediated signallling. cell microbiol. 7: 667-674, 2005. canesi l, betti m, ciacci c, lorusso lc, pruzzo c, gallo g. cell signalling in the immune response of mussel hemocytes. inv. surv. j. 3: 40-49, 2006. cao a, ramos-martı΄nez ji, barcia a. in vitro effects of lps, il-2, pdgf and crf on hemocytes of mytilus galloprovincialis lmk. fish shellfish immunol. 16: 215-225, 2004. cao a, novas a, ramos-martinez ji, barcia r. seasonal variations in haemocyte response in the mussel mytilus galloprovincialis lmk. aquaculture 263: 310-319, 2007. chen jh, bayne cj. hemocyte adhesion in the california mussel (mytilus californianus): regulation by adenosine. biochim. biophys. acta 1268: 178-184, 1995. chen q, powell dw, rane mj, singh s, butt w, klein jb, et al. akt phosphorylates p47phox   18 and mediates respiratory burst activity in human neutrophils. j. immunol. 170: 53025308, 2003. chou mm, hou w, johnson j, graham lk, lee mh, chen cs, et al. regulation of protein kinase cζ by pi 3-kinase and pdk-1. curr. biol. 8: 10691077, 1998. corbetta s, bairati a, vitellaro zuccarello l. immunohistochemical study of subepidermal connective of molluscan integument. eur. j. histochem. 46: 259-272, 2002. cross ar, segal aw. the nadph oxidase of professional phagocytes-prototype of the nox electron transport chain systems. biochim. biophys. acta 1657: 1-22, 2004. dailianis s, kaloyianni m. cadmium induces both pyruvate kinase and na+/h+ exchanger activity through protein kinase c mediated signal transduction, in isolated digestive gland cells of mytilus galloprovincialis (l.). j. exp. biol. 207: 1665-1674, 2004. dailianis s, piperakis sm, kaloyianni m. cadmium effects on ros production and dna damage via adrenergic receptors stimulation: role of na+/h+ exchanger and pkc. free radic. res. 39: 1059-1070, 2005. duvaux-miret o, stefano gb, smith em, dissous c, capron a. immunosuppression in the definitive and intermediate hosts of the human parasite schistosoma mansoni by release of immunoactive neuropeptides. proc. natl. acad. sci. usa 89: 778-781, 1992. fawcett lb, tripp mr. chemotaxis of mercenaria mercenaria hemocytes to bacteria in vitro. j. invertebr. pathol. 63: 275-284, 1994. garcia-garcia e, prado-alvarez m, novoa b, figueras a, rosales c. immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the pi 3k, pkc, and erk kinase families. dev. comp. immunol. 32: 637-653, 2008. gourdon i, guerin mc, torreilles j, roch p. nitric oxide generation by hemocytes of the mussel mytilus galloprovincialis. biol. chem. 5: 1-6, 2001. guan jl, chen hc. signal transduction in cellmatrix interactions. int. rev. cytol. 168: 81-121, 1996. heino j, huhtada m, kapyla j, johnson ms. evolution of collagen-based adhesion systems. inter. j. biochem. cell biol. 41: 341-348, 2009. heldin ch, purton m. signal transduction. chapman & hall, ltd., london, 1996. hine pm. the inter-relations of bivalve hemocytes. fish shellfish immunol. 9: 367-385, 1999. howe a, aplin ae, alahari sk, juliano rl. integrin signalling and cell growth control. curr. opin. cell biol. 10: 220-231, 1998. hughes tkjr, smith em, chin r, cadet p, sinisterra j, leung mk, et al. interaction of immunoreactive monokines (interleukin 1 and tumor necrosis factor) in the bivalve mollusc mytilus edulis. proc. natl. acad. sci. usa 87: 4426-4429, 1990. humphries je, yoshino tp. cellular receptors and signal transduction in molluscan hemocytes: connections with the innate immune system of vertebrates. integr. comp. biol. 43: 305-312, 2003. hynes ro. integrins: versatility, modulation and signalling in cell adhesion. cell 69: 11-25, 1992. hynes ro, lander ad. contact and adhesive specificities in the associations, migrations and targeting of cells and axons. cell 68: 303-322, 1992. hynes ro, zhao q. the evolution of cell adhesion. j. cell biol. 150: 89-96, 2000. johansson mw. cell adhesion molecules in invertebrate immunity. dev. comp. immunol. 23: 303-315, 1999. kaloyianni m, stamatiou r, dailianis s. zinc and 17beta-estradiol induce modifications in na+/h+ exchanger and pyruvate kinase activity through protein kinase c in isolated mantle/gonad cells of mytilus galloprovincialis. comp. biochem. physiol. 141c: 257-266, 2005. kaloyianni m, ragia v, tzeranaki i, dailianis s. the influence of zn on signaling pathways and attachment of mytilus galloprovincialis hemocytes to extracellular matrix proteins. comp. biochem. physiol. 144c: 93-100, 2006. kletsas d, sassi d, franchini a, ottaviani e. pdgf and tgf-beta induce cell shape changes in invertebrate immunocytes via specific cell surface receptors. eur. j. cell biol. 75: 362366, 1998. koliakos g, trachana v, gaitatzi m, dimitriadou a. phosphorylation of laminin-1 by protein kinase c. mol. cells 11: 179-185, 2001. koliakos g, paletas k, kaloyianni m. nhe-1: a molecular target for signalling and cell matrix interactions. connect. tissue res. 49: 157-161, 2008. konstantinidis d, paletas k, koliakos g, kaloyianni m. signaling components involved in leptininduced amplification of the atherosclerosisrelated properties of human monocytes. j. vasc. res. 46: 199-208, 2009. kostidou e, koliakos g, alamdari d, paletas k, tsapas a, kaloyianni m. enhanced laminin carbonylation by monocytes in diabetes mellitus. clin. bioch. 40: 671-679, 2007. koutsogiannaki s, evangelinos n, koliakos g, kaloyianni m. cytotoxic mechanisms of zn and cd involve na/h exchanger (nhe) activation by ros. aquat. toxicol. 78: 315-324, 2006. koutsogiannaki s. signal transduction pathway induced by cadmium in mussel haemocytes. postgraduate diploma thesis, aristotle university of thessaloniki, 2008. lardans v, coppin jf, vicogne j, aroca e, delcroix m, dissous c. characterization of an insulin receptor-related receptor in biomphalaria glabrata embryonic cells. biochem. biophys. acta 1510: 321-329, 2001. leonard pm, adema cm, zhang sm, loker es. structure of two frep genes that combine igsf and fibrinogen domains, with comments on diversity of the frep gene family in the snail biomphalaria glabrata. gene 269: 155165, 2001. li j, zhang y, kirsner rs. angiogenesis in wound repair: angiogenic growth factors and the   19 http://www.ncbi.nlm.nih.gov/pubmed?term=%22konstantinidis%20d%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22paletas%20k%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22koliakos%20g%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22kaloyianni%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22kaloyianni%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract extracellular matrix. microsc. res. tech. 60: 107-114, 2003. little tj, hultmark d, read af. invertebrate immunity and the limits of mechanistic immunology. nat. immunol. 6: 651-654, 2005. macdonald ja, storey kb. cyclic amp-dependent protein kinase: role in anoxia and freezing tolerance of the marine periwinkle littorina littorea. mar biol. 133: 193-203, 1999. malagoli d, casarini l, sacchi s, ottaviani e. stress and immune response in the mussel mytilus galloprovincialis. fish shellfish immunol. 23: 171-177, 2007. malagoli d, casarini l, ottaviani e. effects of the marine toxins okadaic acid and palytoxin on mussel phagocytosis. fish shellfish immunol. 24: 180-186, 2008. massova i, kotra lp, fridman r, mobashery s. matrix metalloproteinases: structures, evolution, and diversification. faseb j. 12: 1075-1095, 1998. mix mc. a general model for leukocyte cell renewal in bivalve molluscs. mar. fish. rev. us natl. mar. fish. serv. 38: 3741, 1976. mydlarz ld, jones le, harvell cd. innate immunity, environmental drivers, and disease ecology of marine and freshwater invertebrates. annu. rev. ecol. evol. s. 37: 251-288, 2006. novas a, cao a, barcia r, ramos-martı΄nez ji. nitric oxide release by hemocytes of the mussel mytilus galloprovincialis lmk was provoked by interleukin-2 but not by lipopolysaccharide. int. j. biochem. cell biol. 36: 390-394, 2004. novas a, barcia r, ramos-martinez ji. implication of pkc in the seasonal variation of the immune response of the hemocytes of mytilus galloprovincialis lmk. and its role in interleukin2-induced nitric oxide synthesis. iubmb life 59: 659-663, 2007. ottaviani e, paemen lr, cadet p, stefano gb. evidence for nitric oxide production and utilization as a bactericidal agent by invertebrate immunocytes. eur. j. pharmacol. 248: 319-324, 1993. ottaviani e, franchini a, cassanelli s, genedani s. cytokines and molluscan immune responses. biol. cell 85: 87-91, 1995. ottaviani e., franchini a, kletsas d, bernardi m, genedani s. involvement of pdgf and tgfbeta in cell migration and phagocytosis in invertebrate immunocytes. anim. biol. 6: 91-95, 1997a. ottaviani e, franchini a, franceschi c. proopiomelanocortin-derived peptides, cytokines and nitric oxide in immune responses and stress: an evolutionary approach. int. rev. cytol. 170: 79-141, 1997b. ottaviani e, franchini a, barbieri d, kletsas d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two aging-related hemocyte stages. ital. j. zool. 65: 349-354, 1998a. ottaviani e, franchini a, hanukoglu i. in situ localisation of acth receptor-like mrna in molluscan and human immunocytes. cell mol. life sci. 54: 139-142, 1998b. ottaviani e, franchini a, malagoli d, genedani s. immunomodulation by recombinant human interleukin-8 and its signal transduction pathways in invertebrate hemocytes. cell mol. life sci. 57: 506-513, 2000. ottaviani e. molluscan immunorecognition. inv. surv. j. 3: 50-63, 2006. parson jt. integrins-mediated signaling: regulation by protein tyrosine kinases and small gtpbinding proteins. curr. opin. cell biol. 8: 146150, 1996. patel kd, cuvelier sl, wiehler s. selectins: critical mediators of leukocyte recruitment. sem. immunol. 14: 73-81, 2002. peteiro lg, labarta u, fernandez-reiriz mj. variability in biochemical components of the mussel (mytilus galloprovincialis) cultured after prestige oil spill. comp. biochem. physiol. 145c: 588-594, 2007. pipe rk. hydrolytic enzymes associated with the granular haemocytes of the marine mussel mytilus edulis. histochem. j. 22: 595-603, 1990. pipe rk. generation of reactive oxygen metabolites by the haemocytes of the mussel mytilus edulis. dev. comp. immunol. 16: 111-122, 1992. renwrantz l. internal defense system of mytilus edulis. in: stefano gb (ed), neurobiology of mytilus edulis, manchester university press, manchester, pp 256-275, 1990. rinkevich b, muller weg. invertebrate immunology, springer, berlin, 1996. ross rs, borg tk. integrins and myocrdium. circ. res. 88: 1112-1119, 2001. sassi d, kletsas d, ottaviani e. interactions of signalling pathways in (1-24)-induced cell shape changes in invertebrate immunocytes. peptides 19: 1105-1110, 1998. serpentini a, ghayor c, poncet jm, hebert v, galéra p, pujol jp, et al. collagen study and regulation of the de novo synthesis by igf-i in hemocytes from the gastropod mollusc, haliotis tuberculata. j. exp. zool. 287: 275-284, 2000. stefano gb, leung mk, zhao x, scharrer b. evidence for the involvement of opioid neuropeptides in the adherence and migration of immunocompetent invertebrate hemocytes. proc. natl. aca. sci. usa 86: 626-630, 1989. stefano gb, kim e, liu y, zhu w, casares f, mantiene kj, et al. nitric oxide modulates microglial activation. med. sci. monit. 10: 1722, 2004. stock cgb, hauck cr, arnold h, mally s, eble ja, dieterich p, et al. migration of human melanoma cells depends on extracellular ph and na+/h+ exchange. j. physiol. 567: 225-238, 2005. tanzer ml. current concepts of extracellular matrix. j. orthop. sci. 11: 326-331, 2006. timpl r, brown jc. the laminins. matrix biol. 14: 275-281, 1994. tiscar pg, mosca f. defense mechanisms in farmed marine molluscs. vet. res. commun. 28: 57-62, 2004. verfaillie c, benis a, iida j, mcglave p, mccarthy j. adhesion of committed human hematopoietic progenitors to synthetic peptides from the c  20 terminal heparin-binding domain of fibronectin: cooperation between the integrin alpha2beta1 and the cd44 adhesion receptor. blood 84: 1802-1811, 1994. webb d, parsons j, horwitz a. adhesion assembly, disassembly and turnover in migrating cell-over and over and over again. nat. cell biol. 4: 97100, 2002. wei j, shaw lm, mercurio am. integrin sgnaling in leukocytes: lessons from the α6β1 integrin. j. leukoc. biol. 61: 397-407, 1997. wildering wc, hermann pm, bulloch ag. neurite outgrowth, rgd-dependent, and rgdindentified molluscan motoneurons on selected substrates. j. neurobiol. 35: 37-52, 1998. wright b, lacchini ah, davies aj, walker aj. regulation of nitric oxide production in lymnaea stagnalis defence cells: a role for protein kinase c and extracellular signalregulated kinase signalling pathways. biol. cell 98: 265-278, 2006. yoshino tp. integrin adhesion receptors in molluscan cells. viith international colloquium on invertebrate pathology and microbial control and ivth international conference on bacillus thuringiensis, sapporo, japan, pp 277-283, 2328 august 1998. yurchenco pd, cheng ys. laminin self-assembly: a three-arm interaction hypothesis for the formation of a network in basement membranes. contrib. nephrol. 107: 47-56, 1994. yurchenco pd, o'rear jj. basement membrane assembly. methods enzymol. 245: 489-518, 1994. zampini m, canesi l, betti m, ciacci c, tarsi r, gallo g, et al. role for mannose-sensitive hemagglutitin in promoting interactions between vibrio cholerae el tor and mussel hemolymph. appl. environ. microbiol. 69: 5711-5715, 2003.     21 isj098.pdf 54 isj 2: 54-59, 2005 issn 1824-307x short communication calcium/calmodulin dependence of nitric oxide synthase from viviparus ater immunocytes d tagliazucchi, a conte department of agricultural sciences, university of modena and reggio emilia, italy accepted april 18, 2005 abstract the calcium ion dependence of soluble and particulate nitric oxyde synthase (nos) activity from viviparus ater immunocytes was investigated. at a calcium ion concentration of 2 nm, the nos activity measured by citrulline formation was 27.1 ± 2.2 and 9.3 ± 0.8 pmol/min/106cell for soluble and particulate nos, respectively. the increase in free calcium ion concentration to 300 nm increases enzyme activity to 57.5 ± 4.1 and 23.5 ± 1.2 pmol/min/106cell, respectively. the 50 % activation of the calcium-dependent activity is 91 and 97 nm ca2+ for soluble and particulate enzymes. trifluoperazine, an inhibitor of the calmodulin-dependent enzyme, partially inhibits both activities. soluble nos is five times more sensitive than particulate nos. the behaviour of both activities with three nos inhibitors (7-nitroindazole, s-methylisothiourea sulphate, diphenyleneiodonium) is very similar, with ic50 values that are not significantly different. the calcium ion dependence of nos activities, in a range of free calcium ion variations, which are transiently observed in receptor-stimulated cells, suggests that nitric oxyde in v. ater immunocytes not only has a defensive role but also signalling relevance in crosstalking between immunocytes and other cells. key words: mollusc; viviparus ater; immunocytes; nitric oxide synthase; calcium ion dependence introduction immunocytes are the cells of the immune response in molluscs and other invertebrates against not-self materials. recognition, phagocytosis and killing of virus and bacteria are one of the most important functions of invertebrate immunocytes (ottaviani, 1992). in mammalian phagocytic cells, oxygen reactive species (ros), such as superoxide ions and hydrogen peroxide, hypochlorous acid and nitric oxide (no), are produced to kill phagocyted organisms. from no and superoxide ions, the more reactive peroxynitrite ion and other oxygen radicals are formed (beckman et al., 1990; porasuphatana et al., 2001; heales et al., 1999). the first evidence for no production and utilisation as a bactericidal agent by invertebrate mollusc immunocytes was reported by ottaviani et al. (1993). t h e n i t r i c o x i d e s y n t h a s e ( n o s ) a c t i v i t y o f corresponding author: davide tagliazucchi university of modena and reggio emilia, department of agricultural sciences, via kennedy 17, 42100 reggio emilia, italy e-mail: tagliazucchi.davide@unimore.it immunocytes of the fresh water snail viviparus ater has been partially characterised biochemically (conte and ottaviani, 1995). nos activity shows a partial dependence on calcium ions. it is also present in particulate fractions and is induced by lipopolysaccharides (lps) (conte and ottaviani, 1995). three main different forms of nos have been isolated from different cell types: neuronal nos (nnos), endothelial nos (enos) and inducible nos (inos) from macrophages (pollock et al., 1991; lamas et al., 1992; bredt et al., 1991; stuehr et al., 1991). the constitutive enos and nnos have an absolute requirement for calcium ions and calmodulin. enos is mainly, if not completely, membrane associated. the presence of covalentlybound myristoyl and palmitoyl residues on protein molecules have an important role in binding to membranes. nnos is mainly cytosolic, but may be bound to cell membranes. inos is cytosolic and is expressed upon immunological and inflammatory stimulation. this form is independent of calcium ions and calmodulin (cho et al., 1992; steven-truss and marletta, 1995). inos may produce toxic and lethal no concentrations. 55 the no produced by nnos and enos has a signalling role under the strict control of intracellular calcium ions. alternative spliced forms of nos have been demonstrated (silvagno et al., 1996; magee et al., 1996). the nos enzyme(s) from immunocytes of v. ater combine the properties of different noss (conte and ottaviani, 1995) and cannot be identified with any of the enzymes studied so far, in particular the inos of mammalian phagocytic cells. in the immunocytes of mytilus edulis, morphine induces a transient increase in intracellular calcium ions, followed by no release (nieto-fernandez et al., 1999) suggesting that the immunocytes of this invertebrate have nos with properties that are similar to those of v. ater. the aim of this study was the characterisation of calcium ion dependence of soluble and particulate nos of the snail v. ater. materials and methods reagents (6r)–5,6,7,8-tetrahydro-l-biopterin dihydrochloride was purchased from dr. b. schircks laboratories (jona, switzerland) and l-[2,3,4,5-3h]arginine monohydrochloride (58 ci/mmol) from amersham (buckinghamshire, england). the ion-exchange resin ag50wx-8 was supplied by bio-rad (milan, italy). calmodulin, glucose-6-phosphate dehydrogenase from yeast (ec 1.1.1.49) and glucose-6-phosphate were from serva (heidelberg, germany). 7-nitroindazole (7ni), s-methylisothiourea sulphate (smt), diphenyleneiodonium (dpi), and trifluoperazine (tfp) were from calbiochem (darmstadt, germany). nitrate reductase from aspergillus spp. (ec 1.6.6.2), lipopolysaccharide (lps) from escherichia coli, ngmonomethyl-l-arginine (l-nmma) and all other biochemicals were obtained from sigma (milan, italy). snails adult specimens of viviparus ater were collected from a freshwater canal near modena (italy), in spring and early summer. the animals were maintained at room temperature in de-chlorinated freshwater, for at least a week before the experiments. snail haemolymph was obtained by prodding the animal’s foot and collected with a pasteur pipette. immunocytes were obtained by centrifugation at 600 x g. the cells were washed twice with snail saline solution (ottaviani, 1983), counted and collected using centrifugation. determination of nos activity immunocytes were homogenised with ultra-turrax (ika-werk, germany) in 5 vols. of ice-cold solution, containing 320 mm sucrose, 50 mm tris, 1 mm edta, 1mm dithiothreitol (dtt), 1mm phenylmethylsulphonyl fluoride, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml antipain and 10 µg/ml bestatin, brought to ph 7.0 at 20°c with hcl. the homogenate was centrifuged at 20,000 x g for 30 min at 4 °c, and the supernatant was freed from low molecular mass compounds by sephadex g-25 chromatography (werner-felmayer et al., 1993). the protein fraction was eluted with a buffer containing 50 mm hepes, ph 7.4, 1 mm edta, 1 mm dtt and the above-mentioned protease inhibitors. nos activity was assayed by following the conversion of radiolabelled arginine to citrulline. standard reaction mixtures contained 50 mm hepes, ph 7.4, 0.5 mm edta, 1.4 mm cacl2, 1 mm mgcl2, 1 mm nadph, 1 mm dtt, 12 mm l-valine, 1 mm citrulline, a variable amount of l-arginine, 80,000-100,000 cpm of purified l-[2,3,4,5-3h]arginine monohydrochloride and 5-50 µl of sephadex g-25 eluate in a final volume of 100 µl. after 30 min incubation at 37 °c, [3h]citrulline was quantified by liquid scintillation counting, after separation from [3h]arginine by cation exchange (ag50wx-8) (bredt and snyder, 1989). the nos activity of the nonsoluble fraction was determined by washing the immunocyte pellet twice with pbs, centrifuging at 20,000 x g for 30 min at 4 °c and re-suspending with the buffer used for sephadex g-25 chromatography. nos activity was determined by calculating the difference between the [3h]citrulline produced in the presence and absence of 10 mm ng-monomethyl-larginine (l-nmma, an inhibitor of mammalian nos) in a standard reaction mixture. activity was calculated using the radiochemical method and expressed as pmol of [3h]citrulline formed/min/106 cells. when calcium ion dependence was investigated, variable amounts of cacl2 and egta were added to the reaction mixture. free calcium concentrations were calculated following fabiato (1988) and controlled by the fura 2 method, in accordance with mülsch et al. (1989). in some cases, incubations also contained variable concentrations of the nos inhibitors: 7-ni, smt, dpi or tfp. the results are expressed as means ± sd of at least three independent experiments performed in triplicate. proteins were determined in accordance with the modified lowry method (markwell et al, 1981), with serum albumin as standard. results the dependence of soluble and particulate nos activities on free calcium ion concentrations at ph 7.4 is reported in fig. 1. the soluble enzyme activity (upper line) at a free ca2+ concentration of 2 nm was 27.1 ± 2.2 pmol/min/106 cells and progressively increased to 57.5 ± 4.1 at 300 nm ca2+. the enzyme activity slightly decreased at higher calcium concentrations. the particulate nos activity (iower line), which is about 40 % of the soluble activity, showed similar calcium dependence. fifty percent activation is obtained at a 91 and 97 nm calcium ion concentration for soluble and particulate enzymes, respectively. calcium ions activated both soluble and particulate nos enzymes at concentrations that are physiologically observed during a transient calcium ion increase in cells stimulated by signalling molecules. the calmodulin dependence, evaluated by trifluoperazine inhibition of nos activity, is reported in fig. 2. trifluoperazine is a calmodulin antagonist that inhibits ca2+/calmodulin-dependent enzymes. the nos soluble activity (upper line), measured at a free ca2+ concentration of 300 nm, decreased with increasing concentration of tfp to 55 % of the 56 0 10 20 30 40 50 60 0 1 2 3 4 5 log [trifluoperazine] microm [3 h ] ci tr u lli n e p m o l/m in /1 0 6 c e lls particulate nos soluble nos fig. 1 dependence of soluble and particulate nos activity on free ca2+ concentration. each point is the mean ± sd of three independent experiments performed in triplicate. 0 10 20 30 40 50 60 0 1 2 3 4 5 6 7 log [ca2+] nm particulate nos soluble nos fig. 2 dependence of soluble and particulate nos activity on trifluoperazine concentration. each point is the mean ± sd of three independent experiments performed in triplicate. [3 h ] ci tr u lli n e p m o l/m in /1 0 6 c e lls [3 h ] ci tr u lli n e p m o l/m in /1 0 6 c e lls 57 table 1 ic50 values of some nos activity inhibitors. the inhibitors were included in standard reaction mixtures, containing 1 µm arginine and prepared as described in the text. the data are the means ± sd of three independent experiments performed in triplicate. soluble nos particulate nos tfp (µm) 74 ± 20 383 ± 92 7-ni (nm) 715 ± 134 648 ± 118 dpi (nm) 102 ± 29 130 ± 26 smt (nm) 877 ± 190 1051 ± 276 activity of the control at about 1 mm tfp. no further decrease in the enzyme activity was observed at higher tfp concentrations. the particulate bound activity (lower line) was inhibited by tfp at about a 5-times higher concentration than soluble nos (table 1) similarly to what we found with the carp enzyme (conte and ottaviani, 1998). table 1 reports the ic50 values of the three other inhibitors tested. no significant difference in the ic50 values was observed between the soluble and particulate enzymes. discussion we found that calcium and calmodulin dependence, evaluated by tfp inhibition, of nos activity from immunocytes of v. ater, measured by citrulline formation, is similar to those of the mutant ä 45 of the human enos enzyme (chen and wu, 2003). in this mutant, residues 594-606 and 614-645 of human enos are removed. these residues are not present in human and mouse macrophage inos. the ä 45 mutant is able to form no, measured by citrulline formation, in the absence of calmoduline or calcium ions, at 60% of the rate in their presence. it has been suggested by roman et al. (2000) that the ä 45 segments play a relevant role in ca2+/calmodulin dependence. the presence of these segments decreases or inhibits the electron flow from the reductase domain, which binds nadph, to the oxygenase domain, which binds arginine. ca2+/calmodulin, binding to the enos or nnos, remove inhibition and allow electron transfer from reductase to oxygenase. geller et al. (1993) reported the molecular cloning and expression in 293 embryonic kidney cells of nos induced by lps in human hepatocytes. the chelating agents edta and egta decreased the activity of this enzyme by 30 % but failed to obtain complete inhibition. trifluoperazine decreased activity by 50 %. the amino acid sequence reported by geller et al. (1993) appears to be the same as nos from human macrophages. whatever the molecular mechanism of partial calcium dependence of v. ater, nos raises several questions on its roles and regulation in immunocytes. nos has been demonstrated in the nervous system of several species of snail. no has neurotransmitterlike functions, for example, it is necessary for the transmission of sensory information to the central pattern generator for feeding behaviour (elphick et al., 1995) and the activation of buccal motor patterns (moroz et al., 1993). no is involved in neural transmission to intestinal muscles in helix lucorum but enteric release of no is blocked during snail dormancy (roszer et al., 2004). no has a role in developing the nervous system of the snail llyanassa obsoleta (thavaradhara and leise, 2001) and regulating the early embryonic behaviour in the snail helisoma trivolvis (cole et al., 2002). in all of the above examples, the constitutive calcium-dependent nnos is considered to be the enzyme that produces no. identification of nos enzymes has been carried out mainly by immunocytochemical methods, using antibodies to mammalian nnos. amino acid sequence studies show that neuronal nos from insects (drosophila melanogaster and schistocerca gregaria) and the snail lymnaea stagnalis, share 43-67 % identity with mammalian nnos (ogunshola et al., 1995; regulski and tully, 1995). it has been reported that v. ater soluble nos is inhibited by 70 % by mammalian nnos antibodies (conte and ottaviani, 1995). moroz et al. (1996) demonstrated constitutive calcium-independent nos activity in the nervous system of some molluscan species, which is largely associated with particulate fractions. using immunochemical investigations, xie et al. (2002) showed that the substrate arginine and the nos enzyme are localised in the separate, but adjacent, neurons of the snail helix pomatia. the product citrulline is observed in the neurons, which contain the nos enzyme, suggesting an unknown signalling pathway between neurons to maintain arginine and no homeostasis. the examples reported above show the complexity of the properties and regulation of nos activity in neuronal cells of snails, as well the expanding roles of no. the defense role of snail immunocyte nos has been demonstrated (ottaviani et al., 1993). during phagocytosis, these cells also produce oxygen species that may combine with no to form the more reactive peroxynitrite, increasing killing capacity. however, it seems that immunocytes are not only of immunological relevance, as they also synthesise signalling peptides (ottaviani et al., 1992). invertebrate immunocytes respond to multiple signal molecules that form the immunoregulatory network. cytokine-like factors affect immune functions such as cell motility, chemotaxis, phagocytosis and cytotoxicity in invertebrate immunocytes and are able to induce no (ottaviani et al., 2004). cytokinestimulated immunocytes in m. edulis modulate ganglionic no release, which later affects their activity level, demonstrating that ganglionic no is involved in down-regulating immunocyte activity (stefano et al., 2004). it has been demonstrated that m. edulis immunocytes have a µ3 morphine receptor, coupled to no release in an intracellular calciummediated manner (nieto-fernandez et al., 1999). no release may mediate morphine-induced changes in immunocyte conformation, lowering chemotactic activity, cellular velocity, and adherence (stefano et al., 1993). furthermore, naturally occurring cannabinoids may share the no-producing effector system with opiate alkaloids in these cells (stefano et al., 1996). not only can morphine-like and 58 cannabinoid substances be found in invertebrate immunocytes, there is also a morphine-like precursor that provides proof of the presence of opiates in these animals (stefano et al., 1993). this evidence, supporting the complexity of the immunoregulatory processes, enables us to assume that the binding of signalling molecules to v. ater immunocyte receptors may determine a calcium-dependent release of no, which allows complex and physiologically relevant cross-talking between immunocytes and other cells in molluscs. acknowledgements this work was supported by a miur (italy) grant to ac. references beckman js, beckman tw, chen j, marshall pa, freeman ba. apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. proc. natl. acad. sci. usa 87: 16201624,1990. bredt ds, hwang pm, glatt ce, lowenstein c, reed rr, snyder sh. cloned and expressed nitric oxide synthase structurally resembles cytochrome p-450 reductase. nature 351: 714-718, 1991. bredt ds, snyder sh. nitric oxide mediates glutamate-linked enhancement of cgmp levels in the cerebellum. proc. natl. acad. sci. usa 86: 9030-9033, 1989. chen pf, wu kk. structural elements contribute to the calcium/calmodulin dependence on enzyme activation in human endothelial nitric-oxide synthase. j. biol. chem. 278: 52392-52400, 2003. cho hj, xie qw, calaycay j, mumford ra, swiderek km, lee td, nathan c. calmodulin is a subunit of nitric oxide synthase from macrophages. j. exp. med. 176: 599-604, 1992. cole ag, mashkournia a, parries sc, goldberg ji. regulation of early embryonic behaviour by nitric oxide in the pond snail helisoma trivolvis. j. exp. biol. 205: 3143-3152, 2002. conte a, ottaviani e. characterization of cyprinus carpio brain nitric oxide synthase. neurosci. lett. 242: 155-158, 1998. conte a, ottaviani e. nitric oxide synthase activity in molluscan immunocytes. febs lett. 365: 120-124, 1995. elphick mr, kemenes g, staras k, o'shea m. behavioral role for nitric oxide in chemosensory activation of feeding in a mollusc. j. neurosci. 15: 7653-7664, 1995. fabiato a. computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solution containing multiple metals and ligands. methods enzymol. 157: 379-417, 1988. geller da, lowenstein cj, shapiro ra, nussler ak, di silvio m, wang sc, nakayama dk, simmons rl, snyder sh, billiar tr. molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. proc. natl. acad. sci. usa 90: 3491-1495, 1993. heales sj, bolanos jp, stewart vc, brookes ps, land jm, clark jb. nitric oxide, mitochondria and neurological disease. biochim. biophys. acta 1410: 215-228, 1999. knowles g, moncada s. nitric oxide synthase in mammals. biochem. j. 298: 249 258, 1994. lamas s, marsden pa, li gk, tempst p, michel t. endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. proc. natl. acad. sci. usa 89: 6348-6352, 1992. magee t, fuentes am, garban h, rajavashisth t, marquez d, rodriguez ja, rajfer j, gonzalez-cadavid nf. cloning of a novel neuronal nitric oxide synthase expressed in penis and lower urinary tract. biochem. biophys. res. commun. 226: 145-151,1996. markwell mak, haas sm, tolbert ne, bieber ll. protein determination in membrane and lipoprotein samples: manual and automated procedures. methods enzymol. 72: 296-303, 1981. moroz ll, chen d, gillette mu, gillette r. nitric oxide synthase activity in molluscan cns. j. neurochem. 66: 873-876, 1996. moroz ll, park jh, winlow w. nitric oxide activates buccal motor patterns in limnea stagnalis. neuroreport 4: 643646, 1993. mülsch a, bassenge e, busse r. nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism. naunynschmiedeberg’s arch. pharmacol. 340: 767-770, 1989. nieto-fernandez fe, mattocks d, cavani f, salzet m, stefano gb. morphine coupling to invertebrate immunocyte nitric oxide release is a dependent on intracellular calcium transients. comp. biochem. physiol. 123b: 295-299, 1999. ogunshola o, picot j, piper m, korneev s, o'shea mr. molecular analysis of the nocgmp signaling pathway in insect and molluscan cns. soc. neurosci. abstr. 21: 631, 1995. ottaviani e. immunorecognition in the gastropod molluscs with particular reference to the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). boll. zool. 59: 129-139, 1992. ottaviani e. the blood cells of the freshwater snail planorbarius corneus (gastropoda, pulmonata). dev. comp. immunol. 7: 209-216, 1983. ottaviani e, franchini a, fontanili p. the presence of immunoreactive vertebrate bioactive peptide substances in immunocytes of the freshwater snail viviparus ater (gastropoda, prosobranchia). cell. mol. neurobiol. 12: 455-462, 1992. ottavianie, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. ottaviani e, paemen lr, cadet p, stefano gb. evidence for nitric oxide production and utilization as a bactericidal agent by invertebrate immunocytes. eur. j. pharmacol. 248: 319-324, 1993. pollock js, forstermann u, mitchell ja, warner td, schmidt hhhw, nakane m, murad f. purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. proc. natl. acad. sci. usa 88: 10480-10484, 1991. porasuphatana s, tsai p, pou s, rosen gm. involvement of the perferryl complex of nitric oxide synthase in the catalysis of secondary free radical formation. biochim. biophys. acta 1526: 95-104, 2001. regulski m, tully t. molecular and biochemical characterization of dnos: a drosophila ca2+/calmodulin-dependent nitric oxide synthase. proc. natl. acad. sci. usa 92: 9072-9076, 1995. roman lj, martasek p, miller rt, harris de, de la garza ma, shea tm, kim jjp, master bs. the c termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin. j. biol.chem. 275: 2922529232, 2000. roszer t, czimmerer z, szentmiklosi aj, banfalvi g. nitric oxide synthesis is blocked in the enteral nervous system during dormant periods of the snail helix lucorum. cell. tissue res. 316: 255-262, 2004. silvagno f, xia h, bredt ds. neuronal nitric-oxide synthase-ì, an alternative spliced isoform expressed in differentiated skeletal muscle. j. biol. chem. 271: 11204-11208, 1996. stefano gb, digenis a., spector s., leung mk, bilfinger tv, makman mh, scharrer b, abumrad nn. opiate-like substance in an invertebrate, an opiate receptor on 59 invertebrate and human immunocytes, and a role in immunosuppression. proc. natl. acad. sci. usa 90: 11099-11103, 1993. stefano gb, liu y, goligorsky ms. cannabinoid receptors are couplet to nitric oxide release in invertebrate immunocytes, microglia, and human monocytes. j. biol. chem. 271: 19238-19242, 1996. stefano gb, mantione k, jones d, zhu w, casares f, cadet p. immunocytes modulate ganglionic nitric oxide release which later affects their activity level. neuro. endocrinol. lett. 25: 57-61, 2004. stevens-truss r, marletta ma. interaction of calmodulin with the inducible murine macrophage nitric oxide synthase. biochemistry 34: 15638-45, 1995. stuehr dj, cho hj, kwon ns, weise mf, nathan cf. purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an fad-and fmncontaining flavoprotein. proc. natl. acad. sci. usa 88: 7773-7777, 1991 thavaradhara k, leise em. localization of nitric oxide synthase-iike immunoreactivity in the developing nervous system of the snail llyanassa obsoleta. j. neurocytol. 30: 449-456, 2001. werner-felmayer g, werner er, fuchs d, hausen a, mayer b, reibnegger g, weiss g, wachter h. ca2+/calmodulin-dependent nitric oxide synthase activity in the human cervix carcinoma cell line me-180. biochem. j. 289: 357-361, 1993. xie m, hermann a, kerschbaum hh. complementary distribution of nadph-diaphorase and l-arginine in the snail nervous system. cell. tissue res. 307: 393-400, 2002. the hemocytes of polyandrocarpa mysakiensis and their role in defense reactions isj 6: 154-161, 2009 issn 1824-307x research report the hemocytes of polyandrocarpa mysakiensis: morphology and immune-related activities l ballarin1, k kawamura2 1department of biology, university of padua, padua, italy 2laboratory of cellular and molecular biotechnology, faculty of science, kochi university, japan accepted november 20, 2009 abstract a preliminary study of the hemocytes of developing buds of the compound ascidian polyandrocarpa misakiensis was carried out at the light microscope level for a better understanding of their biological role. similarly to other ascidians, p. misakiensis immunocytes are represented by phagocytes and morula cells. phagocytes include hyaline amoebocytes and round, giant phagocytes, the former the probable precursors of the latter. hyaline amoebocytes showed high macropinocytotic activity in the presence of bacteria, whereas yeast cells were ingested by phagocytosis. morula cells contain the enzyme phenoloxidase inside their vacuoles, probably stored as pro-enzyme, which is released upon the recognition of non-self. together with macrogranular leukocytes, morula cells were the most abundant hemocyte-types which stresses the importance of these cells in polyandrocarpa biology. macrogranular leukocytes are frequently found inside the vacuoles of phagocytes and were recognized by a polyclonal antibody raised against an opsonin purified from the colonial ascidian botryllus schlosseri, which suggests that a similar lectin can be involved in the interaction between these cells and phagocytes. key words: polyandrocarpa misakiensis; colonial ascidians; hemocytes; morphology; immunity introduction invertebrate chordates or protochordates are represented by cephalochordates and tunicates, the latter being the sister group of vertebrates (delsuc et al., 2006). the peculiar phylogenetic position of tunicates explains the increasing interest towards their biology, in particular, developmental biology and immunobiology. the majority of tunicates are represented by ascidians, sessile filter-feeding marine animals which include both solitary and colonial species. many types of hemocytes are found in ascidians. their morphology has been described by many authors (pérès, 1943; endean, 1955; sabbadin, 1955; andrew, 1961; overton, 1966; smith, 1970a, b; milanesi and burighel, 1978; scippa et al., 1982; burighel et al., 1983; schlumpberger et al., 1984; sawada et al., 1991, 1993; zhang et al., 1992; azumi et al., 1993; dansohkawa et al., 1995; arizza and parrinello, 2009) and various classification criteria have been ___________________________________________________________________________ corresponding author: loriano ballarin department of biology university of padua via u. bassi 58/b, 35100 padua, italy email: loriano.ballarin@unipd.it proposed (goodbody, 1974; wright, 1981; rowley et al., 1984; de leo, 1992; burighel and cloney, 1997). however, uncertainties still exist on their functions, mutual relationships and differentiation pathways. polyandrocarpa misakiensis (fig. 1) is a polystyelid compound ascidian, common along the coasts of the temperate regions of japan, which can reproduce asexually through continuous budding from parental zooids (kawamura and fujiwara, 1994; kawamura et al., 2008). the morphological, biochemical and molecular events occurring during bud differentiation and maturation have been widely studied (kawamura and fujiwara, 1994; hisata et al., 1998; kawamura and sugino, 1999; kamimura et al., 2000; matsumoto et al., 2001; sunanaga et al., 2007; kawamura et al., 2006, 2008). nevertheless, few data are available on polyandrocarpa hemocytes: their morphology has been studied at the electron microscope, but scanty data are available for light microscopy. in addition, the abundance of the different hemocyte types and their possible roles in immune defense have been little investigated. in order to fill this gap, we carried out a preliminary investigation aimed to a better characterization of polyandrocarpa hemocytes at the 154 fig. 1 colony of p. misakiensis. adult zooids (z) bear many buds (b). developing buds are indicated by asterisks. bar = 0.5 mm. light microscope, with particular reference to immunocytes, a well-defined class of circulating hemocytes responsible of both cellular and humoral (through their secretions) immune responses. results confirm the presence of phagocytes, able to quickly ingest foreign particles, through phagocytosis and macropinocytosis, and morula cells which, like many other compound ascidians, are probably involved in cytotoxic immune reactions. in addition, granular leukocytes, which are circulating trophocytes, are recognized by a polyclonal antibody, raised against a lectinic opsonin from botryllus schlosseri, which represents a good and specific marker for this cell type. materials and methods animals colonies of polyandrocarpa misakiensis were reared in the field, attached to glass plates, near the usa marine biological institute, kochi university, japan. when required, they were brought in the laboratory of cellular and molecular biotechnology of the faculty of science and kept at room temperature for few days before their use in a 20-l aerated aquarium filled with seawater. hemocyte collection and culture hemolymph was collected from developing buds (fig. 1) of colonies, previously immersed for few min in 0.38 % na-citrate in artificial seawater (asw), ph 7.5, in order to prevent cell clotting and then blotted dry. buds, already detached from the parent zooid, were punctured with a fine tungsten needle and hemolymph was collected with a glass micropipette and centrifuged at 700xg for 10 min at 4 °c; the pellet was re-suspended in asw to a final concentration of 106 cells/ml. fifty μl of hemocyte suspension were placed in the center of a glass coverslip, previously coated with poly-l-lysine (50 μg/ml), and left to adhere, for 60 min, at room temperature (rt) in a moist chamber. hemocytes were then observed under the light microscope or, alternatively, fixed in 1 % glutaraldehyde in asw containing 1 % sucrose, washed in phosphatebuffered saline (pbs: 8 g/l nacl, 0.2 g/l kcl, 0.2 g/l kh2po4, 1.15 g/l, ph 7.4) and stained for 5 min in 10 % giemsa’s solution. coverslips were then mounted on microscope slides with 80 % glycerol. hemocytes from at least 10 different colonies were observed and counted. blood plasma and hemocyte lysate preparation freshly collected hemolymph was centrifuged at 700xg for 10 min at 4 °c. the resulting supernatant was referred to as blood plasma (bp), whereas the pellet was re-suspended in an equal volume of distilled water, subjected to sonication for 20 s at 0 °c in a braun labsonic u sonifier at 50 % duty cycles and centrifuged at 10,000xg for 10 min in order to get hemocytes lysate (hl) as supernatant. phagocytosis and macropinocytosis assays after adhesion, hemocytes were incubated at rt, in a moist chamber, with 50 µl of a yeast (saccharomyces cerevisiae) suspension in asw (yeast/hemocyte ratio = 10:1) for 60 min, and the uningested yeast was then removed by dipping the coverslips repeatedly in a large volume of asw. living hemocytes were then observed under the light microscope. alternatively, monolayers were fixed and stained as described above before their observation. in another series of experiments, hemocytes were incubated for 60 min with a suspension of living escherichia coli in asw (109 cells/ml). 155 coverslips were then washed by repeated dipping in asw and cells were fixed and stained as described above before their observation under the light microscope. immunocytochemical analysis fixed hemocytes were incubated for 30 min in 1 % h2o2 to block endogenous peroxidase activity, washed in pbs, treated with 2 % powdered milk for 30 min and then incubated for 1 h with 50 μg/ml of purified polyclonal antibody raised against b. schlosseri rhamnose-binding lectin (bsrbl; ballarin et al., 2000; gasparini et al., 2008) in a moist chamber; pre-immune serum was used in controls. after washing in pbs containing 0.1 % tween 20, they were incubated for 30 min in 1 μg/ml of horseradish peroxidase-labelled mouse anti-rabbit secondary antibody (vector laboratories), washed in distilled water and treated with true blue (kpl), which stains positive sites blue, according to the manufacturer’s instructions. assay for phenoloxidase (po) activity on hemolymph, bp, hl and hemocyte incubation medium twenty μl of hemolymph, collected as described above, bp or hl were added to 180 μl of a saturated solution of dihydroxyphenyl-l-alanine (l-dopa) in pbs in the wells of flat bottomed, 96well microtiter plates and the time course of the reaction was read at 490 nm for 5 min with a biorad imark microplate reader. five mm na2so3, a po inhibitor (kong et al., 2000; cong et al., 2005), were added to the hemolymph in negative controls. one relative unit (ru) of po activity was defined as the increase in absorption of 0.001/min in the reaction mixture (söderhäll and smith, 1983). protein concentrations of the supernatants were determined according to lowry et al. (1951) and results were expressed as ru/mg protein. in another series of experiments, 100 μl of hemocytes suspension (106 cells/ml) were incubated for 60 min in asw or, alternatively in yeastcontaining asw (yeast:hemocyte ratio = 10:1). the supernatants were then collected by centrifugation at 700xg at 4 °c and assayed for po activity as described above. cytoenzymatic assay for po activity after fixation, hemocytes were incubated for 1h in saturated dihydroxy-l-phenylalanine (ldopa) solution in pbs in the presence or in the absence of 5 mm na2so3, washed in distilled water, and mounted in acquovitrex. positive cells converted l-dopa to dopachrome and stained blackish-brown. statistical analysis each experiments was repeated at least three times. po activity data were compared with the student’s t test. results the hemocytes of p. misakiensis the following main cell-types could be recognized: i) undifferentiated cells, 4-6 μm in diameter, with a high nucleus-cytoplasm ratio. they amounted to 4.4 ± 0.7 % of circulating cells (figs 2a, b); ii) hyaline amoebocytes, 6-12 μm in length, have a variable shape, homogeneous cytoplasm with various cytoplasmic protrusions (pseudopods), and a roundish nucleus (figs 2c, d). they represent 4.4 ± 0.4 % of the hemocytes; iii) round phagocytes, or macrophage-like cells, 8.1 ± 2.9 % of the hemocytes. they have a spheroidal shape, 10-15 µm in diameter; their cytoplasm can be stained metachromatically by giemsa’s dye, and shows one or few large vacuole(s) containing ingested material which occupy most of the cell volume (figs 2h-k); iv) microgranular leukocytes, 15.3 ± 3.5 % of the total hemocytes, 4-6 µm in diameter. they frequently show an amoeboid form and are characterized by the presence of small granules in their cytoplasm which frequently assume a metachromatic red color with giemsa’s dye (fig. 2e); v) macrogranular (granular) leukocytes, 10-15 µm in size, one of the most abundant circulating cell type in polyandrocarpa, representing 33.9 ± 3.1 % of the hemocytes. they are giant round cells with the nucleus usually found at the periphery of the cell and the cytoplasm filled with many granules, of variable size (up to 2-3 μm in diameter), which appear translucent in living cells and almost empty after fixation (figs 2i, l, m); vi) morula cells (mcs), 10-15 μm in diameter, 32.9 ± 4.4 % of the circulating cells. they are also round cells characterized by the presence of many vacuoles, variable in size, which appear yellowish in living cells and acquire a green color after aldehyde fixation (figs 2i, q) and vii) pigment cells,1.2 ± 0.2 % of the total hemocytes number, 10-15 µm in size. they are characterized by the presence of many small vacuoles filled with red pigment and a nucleus located at the periphery of the cell. their morphology is rarely preserved in fixed samples (fig. 2l). polyandrocarpa phagocytes behave differently in the presence of yeast and bacteria when hemocytes were incubated in the presence of yeast, phagocytes, mainly round cells, filled with yeast cells were frequently found after 60 min of incubation (figs 2h, k). no changes in mc morphology were observed. conversely, in the presence of bacteria most of hyaline amoebocytes showed heterogeneous macropinocytotic vesicles inside their cytoplasm which appeared empty under the light microscope (fig. 2f). most of the microbes resulted agglutinated outside the cells (fig. 2f) and in few cases they were visible inside phagocytes (fig. 2g). 156 fig. 2 hemocytes of p. misakiensis. a, b: living (a) and giemsa-stained (b) undifferentiated cells. c, d: living (c) and stained (d) hyaline amebocytes. e: microgranular leukocyte fixed and stained with giemsa. f: fixed and stained hyaline amebocyte after exposure to e. coli; many micropinocytotic vesicles (mv) are visible as well as agglutinated bacteria (arrowhead) outside the cell. g: fixed and stained hyaline amebocyte with ingested bacteria (arrowheads). h: living phagocytes with ingested yeast cells (arrows). i: living round phagocyte (ph), macrogranular leukocyte (ml) and morula cell (mc). j, k: fixed and stained round phagocytes with vacuoles containing ingested materials (yeast cells in k). l: living macrogranular leukocytes (ml) and pigment cell (p). m: fixed and stained macrogranular (ml) and microgranular (mi) leukocytes. n: fixed macrogranular leukocytes showing immunopositivity to anti-bsrbl antibody on their surface. o: round phagocyte having ingested a immunopositive to anti-bsrbl antibody (arrowhead). p: living macrogranular leukocyte (ml) and morula cells (mc). q: aldehyde-fixed (unstained) morula cells: vacuoles assume a green color. r: fixed hemocytes treated with l-dopa: stain for dopachrome production is evident in morula cells (mc) but not in macrogranular leukocytes (ml). bar = 5 µm. macrogranular leukocytes are recognized by the anti-bsrbl antibody the anti-bsrbl antibody recognized specifically the surface of macrogranular leukocytes (fig. 2n). in developing buds, these cells are frequently found inside phagocyte vacuoles and, in some cases, we could observe labeled cells inside round phagocytes (fig. 2o). po activity is located in mcs when the po activity of whole hemolymph (wh) and bp were compared, the former resulted more than three times higher than the latter, suggesting that the majority of the enzyme was present, in normal conditions, inside the hemocytes. this was confirmed by the higher enzyme activity (3 times that of the hemolymph) of hl. the addition of 157 table 1 po activity of whole hemolymph (wh), blood plasma (bp) and hemocyte lysate (hl) po source po activity (ru/mg protein) wh 1239.2 ± 53.1 wh + 5mm na2so3 0.7 ± 0.2 *** bp 374.3 ± 40.4 *** hl 3720.5 ± 99.3 *** *** p < 0.001 with respect to wh na2so3 to the hemolymph completely abolished the oxidation of l-dopa (table 1). after 60 min of incubation of hemocytes in asw, the po activity of the culture medium amounted to 29.8 ± 4.0 ru/mg protein. conversely, when blood cells were incubated in a suspension of yeast in asw, the resulting enzyme activity of the medium was significantly (p < 0.001) increased and reached the value of 63.3 ± 10.9 ru/mg protein. cytoenzymatic analysis in the presence of ldopa clearly showed that the only mcs, and no other cells type, were labeled in the presence of ldopa (fig. 2r). discussion colonies of the ascidian p. misakiensis continuously form new buds as outgrowths of the parental body which soon separate so that morphogenesis occurs without any contact with the parental organism. for these reasons, this species is considered an excellent model organism for the study of stem cell differentiation during asexual reproduction and regeneration (kawamura and fujiwara, 1994; hisata et al., 1998; kawamura and sugino, 1999; kamimura et al., 2000; matsumoto et al., 2001; sunanaga et al., 2007; kawamura et al., 2006, 2008). hemocytes have been claimed to take part in polyandrocarpa development to adulthood as both a source of undifferentiated cells (kawamura et al., 1991, 2008) and a reservoir of nutrients required for the completion of bud morphogenesis when the young individuals are not yet feeding (kawamura and nakauchi, 1986; kawamura et al., 1991, 1992). however, despite their importance, there are few data in the literature describing polyandrocarpa blood cells, their abundance and behavior (kawamura et al., 1992; sugino et al., 1993). in the present work, we carried out a light microscope morphological study on p. misakiensis hemocytes as a further contribution for better understanding the biological roles of these cells. undifferentiated hemocytes represent less than 5 % of the circulating cells, in agreement with what found in other compound ascidians (cima et al., 2001; ballarin et al., 2005). the presence of circulating undifferentiated cells, or hemoblasts, involved in asexual reproduction is a common feature of colonial ascidians. analogously to what described in stolonal budding of perophora (freeman, 1964) and in vascular budding of botryllid (oka and watanabe, 1957, 1959; sabbadin et al., 1975; rinkevich et al., 1995; rinkevich et al., 2007; voskoboynik et al., 2007), these cells exert a fundamental role in polyandrocarpa bud morphogenesis (kawamura and nakauchi, 1991; kawamura et al., 1991). hyaline amebocytes and round phagocytes have been previously included in the same category of hyaline leukocytes, involved in phagocytosis (sugino et al., 1993). indeed, like in botryllid ascidians, they are probably different morphs of a single phagocyte type which can actively move towards foreign cells or particles by ameboid progression and, upon the ingestion of non-self material, withdraws its cytoplasmic projections assuming a globular shape (cima et al., 2001; ballarin and cima, 2005). hyaline amebocytes of the compound ascidian botryllus schlosseri are capable of constitutive macropinocytosis (ballarin and burighel, 2006): the same process is probably responsible of the abundance of hollow vesicles observed in the cytoplasm of polyandrocarpa hyaline leukocytes (sugino et al., 1993). a clear macropinocytotic activity was observed in the presence of bacteria, in accordance with the general view of macropinocytosis as a process generally responsible of the ingestion of bacteria and necrotic material (krysko et al., 2003). the presence of agglutinated bacteria outside the cells suggests the release of agglutinins, likely lectins, by activated phagocytes. a similar agglutinating ability towards some bacterial strains have been recently demonstrated for bsrbl (unpublished data). conversely, no increase in macropinocytotic activity was observed when yeast cells were used as foreign particles: in this case, analogously to what described in other colonial ascidians (ballarin et al., 1994), phagocytosis occurred and phagocytes turned to large, round cells filled with yeastcontaining vacuoles. mcs represent one of the most abundant circulating hemocyte types. in botryllid ascidians, their frequency ranges from 20 to 60 % (ballarin, 2008) and they are important mediators of the response to non-self as both effectors of allorejection between contacting, genetically incompatible colonies (hirose et al., 1990; ballarin et al., 1995, 1998; rinkevich et al., 1998; shirae et 158 al., 1999, 2002; cima et al., 2004), and sites of synthesis of cytokine(s), able to influence phagocyte activity, in response to non-self recognition (ballarin et al., 2001; menin et al., 2005, menin and ballarin, 2008). in b. schlosseri, one of the first event consequent to the recognition of foreign molecules by mcs is their degranulation and the release of their vacuolar content, mainly po and its polyphenol substrates. like botryllid ascidians, in polyandrocarpa, po is contained inside hemocytes, probably as a precursor which can be readily activated by cell manipulation (ballarin et al., 1998), and mcs are the only cell type showing the enzyme activity. in addition, the enzyme can be released in the medium upon the recognition of foreign cells such as yeast cells. botryllid po is involved in the induction of cytotoxicity consequent to the recognition of non-self in both allorejection (ballarin et al., 1995, 1998; shirae et al., 2002) and responses towards foreign cells or particles (ballarin et al., 1998, 2005): as no allorecognition phenomena are known in polyandrocarpa, we can hypothesize that, in this species, mcs exert a general immunosurveillance role, being capable to induce cytotoxicity through the release of their vacuolar content, both po (present data) and peroxidase (kawamura et al., 1992), upon the recognition of non-self, even if no apparent signs of degranulation are visible in blood smears. macrogranular leukocytes constitute the other most common circulating cell-type in polyandrocarpa developing buds. their abundance suggests an important role for these cells and, indeed, there is a general agreement on the fact that they are trophocytes, involved in the uptake and storage of nutrients useful for bud morphogenesis (kawamura and nakauchi, 1986; kawamura et al., 1992); the alkaline phosphatase activity associated with their plasma membrane could be involved in this process (kawamura et al., 1992). this is in agreement with the observation that, in developing buds, they are frequently found inside phagocytes. the antibody raised against bsrbl recognized specifically the surface of macrogranular leukocytes suggesting the presence of molecules sharing some degree of similarity with bsrbl. a lectin with galactose specificity has already been demonstrated in these cells (kawamura et al., 1991). since the lectin from b. schlosseri has been demonstrated to exert an opsonic role through the coating of foreign cells before their phagocytosis and immunopositive macrogranular leukocytes are sometimes present in vacuoles of round phagocytes, we can hypothesize: i) that the molecule recognized by the anti-bsrbl antibody can have a role in the recognition of trophocytes by phagocytes and ii) that in polyandrocarpa asexual development, like in botryllus take-over (lauzon et al., 2002; cima et al., 2003, 2009; ballarin et al., 2008), phagocytosis is the main process which render available nutrients stored in trophocytes and dying cells, respectively. collectively, our results indicate or suggest some possible biological roles for polyandrocarpa hemocytes. however, many questions remain unanswered, such as the molecular mechanism of trophocyte recognition by phagocytes in order to render nutrients available to developing buds, or the role of mc and po in a species where allorecognition phenomena are not known, or the influence of mc on phagocyte activity. future research will help in clarifying the above points. acknowledgements this work was partially supported by a grant (fy2009) from the japan society for the promotion of science to one of us (lb) for working at kochi university. authors wish to thank the personnel at usa marine biological institute for technical help. references andrew w. phase microscope studies of living blood-cells of the tunicates under normal and experimental conditions, with a description of a new type of motile cell appendage. quart. j. microsc. sci. 102: 89-105, 1961. arizza v, parrinello d. inflammatory hemocytes in ciona intestinalis innate immune response. inv. surv. j. 6: s58-s66, 2009. azumi k, satoh n, yokosawa h. functional and structural characterization of hemocytes of the solitary ascidian, halocynthia roretzi. j. exp. zool. 265: 309-316, 1993. ballarin l. immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art. inv. surv. j. 54-74, 2008. ballarin l, cima f. cytochemical properties of botryllus schlosseri haemocytes: indications for morpho-functional characterisation. eur. j. histochem. 49: 255-264, 2005. ballarin l, burighel p. rgd-containing molecules induce macropinocytosis in ascidian hyaline amoebocytes. j. invertebr. pathol. 91: 124-130, 2006. ballarin l, cima f, sabbadin a. phagocytosis in the colonial ascidian botryllus schlosseri .dev. comp. immunol. 18: 467-481, 1994. ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri .dev. comp. immunol. 22: 479-492, 1998. ballarin l, tonello c, sabbadin a. humoral opsonin from the colonial ascidian botryllus schlosseri as a member of the galectin family. mar. biol. 136: 813-822, 2000. ballarin l, franchini a, ottaviani e, sabbadin a. morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species botryllus schlosseri. biol. bull. 201: 59-64, 2001. ballarin l, menin a, franchi n, bertoloni g, cima f. morula cells and non-self recognition in the compound ascidian botryllus schlosseri. inv. surv. j. 2: 1-5, 2005. ballarin l, menin a, tallandini l, matozzo v, burighel p, basso g, et al. haemocytes and blastogenetic cycle in the colonial ascidian botryllus schlosseri: a matter of life and death. cell tissue res. 331: 555-564, 2008. 159 burighel p, cloney ra. urochordata: ascidiacea. in: harrison fw and ruppert ee (eds), microscopic anatomy of invertebrates, vol. 15, wiley-liss, new york, pp 221-347, 1997. burighel p, milanesi c, sabbadin a. blood cell ultrastructure of the ascidian botryllus schlosseri. ii. pigment cells. acta zool. (stockh.) 64: 15-23, 1983. cima f, perin a, burighel p, ballarin l. morphofunctional characterisation of haemocytes of the compound ascidian botrylloides leachi (tunicata, ascidiacea). acta zool. 82: 261-274, 2001. cima f, basso g, ballarin l. apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian botryllus schlosseri. cell tissue res. 312: 369-376, 2003. cima f, sabbadin a, ballarin l. cellular aspects of allorecognition in the compound ascidian botryllus schlosseri. dev. comp. immunol. 28: 881-889, 2004. cima f, manni l, menin a, basso g, fortunato e, accordi b, et al. hovering between death and life: haemocytes and natural apoptosis in the blastogenetic cycle of the colonial ascidian botryllus schlosseri. dev. comp. immunol. doi:10.1016/j.dci.2009.10.005. cong r, sun w, liu g, fan t, meng x, yang l, et al. purification and characterization of phenoloxidase from clam ruditapes philippinarum. fish shellfish immunol. 18: 61-70, 2005. dan-sohkawa m, morimoto m, mishima h, kaneko h. characterization of coelomocytes of the ascidian halocynthia roretzi based on phasecontrast, time-lapse video and scanning electron microscopic observations. zool. sci. 12: 289-301, 1995. de leo g. ascidian hemocytes and their involvement in defense reactions. boll. zool. 59: 195-213, 1992. delsuc f, brinkmann h, chourrout d, philippe h. tunicates and not cephalochordates are the closest living relatives of vertebrates. nature 439: 965-958, 2006. endean r. studies of the blood and tests of some australian ascidians. i. the blood of pyura stolonifera (heller). austr. j. mar. freshwat. res. 6: 35-59, 1955. gasparini f, franchi n, spolaore b, ballarin l. novel rhamnose-binding lectins from the colonial ascidian botryllus schlosseri. dev. comp. immunol. 32: 1177-1191, 2008. goodbody i. the physiology of ascidians. adv. mar. biol. 12: 1-149, 1974. hirose e, saito y, watanabe h. allogeneic rejection induced by cut surface contact in the compound ascidian, botrylloides simodensis. invertebr. reprod. dev. 17: 159-164, 1990. hisata k, fujiwara s, tsuchida y, ohashi m, kawamura k. expression and function of a retinoic acid receptor in budding ascidians. dev. genes evol. 208: 537-546, 1998. kamimura m, fujiwara s, kawamura k, yubisui t. functional retinoid receptors in budding ascidians. develop. growth differ. 42: 1-8, 2000. kawamura k, nakauchi m. mitosis and body patterning during morphallactic development of palleal buds in ascidians. dev. biol. 116: 39-50, 1986. kawamura k, nakauchi m. homeostatic integration of stem cells dynamics during palleal budding of ascidians. zool. sci. 8: 11-22, 1991. kawamura k, fujiwara s. transdifferentiation of pigmented multipotent epithelium during morphallactic development of budding tunicates. int. j. dev. biol. 38: 369-377, 1994. kawamura k, sugino ym. cell adhesion in the process of asexual reproduction of tunicates. microsc. res. tech. 44: 269-278, 1999. kawamura k, fujiwara s, sugino ym. buddingspecific lectin induced in epithelial cells is an extracellular matrix component for stem cell aggregation in tunicates. development 113: 995-1005, 1991. kawamura k, yamamoto t, sugino ym, fujiwara s. alkaline phosphatase and peroxidase activities of blood cells in the polystyelid ascidian, polyandrocarpa misakiensis. mem. fac. sci. kochi univ. (ser. d) 13: 9-17, 1992. kawamura k, kariya y, ono y, muramoto a, ohta k, fujiwara s. molecular collaborations between serpins and trefoil factor promote endodermal cell growth and gastrointestinal differentiation in budding tunicates. develop. growth differ. 48: 309-322, 2006. kawamura k, sugino y, sunanaga t, fujiwara s. multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates. develop. growth differ. 50: 1-11, 2008 kong kh, hong mp, choi ss, kim yt. purification and characterization of a highly stable tyrosinase from thermomicrobium roseum. biotechnol. appl. biochem. 31: 113-118, 2000. krysko dv, brouckaert g, kalai m, vandenabeele p, d'herde k. mechanisms of internalization of apoptotic and necrotic l929 cells by a macrophage cell line studied by electron microscopy. j. morphol. 258: 336-345, 2003. lauzon rj, shizuka kj, weissman il. cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration. dev. biol. 249: 333-348, 2002. lowry oh, rosenbrough nj, farr al, randall rj. protein measurement with the folin phenol reagent. j. biol. chem., 193: 265-275, 1951. matsumoto j, nakamoto c, fujiwara s, yubisui t, kawamura k. a novel c-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates. development 128: 33393347, 2001. menin a, ballarin l. immunomodulatory molecules in the compound ascidian botryllus schlosseri: evidence from conditioned media. j. invertebr. pathol. 99: 275-280, 2008. menin a, del favero m, cima f, ballarin l. release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian. mar. biol. 148: 225230, 2005. 160 milanesi c, burighel p. blood cell ultrastructure of the ascidian botryllus schlosseri. i. hemoblast, granulocytes, macrophage, morula cell and nephrocyte. acta zool. (stockh.) 59: 135-147, 1978. oka h, watanabe h. vascular budding, a new type of budding in botryllus. biol. bull. 112: 225-240, 1957. oka h, watanabe h. vascular budding in botrylloides. biol. bull. 117: 340-346, 1959. overton j. the fine structure of blood cells in the ascidian perophora viridis. j. morph. 119: 305326, 1966. pérès jm. recherches sur le sang et les organes neuraux des tuniciers. ann. inst. ocean. (monaco) 21: 229-359, 1943. rinkevich b, shlemberg z, fishelson l. whole-body protochordate regeneration from totipotent blood cells. proc. nat. acad. sci. usa 92: 7695-7699, 1995. rinkevich b, tartakover s, gershon h. contribution of morula cells to allogeneic responses in the colonial urochordate botryllus schlosseri. mar. biol. 131: 227-236, 1998. rinkevich y, paz g, rinkevich b, reshef r. systemic bud induction and retinoic acid signaling underlie whole body regeneration in the urochordate botrylloides leachi. plos biol. 5, e71, 2007. rowley af, rhodes cp, ratcliffe na. protochordate leucocytes: a review. zool. j. linn. soc. (london) 80: 283-295, 1984. sabbadin a. studio sulle cellule del sangue di botryllus schlosseri (pallas) (ascidiacea). arch. ital. anat. embriol. 60: 33-67, 1955. sabbadin a, zaniolo g, majone f. determination of polarity and bilateral asymmetry in palleal and vascular buds of the ascidian botryllus schlosseri. dev. biol. 46: 79-87, 1975. sawada t, fujikura y, tomonaga s, fukumoto t. classification and characterization of ten hemocyte types in the tunicate halocynthia roretzi. zool. sci. 8: 939-950, 1991. sawada t, zang j, cooper el. classification and characterization of hemocytes in styela clava. biol. bull. 184: 87-96, 1993. schlumpberger jm, weissman il, scofield vl. separation and labeling of specific subpopulations of botryllus blood cells. j. exp. zool. 229: 401-411, 1984. scippa s, botte l, de vincentiis m. ultrastructure and x-ray microanalysis of blood cells of ascidia malaca. acta zool. (stockh.) 63: 121131, 1982. shirae m, hirose e, saito y. behavior of hemocytes in the allorejection reaction in two compound ascidians, botryllus scalaris and symplegma reptans. biol. bull. 197: 188-197, 1999. shirae m, ballarin l, frizzo a, saito y, hirose e. involvement of quinones and phenoloxidase in the allorejection reaction in a colonial ascidian, botrylloides simodensis: histochemical and immunohistochemical study. mar. biol. 141: 659-665, 2002. smith mj. the blood cells and tunic of the ascidian halocynthia aurantium (pallas). i. hematology, tunic morphology, and partition of cells between blood and tunic. biol. bull. 138: 354-378, 1970a. smith mj. the blood cells and tunic of the ascidian halocynthia aurantium (pallas). ii. the histochemistry of blood cells and tunic. biol. bull. 138: 379-388, 1970b. sugino ym, tsuji y, kawamura k. an ultrastructural study of blood cells in the ascidian, polyandrocarpa misakiensis: their classification and behavioral characteristics. mem. fac. sci. kochi univ. (ser. d) 14: 33-41, 1993. sunanaga t, watanabe a, kawamura k. involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates. dev. genes evol. 217: 1-11, 2007. voskoboynik a, simon-blecher n, soen y, rinkevich b, de tomaso aw, ishizuka kj, et al. striving for normality: whole body regeneration through a series of abnormal generations. faseb j. 21: 1335-1344, 2007. wright rk. urochordates. in: ratcliffe na and rowley af (eds), invertebrate blood cells, vol. 2, academic press, new york, london, pp 565626, 1981. zhang h, sawada t, cooper el, tomonaga s. electron microscopic analysis of tunicate (halocynthia roretzi) hemocytes. zool. sci. 9: 551-562, 1992. 161 isj116.pdf 40 isj 3: 40-49, 2006 issn 1824-307x review cell signalling in the immune response of mussel hemocytes l canesi1, m betti2, c ciacci2, lc lorusso2, c pruzzo1, g gallo1 1dipartimento di biologia, università di genova, genova, italy 2istituto di scienze fisiologiche, università di urbino, italy accepted may 24, 2006 abstract in this work data on immune cell signallling in the circulating hemocytes of the edible bivalve, the mussel mytilus spp, are summarized. studies with different bacterial species and strains, heterologous cytokines and natural hormones, as well as with organic environmental chemicals, led to the identification of the role of conserved components of kinase-mediated transduction pathways, including cytosolic kinases (such as mapks and pkc) and kinase-activated transcription factors (such as stats, creb, nf-kb), in the immune response. from these data a general scenario emerged indicating that close similarities exist in the signalling pathways involved in cell mediated immunity in bivalve and mammalian immunocytes. in particular, the results indicate that both the extent and duration of activation of components of kinase-mediated cascades are crucial in determining the hemocyte response to extracellular stimuli. the identification of the basic mechanisms of immunity and its modulation in mussels can give important information for the possible utilization of these species as an invertebrate model for studies on innate immunity. moreover, the application of this knowledge to the understanding of the actual adaptive responses of bivalves when exposed to microorganisms in their natural environment can represent significant ecological, economical and public health-related interest. key words: mytilus; hemocytes; innate immunity; kinase-mediated cell signalling introduction innate immunity has recently received a renewed interest, in particular due to genetic and molecular evidence supporting the hypothesis that it represents an ancient and evolutionary conserved defense system (cooper et al., 2002, 2006). in particular, studies from the invertebrate model drosophila have been crucial in the identification of key molecular components (such as the toll-like receptors) of cell-mediated immunity also in vertebrates (miester and lagueux, 2003; nappi et al., 2004). however, invertebrates represent about 95 % of total species in animal kingdom and only for a few other invertebrate models information is becoming available on sequences coding for immune genes and genes corresponding author: laura canesi dipartimento di biologia, università di genova corso europa 26, 16132 genova, italy e-mail: laura.canesi@unige.it involved in immune cell signalling (gueguen et al., 2003; shida et al., 2003; venier et al., 2003; kim and ausuvel, 2005). therefore, for many invertebrate species, information on the mechanisms of activation of the innate immune response, and in particular on the signall transduction pathways involved, has been largely obtained by indirect means, in functional studies utilizing antibodies and pharmacological inhibitors directed towards the mammalian counterparts of signalling components. the scenario emerging from these studies indicates that a high degree of conservation occurs between the pathways involved in the activation of immunocytes from molluscan species (mainly gastropods and bivalves) and those of mammals. in bivalves (mussels, oysters, etc.) a few molecular studies have identified sequences potentially involved in immune signalling (escoubas et al., 1999; gueguen et al., 2003; montagnani et al., 2004). here we will summarize the results obtained on the signall transduction pathways involved in mediating the immune function of the edible marine 41 bivalve, the mussel mytilus galloprovincialis lamk. mussels are adapted to the natural exposure to a variety of microorganisms, and can accumulate in their tissues huge quantities of bacteria, mainly gram negative enterobacteria and vibrios, that can be pathogenic both to the mussels and to humans. although mussels are extremely simple organisms, they have a long life cycle (about 10 years) during which they are constantly exposed to microbes, and thus probably require rather complex homeostatic mechanisms to integrate both environmental and endogenous signalls. functional and molecular characterization of the basic mechanisms underlying the innate immune response in bivalves can give important comparative information on the immune response and on its modulation in this invertebrate group that can be applied to the understanding of the actual adaptive responses of these molluscs exposed to microorganisms in their natural environment. the immune system of bivalve mollusks the immune system of bivalves consists of the blood cells, the hemocytes, and of soluble hemolymph factors, that operate in a co-ordinated way to provide protection from invading micro-organisms (renwrantz, 1990; rinkevich and muller, 1996; hine, 1999). bivalve hemocytes are responsible for cell-mediated immunity through phagocytosis and various cytotoxic reactions (such as lysosomal enzyme and antimicrobial peptide release and oxyradical production); hemolymph serum contains humoral defense factors, such as soluble lectins, hydrolytic enzymes and antimicrobial peptides (canesi et al., 2002a; pruzzo et al., 2005) (fig. 1). bivalve hemocytes are extremely heterogeneous; classifications proposed for different bivalve species are reported elsewhere (fryer and bayne, 1996; ottaviani et al., 1998; hine 1999; wootton et al., 2003). in mytilus spp. granular hemocytes represent the dominant cell type in the hemolymph; they are characterised by a low nucleus/cytoplasm ratio, high phagocytic activity and capacity for oxyradical production (wottoon et al., 2003). survival of bacteria to the hemolymph microbicidal activity may depend on their different ability to attract phagocytes, to interact with opsonizing molecules, to bind to hemocyte surface, thus favouring or inhibiting the response of the host (canesi et al., 2002a; pruzzo et al., 2005). bacteria could evade hemocyte killing by avoiding phagocytosis, generally by inducing damage in the hemocyte, as well as by preventing the oxidative burst associated with the phagocytic process. finally, in analogy with host-pathogen interactions in mammalian models of infection (rosenberger and finlay, 2003), bacteria may undermine the hemocyte function through disregulation of the signalling pathways involved in hemocyte activation. cell signalling involved in the responses of mytilus hemocytes to bacterial challenge: the role of mapks and pkc in our first studies on the immune response of mytilus hemocytes to bacterial challenge we utilized an in vitro model of hemocyte monolayers incubated in the presence of the soluble hemolymph fraction (hemolymph serum) with e. coli mg155, a wild type (wt) strain carrying the type 1 fimbriae (canesi et al., 2001a). this strain was shown to adhere to and to be internalized by hemocytes through mannosesensitive interactions that were dependent on the presence of soluble serum components. the extent and time course of the hemocyte bactericidal were similar in vitro and in vivo (canesi et al., 2001a); therefore, the in vitro model was subsequently utilized for investigating the signall transduction pathways involved in the immune response. in mammalian systems, activation of a complex network of signalling pathways, that include proteinkinase and lipid-kinase driven cascades, is critical in innate immunity. in particular, one of these pathways, involving members of the highly conserved mitogen activated protein kinase (mapk) superfamily, plays a key role in both macrophage and neutrophil activation (caffrey et al., 1999). mapks are a family of serine-threonine kinases that are activated by phosphorylation in responses to different extracellular stimuli, with each member being activated by a distinct kinase cascade (caffrey et al., 1999). the extracellularly regulated mapks (erk mapks) are regulated by mitogens and growth factors; the stress-activated p38 and the c-jun n-terminal kinases (jnks) are activated by stress signalls such as uv, cytokines, heat and osmotic shock, endotoxin. a conserved role of mapk members in mediating the pleiotropic response to growth factors was demonstrated in mytilus cells (canesi et al., 2001b) cell signalling involved in the hemocyte response to bacterial challenge was first investigated by use of pharmacological inhibitors. hemocyte bactericidal activity (i.e. percentage of bacterial killing) towards wt e. coli mg155 was prevented by cell pre-treatment with specific inhibitors of the stress-activated p38 mapk and of pi-3kinase (phosphatidyl inositol 3 kinase), sb203580 and wortmannin, respectively. a significant decrease in the hemocyte response was also observed with inhibitors of enzymes involved in arachidonic acid production and metabolism, indicating a role for prostaglandins and leukotrienes in mediating the hemocyte response to bacterial challenge (canesi et al., 2002b). therefore, we first focused our attention on the involvement of mapks in hemocyte immune signalling. the presence and phosphorylation state (activation) of different mapk-like proteins were evaluated by sds-page electrophoresis and western blotting of hemocyte protein extracts with specific anti-phospho-antibodies directed against mammalian erk mapks and p38 and jnk mapks. the key role of mapks, in particular of the stressactivated mapks, in the immune response of mussel hemocytes towards the wt e. coli strain was confirmed by the rapid increase in phosphorylation of p38 and jnk mapks induced upon bacterial challenge in the presence of hemolymph serum; activation of erk mapks was also observed (canesi et al., 2002c). further information on the kinase-mediated pathways involved in the hemocyte response came from studies on the interactions with different bacterial species and strains; vibrio cholerae strains were utilized as a model of autoctonous marine 42 fig. 1 mechanisms involved in the bactericidal activity of bivalve hemolymph. vibrios that are particularly accumulated in mussel tissues, where they can persist after depuration processes in controlled waters (pruzzo et al., 2005). different strains of e. coli and v. cholerae incubated with hemocytes showed differences in adhesion and internalization (canesi et al., 2001a; zampini et al., 2003). these differences were associated with different sensitivities to the overall hemocyte bactericidal activity, with wt e. coli > mutant e. coli ≅ wt v. cholerae > mutant v. cholerae. we subsequently showed that different strains of e. coli and v. cholerae induced distinct patterns of mapk phosphorylation in mussel hemocytes within a narrow time range (5-60 min) (canesi et al., 2005a). although both wt and mutant e. coli strains induced an increase in the phosphorylation of the stressactivated p38 and jnk mapks, a different time course was observed in hemocytes incubated with the mutant e. coli strain compared to that observed with the wt e. coli: the increase in p38 mapk phosphorylation was delayed, and jnk phosphorylation was only transient. these differences may be ascribed to differences in adhesion between the two strains to hemocytes in the presence of serum; since the higher adhesion of the wild strain was due to serum opsonins (canesi et al., 2001a), different mechanisms in binding to hemocytes of the two strains may involve differential activation of receptors, receptor complexes and signalling pathways, this resulting in different patterns of mapk phosphorylation and leading to differential activation of the hemocyte response. interestingly, wt v. cholerae had little effect on mapk phosphorylation, in particular on the stressactivated mapks; on the other hand, this strain induced a rapid and large protein kinase c (pkc) phosphorylation. a distinct scenario was observed with the v. cholerae mutant, that, among the tested strains, showed the lowest sensitivity to hemocyte microbicidal activity (zampini et al., 2003). moreover, this strain induced severe cellular stress in the hemocytes, as indicated by the large destabilisation of lysosomal membranes, and the decrease in the phosphorylation state of all mapk members (erk, p38, jnks), as well as of pkc (canesi et al., 2005a). these data suggested that the inability of mussel hemocytes to mount an efficient defense response against the v. cholerae mutant may be related to down-regulation of the hemocyte signalling pathways, in particular through interruption of signalling cascades upstream of mapk activation, as previously described in mammalian host cells infected with pathogens (rosemberger and finlay, 2003). overall, the results demonstrated that, in bivalve hemocytes like in mammalian cells, different bacteria and bacterial strains can differently modulate the host signalling pathways. moreover, these studies indicated that not only the extent, but also the time course of bacteria-induced phosphorylation (activation) of different cytosolic kinases (mapks and pkc) is crucial in determining the hemocyte response. phosphorylation of transcription factors in mammalian immunocytes different stimuli can activate a number of cytosolic kinases, including mapks and pkc, leading to phosphorylation of different transcription factors that can modulate the expression of various genes involved in the immune response (su and karin, 1996; guha and mackman, 2001). among these, stat proteins (signall transducers and activators of transcription) are a family of transcription factors conserved in vertebrates and invertebrates unique in that they act both as signalling molecules and as transcription factors (decker and kovarik, 1999; horvath, 2000); moreover, they are the only transcription factor specifically activated by tyrosine phosphorylation (decker and kovarik, 1999; horvath, 2000). initially identified in interferon signalling (the jak/stat 43 pathway), stats have been recognised as essential components of both adaptive and innate immunity (stark et al., 1998). in particular, stat1 activation plays a critical role in the macrophage response against gram negative bacteria (ohmori and hamilton, 2001). in mussel hemocytes wt e. coli mg155 induced rapid and persistent tyrosine phosphorylation of immunoreactive stat1-, stat3-, and stat5-like proteins (canesi et al., 2003a); the time course of stat phosphorylation was consistent with that of the bactericidal activity, suggesting a physiological role for stat-like proteins in mediating the immune function. we have recently observed that also the wt v. cholerae strain induced significant tyrosine phosphorylation of stat1 and the results are reported in fig. 2a. interestingly, a distinct pattern of phosphorylation of stat1 was observed in response to different bacteria; v. cholerae rapidly induced a large but transient increase in stat1 phosphorylation, whereas the level of p-stat1 steadily increased with time in response to e. coli. we also investigated the effect of bacterial challenge on the phosphorylation state of the transcription factor creb (c-amp responsive element binding protein), whose activation is mediated by serine/threonine kinases. creb activation is an early response transcription factor whose role in inflammatory response has been well established; creb can be phosphorylated at ser133 by both pka and mapks; interactions between creb and other transcription factors such as stats can modulate transcriptional activity (decker and kovarik, 1999). a creb-like protein was identified in mussel hemocytes (canesi et al., 2005b). as shown in fig. 2b, wt e. coli induced a rapid and dramatic increase in creb phosphorylation; the level of p-creb peaked at 5 min after addition of bacteria (reaching about a maximal ten-fold increase with respect to controls) and remained high up to 60 min. also wt v. cholerae induced a significant creb phosphorylation; however, the effect was transient and much smaller than that observed with wt e. coli. in macrophages, differential activation of creb by different bacteria was shown to be dependent on differential activation of both pka and p38 mapk, with consequent effects on tumor necrosis factor α (tnf α) production (roach et al., 2005). these data further support the hypothesis that, like in mammalian macrophages, conserved kinase-mediated signalling cascades lead to phosphorylation of transcription factors, this possibly resulting in modulation of transcriptional responses in the hemocyte. moreover, the results further support the hypothesis that different bacteria have distinct effect on components of kinasemediated cell signalling, this resulting in differential activation of the immune response. cytokine signalling many studies indicate that host defense mechanisms in different invertebrate groups, including molluscs, can be modulated by a cytokine network as in vertebrates; however, most information relies on functional assays, using heterologous cytokines and antibodies directed towards vertebrate cytokines and their receptors (reviewed by beschin et al., 2003; ottaviani et al., 2004). although no molecular evidence for the presence of full length cytokine receptor or cytokine homologues has been provided in the genome of model invertebrates like drosophila melanogaster and caenorhabditis elegans, genes and corresponding proteins expressing domains found in vertebrate cytokines, cytokine receptors and components of cytokine signalling have been described in invertebrates (beschin et al., 2003). in mytilus spp, heterologous cytokines have been shown to modulate the activity of the hemocytes, and functional analogues of different cytokines and cytokine receptors have been described in this species (hughes et al., 1990, 1993; ottaviani et al., 1995, 2000; cao et al., 2004). in mammalian macrophages, complete activation results from stimulation with both bacterial products (lps) and the cytokine interferon γ (ifnγ) that act in concert to generate a maximal capacity to ingest and kill the microbes through activation of signalling pathways, including mapks and stats, leading to modulation of gene expression (su and karin, 1996, guha and mackman, 2001). we therefore tested the possibility that heterologous ifns may modulate mussel hemocyte signalling and function. we observed that short-term hemocyte pre-treatment with human recombinant ifnγ, but not with ifnα, significantly increased the bactericidal activity of mussel hemocytes towards e. coli (canesi et al., 2003a). ifnγ stimulated tyrosine phosphorylation of different stat-like members stat1, stat3 and stat5. in particular, ifnã lead to persistent phosphorylation of immunoreactive stat1; moreover, hemocyte pretreatment with ifnã, but not with ifnα, significantly increased stat1 phosphorylation induced by bacterial challenge with e. coli. ifnγ also affected the phosphorylation state of different mapks; in particular, activation of erk2 mapk and slow downregulation of stress-activate mapks were observed. the extent and time course of mapk phosphorylation induced by ifnγ were distinct from those elicited by either ifnα or bacterial challenge, again indicating a specificity of the hemocyte response to ifnγ. these results indicate that the hemocyte function can be modulated by heterologous cytokines and bacterial signals that act in concert through tyrosine kinasemediated transduction pathways involving both mapkand stat-like members. in particular, in mussel hemocytes, like in mammalian macrophages, both bacterial signals and ifnã converge on activation of stat1, a transcription factor that plays a critical role in the response towards gram negative bacteria. in previous studies, mussel hemocytes were shown to be responsive to heterologous tnfα and produce tnfα–like molecules in response to bacterial components (hughes et al., 1990, 1993; ottaviani et al., 1995).tnfα is a pleiotropic cytokine that plays a pivotal role in orchestrating innate immune responses, as well as in regulation of cell proliferation, differentiation and apoptosis in vertebrates (baud and karin, 2001). tnfα signalling involves binding to members of tnf receptor superfamily (tnfrs) and recruitment of a complex of adapter proteins; among these, tnf-receptorassociated factors (trafs) activate several 44 fig. 2 effects of bacterial challenge on the phosphorylation state of transcription factors of mussel hemocytes. changes in phosphorylation of stat1 (a) and creb (b) are shown in hemocytes incubated with wt e. coli (strain mg155) and v. cholerae (o1 el tor biotype strain n16961) in the presence of hemolymph serum for different periods of time. hemocyte protein extracts were subjected to 12 % and 10 % sds-page, respectively, followed by western blotting using polyclonal phosphospecific antibodies directed against phosphorylated stat1 (tyr701) and creb (ser133) as previously described (canesi et al., 2003a, 2005b). bands were stripped and reprobed with antibodies to the corresponding unphosphorylated stat1 and creb forms. data (mean±sd) of three independent experiments are expressed in % changes in p-stat1/stat1 and p-creb/creb with respect to controls. * = p�0.05, mann whitney u test. relative increases in band optical densities (arbitrary units) were normalised for the control band in each series. intracellular signall transduction pathways, in particular mapks and nf-kb, that lead to modulation of gene expression by different transcription factors (baud and karin, 2001). members of tnfα transduction machinery have been characterized in invertebrates; in drosophila a tnf superfamily ligand, eiger, has been identified, that triggers jnk-dependent apoptosis (cha et al., 2003). in drosophila, dtraf1 is essential for endogenous jnk activation, whereas dtraf2 is required for nf-kb signalling. when we first assayed hemocyte functional parameters in response to heterologous tnfα, distinct effects were observed in the presence and absence of hemolymph serum (betti et al., 2006). when added in the absence of serum (in asw-artificial sea water) tnfα induced cellular stress, as indicated by large lysosomal destabilization and decreased phagocytosis; on the other hand, in the presence of serum, tnfα did not affect lysosomal stability and significantly stimulated phagocytosis. tnfα induced rapid and large phosphorylation of the stress-activated p38 and jnk mapks, as well as of stat1; activation of p38 and jnks in mediating the effects of tnfá was confirmed by the use of specific mapk inhibitors. however, the effects on mapks and stat1 were persistent in asw but transient in serum, a difference that had not been previously observed with ifnã. flow cytometric analysis indicated that tnfα in the presence of serum induced transient phosphatidylserine exposure on the haemocyte surface, evaluated as annexin v binding; in asw, the cytokine resulted in a stable increase in the percentage of both annexin vand propidium iodide-positive cells, indicating possible apoptotic/necrotic processes. more recent data indicate tnfα can also affect nf-kb signalling in the hemocyte. transcription factors of the nf-kb/rel family play a pivotal role in the inflammatory and immune response (gosh et al., 1998). nf-kb/rel is composed of a set of structurally related and evolutionary conserved dna binding proteins: members of class i (p100 and 105 in mammals and relish in drosophila) and members of class ii rela/p65, c-rel, relb in mammals, dorsal, dif in drosophila and gambif in anopheles). a rel homolog, cg-rel, has been characterized in oysters (montagnani et al., 2004). p105 and p100 are activated by proteasomal degradation to p50 and p52 products that form dimeric complexes with rel proteins, which are then able to bind dna and regulate transcription. activation and nuclear translocation of nf-kb is modulated through both phosphorylation and ubiqutination induced by different stimuli (karin and ben-neriah, 2000). western blots of mussel hemocyte extracts with anti-nf-kb p105/p50 and anti-phospho-nf-kb-p65 antibodies showed the presence of immunoreactive protein bands corresponding to the p105 precursor and its cleavage product p50; moreover, a constitutively ser536 phosphorylated p65-like protein was identified (fig. 3). as shown in the figure, addition of tnfα to the hemocytes in the presence of serum did not apparently affect the level of p105 and p50; however, tnfá-induced a significant, rapid and transient increase in the level of phosphorylated p65. inducible phosphorylation of p65 at ser536 has been described in mammalian 45 fig. 3 effect of tnfα (200 nm) on the level and phosphorylation state of nf-kb components in mussel hemocytes. protein extracts from control and tnfα-treated hemocytes were subjected to 12 % sds-page followed by western blotting using polyclonal antibodies to p105 and p50 (a), or polyclonal phosphospecific antibodies to p65 (ser536) (b). bands were detected using enhanced chemioluminescence reagents. results are representative of three independent experiments. c = control. hmw and lmw protein standards. inset: densitometric analysis of blots (p-p65) from three independent experiments (mean±sd). * = p≤0.05, mann whitney u test. relative increases in band optical densities (arbitrary units) were normalised for the control band in each series. cells in response to tnf and lps, leading to increase in nf-kb transcriptional activity (sasaki et al., 2005). our data support the hypothesis that components of nf-kb signalling are present in mytilus hemocytes and that their activity may be modulated by heterologous tnfα. overall, the results indicate that tnfα can affect the function of bivalve haemocytes through conserved transduction pathways involving stress-activated mapks, stats, and components of nf-kb signalling and suggest that the haemocyte response to this cytokine is influenced by soluble hemolymph components. endogenous immunomodulators: 17ββ -estradiol signalling in bivalve molluscs, and mytilus in particular, a few endocrine and immune modulators have been identified so far (lacoste et al., 2001; stefano et al., 46 fig. 4 signalling pathways involved in the immune response of mytilus hemocytes. the main signalling components activated by both bacterial challenge and heterologous cytokines are reported. mapks = mitogen activated protein kinases; erk = extracellularly regulated kinase; p38 = stress-activated p38 mapk; jnk = cjun n-terminal kinase; pkc = protein kinase c; jak = janus activated kinase; stat = signall transducer and activators of transcription; creb =c-amp responsive element binding protein; nf-kb = nuclear factor kb; ros = reactive oxygen species. 2003a). estrogens have been identified in bivalves, where their role has been mainly investigated in the control of gametogenesis (osada et al., 2004; gauthierclerc et al., 2006). however, evidence has been provided that estrogen can represent an important signalling molecule involved in roles other than reproduction in these organisms. in neural tissues of mytilus spp., 17β-estradiol (e2) has been shown to downregulate ganglionic microglial cells after surgical insult; the effect was mediated by rapid induction of a ca2+-induced nitric oxide (no) release by nervous tissue and were antagonized by classical antiestrogens (stefano et al., 2003b). in mytilus, e2 was also shown to affect the digestive cells and circulating hemocytes, in particular at the level of the lysosomal function (moore et al., 1978; burlando et al., 2002). addition of e2 (in the low nm range) to hemocyte monolayers induced a moderate increase in cytosolic [ca2+], destabilisation of lysosomal membranes, morphological changes, hydrolytic enzyme release and stimulated the bactericidal activity towards e. coli (canesi et al., 2004a); all these effects were rapid, occurring from seconds to minutes from e2 addition and were prevented by the antiestrogen tamoxifen (canesi et al., 2004a). the effects of e2 were mediated by rapid activation of the stressactivated p38 mapk. moreover, in mussel hemocytes, e2 induced increased tyrosine phosphorylation of stat3and stat5-like members, as previously observed in mammalian cells. when the effects of e2 on components of the immune response were investigated in more detail, we observed that e2, in a narrow concentration range (5-25 nm), rapidly stimulated phagocytosis and oxyradical production; higher concentrations inhibited phagocytosis (canesi et al., 2006). e2induced oxidative burst was prevented by the nitric oxide synthase inhibitor l-nmma and by sod, indicating the involvement of both no and o2 -; no production was confirmed by nitrite accumulation. also these effects of e2 were prevented by the antiestrogen tamoxifen and by sb203580, further supporting a receptor-mediated event and involvement of the p38 mapk. e2-induced 47 stimulation of phagocytosis and oxidative burst were also prevented by the pkc inhibitor gf109203x, indicating a role also for pkc in mediating the effects of e2. in fact, e2 induced rapid and transient increases in the phosphorylation state of pkc, and in particular of a pkcα/βii-like isoform, as well as of the transcription factor creb. the results demonstrated that the signalling components that play a key role in the immune response of mussel hemocytes represent significant targets for the action of e2. the effects of e2 on the immune function were confirmed in vivo. for longer exposure times (6 and 24 hrs) in the hemocytes of e2-injected mussels lower concentrations resulted in immuno-stimulation, whereas higher concentrations had inhibitory effects (canesi et al., 2006). overall, the obtained data indicate that e2 signalling appears to be conserved from invertebrates to mammals. immune signalling as a target for environmental contaminants different environmental contaminants known as endocrine disrupting chemicals (edcs) (witorsch, 2002) have been shown to affect the hemocyte function through modulation of the signall transduction pathways involved in the activation of the immune response. first data were obtained with polychlorinated biphenyls (pcbs): different ortho-substituted, non coplanar pcb congeners were shown to affect the immune function through disregulation of mapk and stat signalling (canesi et al., 2003b). in particular, the di-orthosubstituted pcbs p47 (2,2’,4,4’-tetrachlorobiphenyl) and p153 (2,2’,4,4’,5,5’-hexachlorobiphenyl) were shown to rapidly increase the phosphorylation state of both mapks and stat members. among the tested congeners p47 showed the strongest immunotoxic effect, resulting in lysosomal destabilization, inhibition of e. coli-induced lysozyme release, decrease in the overall bactericidal activity; the effects were mainly due to a large and persistent phosphorylation of the stressactivated mapks p38 and jnks. other edcs, including synthetic estrogens, phytoestrogens and estrogenic chemicals that represent significant contaminants of the aquatic environment were shown to induce destabilization of lysosomal membranes in mussel hemocytes (canesi et al., 2004b). the effects were prevented by different kinase inhibitors, indicating that the effect of each compound was mediated by activation of different signalling components. when the effects of different edcs on the phosphorylation state of mapks and stats were evaluated, certain synthetic estrogens, like des (diethylstilbestrol) were shown to induce activation of p38 mapk and stats, with effects similar to those observed with the natural estrogen e2, although at higher concentrations; other estrogenic chemicals, such as the alkylphenols bisphenol a and nonylphenol, induced a large decrease in phosphorylation of p38 mapk and stat5. the effects of bpa on hemocyte function and signalling were confirmed in vivo, in the hemocytes of mussels sampled at different times postinjection with the compound (canesi et al., 2005b). the results showed that also in vivo, and at longer exposure times, bpa induced hemocyte lysosomal destabilization and decrease in phosphorylation state of p38 mapk, stat5, and creb, indicating down-regulation of cell signalling and possible immunodepression. another class of contaminants identified as potential hemocyte immunomodulators are certain brominated flame retardants (binmbaum and staskal, 2004). tbbpa (tetrabromobisphenol a) was shown to induce activation of erk, p38 and jnk mapks and pkc. these effects on cell signalling resulted in stimulation of the overall microbicidal activity through increase in phagocytosis and oxidative burst (canesi et al., 2005c). the results so far obtained indicate that different organic contaminants known as endocrine disruptors in vertebrate system can also act as immune disruptors in mussel hemocytes through disregulation of different components of kinase mediated cell signalling, resulting in distinct effects depending on the compound tested. conclusions the signalling pathways involved in the innate immune response show common features in bivalve hemocytes and mammalian immunocytes. from the results obtained so far, the main signalling components that play a role in the immune function of mytilus hemocytes can be summarized as shown in fig. 4. the results obtained with different stimuli (bacteria, cytokines, hormones, environmental chemicals) support the hypothesis that both the extent and duration of activation of components of kinase-mediated cascades are crucial in determining the hemocyte response to extracellular stimuli. in general, sustained but transient phosphorylation of both cytosolic kinases and transcription factors seems to be associated with efficient activation of the immune response; on the other hand, large and persistent activation of signalling components leads to cellular stress and irreversible damage. conversely, down-regulation of hemocyte signalling observed in response to certain bacteria of contaminants impairs hemocyte activation with different consequences ranging from immunodepression to citotoxicity. however, the study of immune signalling in bivalve molluscs, compared to that in other invertebrate models, is still at its infancy. the scheme depicted in fig. 4 does not include components whose involvement in mussel hemocytes has been suggested by indirect observations, such as the camp/protein kinase a (pka) pathway, pi-3 kinase, or enzymes involved in arachidonic acid metabolism. moreover, information immune-related receptors (copper, 2006), on signalling components upstream of mapk activation (caffrey, 1999), as well as on the role of the rho family gtpases, that are involved in many processes essential to the coordination of the complex machinery underlying innate immunity (bokoch, 2005), is still lacking. the identification of the basic mechanisms of immunity and its modulation in mussels can give important information for the possible utilization of this species as an useful invertebrate model for 48 studies on innate immunity. moreover, the application of this knowledge to comprehension of the actual adaptive responses of bivalves when exposed to microorganisms in their natural environment can represent significant interest not only from an ecological, but also a financial point of view. these studies can provide the basis for better understanding the reasons for the persistence of certain pathogenic agents in bivalve tissues, and possibly to prevent diseases in the molluscs themselves, with obvious advantages for the conservation of natural populations. the identification of the processes that influence the elimination of microorganisms by edible bivalves can also give useful information to devise strategies to ameliorate the depuration processes utilized in shellfish farming that are needed to lower the microbial load to levels acceptable for human consumption; these data can therefore be of interest for human health, since they contribute to prevent the risk of disease. a great effort is being dedicated by different groups at identifying immuneand signall transduction-related sequences in different molluscan species, including edible bivalves such as mussels and oysters. in this light, research carried out by the bivalve activity group, within the activities of the ongoing project “improved immunity of aquacultured animalsimaquanim” (sixth framework eu programme area t6.4, project n. 007103), will represent a crucial advance in the identification of the molecular and functional aspects of the immune function in these organisms. references baud v, karin m. signall transduction by tumor necrosis factor and its relatives. trends cell. biol. 11: 372-377, 2001. beschin a, bilej m, lucas r, de baetselier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on similar lectin-like activity. in beschin a (ed), progress in molecular and subcellular biology. invertebrate cytokines, springer-verlag, berlin, germany, pp. 145-163, 2003. betti m, ciacci c, lorusso lc, canonico b, falcioni t, gallo g, canesi l. effects of tumor necrosis factor alpha (tnfalpha) on mytilus hemocytes: role of stress-activated map kinases. biol. cell 98: 233-244, 2006. bimbaum, l.s., staskal, d.f. brominated flame retardants: cause for concern? environ. health perspect. 112: 9-17, 2004. bokock gm. regulaton of innate immunity by rho gtpases. trends cell biol. 15: 163-171, 2005. burlando b, marchi b, panfoli i, viarengo a. essential role of ca2+-dependent phospholipase a2 in estradiol-induced lysosome activation. am. j. physiol. cell physiol. 283: c1461-c1468, 2002. caffrey dr, o’neill laj, shields dc. the evolution of the map kinase pathways: coduplication of interacting proteins leads to new signallling cascades. j mol. evol. 49: 576582, 1999. canesi l, betti m, ciacci c, citterio b, pruzzo c, gallo g. tyrosine kinase mediated cell signallling in the activation of mytilus hemocytes: possibile role of stat proteins. biol. cell. 95: 608-618, 2003a. canesi l, betti m, ciacci c, gallo g. growth factor-mediated cell signallling in bivalve cells. trends comp. biochem. physiol., research trends, trivandrum, 8: 117-127, 2001b. canesi l, betti m, ciacci c, lorusso lc, gallo g, pruzzo c. interactions between mytilus hemocytes and different strains of escherichia coli and vibrio cholerae o1 el tor: role of kinase-mediated signallling. cell. microbiol. 7: 667674, 2005a. canesi l, betti m, ciacci c, scarpato a, citterio b, pruzzo c, gallo g. signalling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 mapk. dev. comp. immunol. 26: 325-334, 2002c. canesi l, betti m, ciacci c, scarpato a, pruzzo c, gallo g. bacterial killing by mytilus hemocytes as a model for investigating the signallling pathways involved in mussel immune defense. mar. environ. res. 54: 547551, 2002b. canesi l, betti m, lorusso lc, ciacci c, gallo g. ‘in vivo’ effects of bisphenol a in mytilus hemocytes: modulation of kinase-mediated signalling pathways. aquat. toxicol. 71: 73-84, 2005b. canesi l, ciacci c, betti m, lorusso lc, marchi b, burattini s, falcieri e, gallo g. rapid effect of 17beta-estradiol on cell signallling and function of mytilus hemocytes. gen. comp. endocrinol. 136: 5871, 2004a. canesi l, ciacci c, betti m, scarpato a, citterio b, pruzzo c, gallo g. effects of pcb congeners on the immune function of mytilus hemocytes: alterations of tyrosine kinase-mediated cell signalling. aquat. toxicol. 63: 293-306, 2003b. canesi l, gavioli m, pruzzo c, gallo g. bacteriahemocyte interactions and phagocytosis in marine bivalves. microsc. res. tech. 57: 469-476, 2002a. canesi l, lorusso lc, ciacci c, betti m, gallo g. effects of the brominated flame retardant tetrabromobisphenol-a (tbbpa) on cell signalling and function of mytilus hemocytes: involvement of map kinases and protein kinase c. aquat. toxicol. 75: 277-287, 2005c. canesi l, lorusso lc, ciacci c, betti m, gallo g. environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinase pathways. gen. comp. endocrinol. 138: 58-69, 2004b. canesi l, pruzzo c, tarsi r, gallo g. surface interactions between escherichia coli and the hemocytes of the mediterranean mussel mytilus galloprovincialis lam. leading to efficient bacterial clearance. appl. environ. microbiol. 67: 464-468, 2001a. canesi l,ciacci c, lorusso lc, betti m, guarnieri t, tavolari s, gallo g. immunomodulation by 17âestradiol in bivalve hemocytes, am j. physiol. integr. resp. comp. physiol, 2006, in press. cao a, ramos martinez ji, barcia r. implication of pka and pkc in the hemocytes of mytilus galloprovincialis lmk by lps and il-2. mol. immunol. 41: 45-52, 2004. cha gh, cho ks, lee jh, kim m, kim e, parl j, lee sb, chung j. discrete functions of traf1 and traf2 in drosophila melanogaster mediated by c-jun nterminal kinase and nf-kb-dependent signallling pathways. mol. cell. biol. 23: 7982-7991, 2003. cooper el, kauscke e, cossarizza a. digging for innate immunity since darwin and metchnikoff. bioessays 24: 319-333, 2002. cooper el, kvell k, engelmann p, nemeth p. still waiting for the toll? immunol. lett. 104: 18-28, 2006. decker t, kovarik p. transcription factor activity of stat proteins: structural requirements and regulation by phosphorylation and interacting proteins. cell. mol. life sci. 55: 1535-1546, 1999. escoubas jm, briant l, montagnani c, hez s, devaux c, roch p. oyster ikk-like protein shares structural and functional properties with its mammalian homologues. febs lett. 453: 293-298, 1999. fryer se, bayne cj. host-parasite interactions in molluscs. in rinkevich b and muller weg (eds), invertebrate immunology, springer verlag, berlin, germany, 131-153, 1996. gauthier-clerc s, pellerin j, amiard jc. estradiol-17â and testosterone concentrations in male and female mya 49 arenaria (mollusca bivalvia) during the reproductive cycle. gen. comp. endocrinol. 145: 133-139, 2006. gosh s, may mj, kopp eb. nf-kappa b and rel proteins: evolutionarily conserved mediators of immune responses. annu. rev.immunol. 16: 225-260, 1998. gueguen y, cadeoret jp, flament d, barreau-romiguere c, girardot al, garnier j, hoareau a, bachere e, escoubas jm. immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, crassostrea gigas. gene 303: 139-145, 2003. guha m, mackman n. lps induction of gene expression in human monocytes. cell signall. 13: 85-94, 2001. hine pm. the inter-relations of bivalve hemocytes. fish shellfish immunol. 9: 367-385, 1999. horvath cm. stat proteins and transcriptional responses to extracellular signalls. trends biochem. sci. 25: 496-502, 2000. hughes tk, smith em, chin r, cadet p, sinisterra j, leung mk, shipp ma, scharrer b, stefano gb. interaction of immunoreactive monokines (interleukin 1 and tumor necrosis factor) in the bivalve mollusc mytilus edulis. proc. natl. acad. sci. usa 87: 4426-4429, 1990. hughestk, smith em, barnett ja, charles b, stefano gb. lps stimulates invertebrate hemocytes: a role for immunoreactive tnf and il-1, dev. comp. immunol. 15: 117-122, 1993. karin m, ben neriah y. phosphorylation meets ubiquitination: the control of nf-kappa b activity. annu. rev. immunol. 18: 621-663, 2000. kim dh, ausuvel fm. evolutionary perspectives on innate immunity from the study of caenorhabditis elegans. curr. opin. immunol., 17: 4-10, 2005. lacoste a, malham sk, cueff a, and poulet sa. noradrenaline modulates oyster hemocyte phagocytosis via a âadrenergic receptor-camp signallling pathway. gen. comp. endocrinol. 122: 252-259, 2001. miester m, lagueux m. drosophila blood cells. cell. microbiol. 5: 573-580, 2003. montagnani c, kappler c, reichhart jm, escoubas jm. cg-rel, the first rel/nf-kb homolog characterized in a mollusk, the pacific oyster crassostrea gigas. febs lett. 561: 75-82, 2004. moore mn, lowe dm, fieth pem. responses of lysosomes in the digestive cells of the common mussel mytilus edulis, to sex steroids and cortisol. cell tissue res. 188: 1-9, 1978. nappi aj, kohler l, mastore m. signallling pathways implicated in the cellular innate immune responses of drosophila. inv. surv. j. 1: 5-33, 2004. ohmori y, hamilton ta. requirement for stat1 in lpsinduced gene expression in macrophages. j. leukoc. biol. 69: 598-604, 2001. osada m, tawarayama h, mori k. estrogens in relation to gonadal development of the japanese scallop, patinopecten yessoensis: gonadal profile and immunolocalisation of p450 aromatase and estrogen. comp. biochem. physiol. 139c: 123-128, 2004. ottaviani e, caselgrandi e, franceschi c. cytokines and evolution, in vitro effects of il-1 alpha, il-1 beta, tnf alpha and tnf beta on an ancestral type of stress response. biochem. biophys. res. commun. 207: 288292, 1995. ottaviani e, franchini a, barbieri d, kletsas d, comparative and morphofunctional studies on mytilus gallorpovincialis hemocytes: presence of two aging-related hemocyte stages. ital. j. zool. 65: 349-354, 1998. ottaviani e, franchini a, malagoli d, genedani s. immunomodulation by recombinant human interleukin-8 and its signall transduction pathways in invertebrate hemocytes, cell. mol. life sci. 57: 506513, 2000. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. pruzzo c, gallo g, canesi l. persistence of vibrios in marine bivalves: the role of interactions with hemolymph components. environ. microbiol. 7: 761772, 2005. renwrantz l. internal defense system of mytilus edulis. in stefano gb (ed), neurobiology of mytilus edulis, manchester university press, manchester, 256-275, 1990. rinkevich, b. and muller w.e.g. invertebrate immunology. berlin, springer, 1996. roach sk, lee s-b, schorey js. differential binding of the transcription factor cyclic amp response element binding protein (creb) in macrophages following infection with pathogenic and non-pathogenic mycobacteria and role for creb in tumor necrosis factor production. infect. immun. 73: 514-522, 2005. rosemberger cm, finlay bb. phagocyte sabotage: disruption of macrophage signallling by bacterial pathogens. mol. cell. biol. 4: 385-396, 2003. sasaki c.y., barberi t.j., ghosh p., longo d.l. phosphorylation of rela/p65 on serine 536 defines an ikbα-independent nf-kb pathway. j. biol. chem. 280: 34538-34547, 2005. shida k, terajima d, uchino r, ikawa s, ikeda m, asano k, watanabe t, azumi k, nonaka m, satou y, satoh n, satake m, kawazoe y, kasuya a. hemocytes of ciona intestinalis express multiple genes invoved in innate immune response. biochem. biophys. res. commun. 302: 207-218, 2003. stark gr, kerr im, williams brg, silverman rh, schreiber rd. how the cells respond to interferons. ann. rev. biochem. 67: 227-264, 1998. stefano gb, cadet p, mantione k, cho jj, jones d, zhu w. estrogen signallling at the cell surface coupled to nitric oxide release in mytilus edulis nervous system. endocrinology 144: 1234-1240, 2003b. stefano gb, cadet p, rialas cm, mantione k, casares f, goumon y, zhu w. invertebrate opiate immune and neural signallling. adv exp med biol. 521: 126-147, 2003a. su b, karin m. mitogen-activated protein kinase cascades and regulation of gene expression. curr. opin. immunol. 8: 402-411, 1996. venier p, pallavicini a, de nardi b, lanfranchi g. towards a catalogue of genes transcribed in multiple tissues of mytilus galloprovincialis. gene 314: 29-40, 2003. witorsch rj. endocrine disruptors: can biological effects be predicted? regul. toxicol. pharmacol. 36: 118130, 2002. w ootton ec, dyrynda ea, ratcliffe na. bivalve immunity: comparisons between the marine mussel (mytilus edulis), the edible cockle (cerastoderma edule) and the razor-shell (ensis siliqua). fish shellfish immunol. 15: 195-210, 2003. zampini m, canesi l, betti m, ciacci c, tarsi r, gallo g, pruzzo c. role for mannose-sensitive hemagglutitin in promoting interactions between vibrio cholerae el tor and mussel hemolymph. appl. environ. microbiol. 69: 5711-5715, 2003. review isj 6: s58-s66, 2009 issn 1824-307x review inflammatory hemocytes in ciona intestinalis innate immune response v arizza, d parrinello laboratory of marine immunobiology, department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 abstract in the present paper an attempt is carried out to revise ciona intestinalis inflammatory hemocytes according to their morphology as formerly observed by light and electron microscopy, and taking in account recent reports on innate immunity gene expression. we also examine hemocyte morphofunctional aspects as derived from previous papers that refer to the tunic and body wall inflammatory responses challenged by corpusculate or soluble agents. lps inoculation into the body wall or treating hemocytes in vitro with lps have also been taken in account. lps inoculation stimulated the expression of citnfα, cifacit-αchain collagen, cic3a, cicd94 and enhanced phenoloxidase activity. these reports allow us to distinguish two main hemocyte types categories: 1. agranular hemocytes, including hemoblasts, circulating lymphocyte-like cells, hyaline amebocytes; 2. granular hemocytes including granulocytes with small granules, granulocytes with large granules, unilocular refractile granulocytes and morula cells. compartment cells and signet ring cells could be intermediate or terminal states presumably involved in releasing inflammatory factors or tunic matrix components. we suggest that the various hemocyte shapes, as shown by light and electron microscopy, could represent functional states as disclosed in inflamed tissues. although it cannot be excluded that a same cell expresses multiple activities, it is likely that several populations of a same cell type can exert distinct roles. key words: tunicate; innate immunity, inflammation, hemocyte, ciona intestinalis evolutionary relevance of ascidian immunology studies the developmental plan, the tadpole-like larva as well as molecular phylogenesis analysis support that tunicates are primitive members of the phylum chordata. ascidians (urochordata) occupy a critical position in the phylogenetic line leading to the vertebrates (swalla et al., 2000; zeng and swalla, 2005). recently delsuc et al. (2006) suggested that ascidians and not cephalochordates are the sister group of vertebrates, consequently urochordates have attained further importance for evolutionary immunology studies. ciona intestinalis is the representative species of the solitary ascidians generally retained a basic model for comparative biology research. the whole genome has been sequenced and several vertebrate homologous genes have been annotated (dehal et al., 2002; satou, 2002, 2003). bioinformatic ___________________________________________________________________________ corresponding author: vincenzo arizza department of animal biology university of palermo via archirafi, 18, 90123 palermo, italy e-mail: arizza@unipa.it approach and extensive in silico search have concerned immunorelevant molecules, gene expression patterns and some immune properties (davidson and swalla, 2002; fujita, 2002; nonaka and miyazawa, 2002; azumi et al., 2003; shida et al., 2003; terajima et al., 2003; du pasquier, 2004; fujita et al., 2004; kasahara et al., 2004). inflammatory reaction in the body wall of c. intestinalis, is a key evolutionary innate immunity model as well as can disclose hemocyte morpho functional aspects and pro-inflammatory products. soluble or particulate materials injected into the body wall are competent in challenging an inflammatory response including encapsulation and, in some cases, a tissue damage (parrinello, 1981; parrinello et al. 1984; parrinello and patricolo, 1984). in a variable time-course, these reactions can be visible through the transparent tunic, and microscopy observations show that in few hours the tunic matrix appears to be densely populated with hemocytes infiltrated through the epidermis(di bella and de leo, 2000), presumably coming from the pharynx and connective tissue close to the epidermis. hematogenic sites (crypts or nodules in the pharynx and emopoietic cells clusters) in the s58 pharynx and connective tissue (ermak, 1976) as well traits of proliferating epidermis have been shown (di bella and de leo, 2005). the nature of the used inflammatory agent as well as seasonal or environmental effects on the naïve ascidian populations could explain the variability in the timecourse and strength of the response. usually, in a few days, corpuscolate materials cause an intense heightened hemocyte populations density in the pharynx vessels, connective tissue, and tunic matrix where undergo differentiation, degranulation, necrosis, apoptosis, contributing in tunic matrix remodelling phase. the inflammatory cell-types show various shapes that could be related to activation and responding state, their products surround and isolate the host tunic containing the foreignness. in a significant number of individuals a degenerative process provokes a tunic wound which, successively, can be repaired (parrinello et al., 1977, 1984). although the timing sequence of the tunic reaction, after a second-set injection, is characterized by a heightened non-specific response, the chronic inflammation due to the first inoculation could maintain high hemocyte number and inflammatory factors level allowing an accelerate secondary response. therefore the presence of committed immunocompetent hemocytes may be excluded (parrinello et al., 1977). morphologically distinguishable hemocytes and their transitional forms, including cells that release their contents, have been observed. few stem cells, granular amebocytes and numerous hemocytes with large granules, signet ring cells and unilocular refractile granulocytes (urgs) have been found in the inflamed tunic. granulocytes degranulate, signet ring cells release their content, and numerous urgs express phenoloxidase (po) activity and release the active enzyme and/or pathway products. in the inflamed tunic matrix, a great amount of free granules and vacuoles, including lysosome particles, appear to be discharged by hemocytes. cellular debris and inflammatory molecules amplify the process also leading to tissue damage. after infection with e. coli, also circulating hemocytes promptly phagocytise the bacteria and excrete lysosome particles, while granular amebocytes liberate a lot of particles (liu et al., 2006). recently, we have examined c. intestinalis body wall inflammatory responses challenged by lipopolysaccharide (lps) (see below). lps is a component of the pathogen-associated molecular patterns (pamps). in organisms, ranging from invertebrates to humans, immune cells bear toll-like receptors (tlrs) that bind pamps (vasselon and detmers, 2002). three tlr distinct genes and the corresponding signal transduction cascades have been recognized in c. intestinalis draft genome (kimbrell and beutler, 2001; azumi et al. 2003; khalturin et al., 2004; roach et al., 2005). therefore it is presumable that lps-tlr binding induces responses including phagocytosis and release of inflammatory agents initiating the inflammatory response. ascidian hemocytes involved in immunity several approaches have been attempted to identify c. intestinalis hemocyte-types by using their morphological features under light or electron microscopy (rowley, 1981, 1982; de leo, 1992). in the last few years, interest in the mutual relationships between ascidians hemocyte types and products of innate immunity gene repertoire has led to a more clear-cut knowledge of these cells and their roles in immunity. this approach could also disclose that differentiation of activated cells yield to morpho-functional shapes of inflammatory hemocyte populations. a technique to classify c. intestinalis hemocytes is to look for the presence of granules, which allows to distinguish cells into two wide categories as agranulocytes (hemoblast, lymphocyte-like cells, hyaline amebocytes), or granulocytes (granulocytes with small or large granules, unilocular refractile granulocytes, morula cells). signet ring cells and compartment cells could be intermediate or final differentiation shapes following a challenge. variable frequency of circulating hemocyte types have been reported, presumably due to the variability within distinct ascidian populations as well as to seasonal factors and sea coastal environmental conditions. although transitional hemocytes have been identified in the hemolymph, they can be mainly found in inflamed tissues. hemoblasts and lymphocyte-like cells hemoblasts in hemopoietic nodules, are considered stem cells (about 3.0 5.0 µm) with a typical high nucleus/cytoplasm ratio (ermak, 1976, 1982). small hematogenic nodules are abundant in the pharyngeal wall and around the gut-loop; a few clusters also occur where the pharynx is attached to the body wall, under the endostyle and mesenteries. in the pharynx, hematogenic clusters are plentiful in transverse bars, spread in longitudinal bars, along the endostyle, and associated with the connective tissue. few hemoblasts with a lesser nucleus/cytoplasm ratio have been found in the circulating hemolymph (rowley, 1981; wright 1981; de leo, 1992). as shown by fine structure observations, the round nucleus contains condensed chromatine adherent to the nuclear envelope, and a characteristic prominent nucleolus. the cytoplasm presents free ribosomes and polyribosomes, mitochondria, occasional rough endoplasmic reticulum, rare golgi cisternae and a few lipid droplets. in different ascidian species, after an allogeneic challenge, circulating hemoblasts could proliferate and/or differentiate diverse hemocyte types (raftos and cooper, 1991). lymphocyte-like cells (llcs) (4.0-5.0 µm) in the hemolymph are similar to the circulating hemoblasts but present a lesser nucleus/cytoplasm ratio, the nucleus does not present a nucleolus, and the basophilic cytoplasm contains few small vesicles. light microscopy studies of unstained circulating cells do not allow a precise distinction between s59 http://en.wikipedia.org/wiki/pathogen-associated_molecular_pattern http://en.wikipedia.org/wiki/pathogen-associated_molecular_pattern http://en.wikipedia.org/wiki/granule_(cell_biology) http://en.wikipedia.org/wiki/granulocyte hemoblasts and llcs, and they are put on a pair with stem cells. frequently, structures recognized in the llcs presumably disclose initial differentiation steps, and some llcs wider in size are similar to small amebocytes containing numerous mitochondria and lysosome-like granules (warr et al,. 1977; fuke and fukumoto, 1993). various llcs frequency have been reported, presumably dependent on environmental conditions and the life cycle phase. frequency ranging from few cells up to about 20 % have been calculated (rowley 1981; wright 1981). recently liu et al. (2006) reported that some llcs can be marked by anti-cd34 monoclonal antibodies in agreement with the potential role of a pluripotent cell able to differentiate cell types. cd34 is a transmembrane protein expressed in mammalian hemopoietic cells (furukawa, 1998) and widely adopted as a marker of the human hemopoietic stem cells. although circulating hemoblasts were not distinguished, the llcs frequency slightly but significantly increases after in vivo bacterial (escherichia coli) challenge suggesting that they could proliferate in the circulatory system or in discrete body wall sites. llcs have been retained a primordial form of vertebrate lymphocyte (peddie and smith, 1995). although the enhanced proliferative activity may be related to their role in immunity, unequivocal evidences on c. intestinalis immunocompetent llcs in allorecognition have not been reported. in other ascidian species, llcs have been linked to non-self recognition and allograft rejection, also claimed as depositary of an adaptive histocompatibility-dependent cellular response including immunocompetent cell proliferation and differentiation of inflammatory hemocytes (raftos et al. 1987; raftos and cooper 1991). hyaline amebocytes in the hemolymph, hyaline amebocytes have been distinguished by electron microscopy as nonvacuolar or vacuolar hyaline amebocytes (rowley, 1982; de leo, 1992). in the inflammatory response, especially examined by light microscopy, immunocytochemistry and in situ hybridization methods, non-vacuolar and vacuolar amoebocytes cannot be distinguished, thus they are considered together as hyaline amoebocytes. electron microscopy observations show the cytoplasm with several vesicles of a variable size containing electron-lucid or a diffuse fibrous material, microtubules and microfilaments. vacuoles contain finely electron-dense granular inclusions, poorly developed endoplasmatic reticulum and golgi cisternae can also be observed. large and small vacuoles have been estimated as derived from expanded endoplasmic reticulum and golgi cisternae. morphologically some vesicles resemble the primary lysosomes of mammalian macrophages, while acpase positivity suggests phagolysosome formation (rowley, 1982). hyaline amebocytes are the most common cell type with phagocytic activity. within few minutes, they attach and, after the formation of pseudopodia, ingest formalinized sheep erythrocytes (more than 3/cell) (rowley, 1981), e. coli (liu et al., 2006) and yeast (personal observations), whereas they are not able to phagocytise polystyrene latex beads (zucchetti et al., 2008). after e. coli inoculation, secondary lysosomes can be formed and lysosomal enzyme particles secreted on the bacteria surface. part of the infected cells undergo cell death, either necrosis or apoptosis. electron microscopy observations show that the necrosed cells lose their membrane integrity, and broke completely. another portion of hemocytes which present integral membrane showed characteristics of apoptotic cells that promptly appear after in vivo infection. the phagocyte activity against yeast can be enhanced when the targets were opsonized with lectins (parrinello et al., 2007) suggesting that a lectin recognition mechanism characterizes these cells. hyaline amebocytes, with the above characters described for circulating cells, have not been observed in the inflamed tunic tissue, although the possibility exists that infiltrated cells undergo morpho-functional differentiation. in a recent paper parrinello et al. (2008) showed that, at 4 h after lps inoculation, circulating hyaline amoebocyte populations contain and presumably release the citnfα cytokine (cloned and sequenced) as shown by in situ hybridization analysis that identified the mrna mainly in the nucleus. granulocytes several circulating granulocytes have been described by light and electron microscopy, and distinguished by the granule size and abundance, shape and electron density of their content (de leo, 1992). in some cases, the granules contain a low electron-dense material and they have been referred as “vesicles” or “vacuoles”. rod-like and refractile granules have been observed upon phase contrast microscopy (rowley, 1981). light and electron microscopy showed that inflamed tissues including pharynx vessels, hemolymph and tunic were enriched with granulocytes containing granules of various shape, size, content density and fine structure organization. granular amebocytes with small granules following an inflammatory challenge numerous granular amebocytes promptly populate the tunic tissue, degranulate (parrinello et al., 1990) and release inflammatory factors. circulating granular amebocytes with small granules challenged in vitro and in vivo by e. coli (liu et al., 2006) lose their membrane integrity and degranulate, and electron microscopy shows small holes in the plasma membrane. after in vivo infection, apoptosis promptly appeared in the granular amebocytes, and massive density of necrotic hemocytes and degranulating granulocytes were also found in the inflamed tunic. in the inflamed tunic, cell functional states could be characterized by various electrondensity of the granular content that in some case appears heterogeneous in its fine structure. some granules present an electron-dense area surrounded with microtubules, whereas small s60 electron-transparent granules are acpase positive. rowley (1981) reports that these cells did not usually spread out to the same extent as the hyaline amebocytes and their cytoplasmatic extensions were often less evident. contrasting data have been reported on their phagocytic activity. although circulating granular amebocytes can fix bacteria, they do not phagocytised e. coli (liu et al., 2006), and it has been shown that after bacterial challenge the activated cells secrete in vitro numerous granules while typical apoptosis bodies emerge in ameboidic granulocytes. on the contrary several reports concern their phagocytic activity. smith and peddie (1992) showed that granular amebocytes collected from a percoll density band, ingest in vitro psychrobacter immobilis and their activity increases when the bacteria were pre-treated with hemocytelysate supernatant. however they were not able to distinguish between granular amebocytes and hyaline amebocytes both contained in the separated band. zucchetti et al. (2008) report that granular amebocytes are able to phagocytise polystyrene latex beads, and, after lps inoculation or in vitro treatment, these cells become more effective (up to 80 % granulocytes) in phagocytising the target. finally, rowley (1981) showed that these hemocytes promptly ingest formalinized sheep erythrocytes, showing indistinct ruffled membranes or spike-like pseudopodial extensions. the attachment of erythrocytes not always results in their internalization presumably due to differences in the recognition pattern. these various behaviours could be in accordance with granular amebocyte populations provided of different target specificity. lps inoculation puts in evidence that granular amebocytes, presumably distinct populations, are engaged in cic3a production and cicd94-1 expression. two c3-like genes, cic3-1 and cic3-2, from hemocyte total rna have been cloned and sequenced (marino et al., 2002). as in mammals, anaphylotoxin cic3a peptide is generated by proteolytic cleavage of the c3α-chain and it may exert proinflammatory activity including hemocyte recruitment (pinto et al., 2003). following lps inoculation, an increased number of these cells contain cic3a fragment with chemotactic activity (pinto et al., 2003), and also constitutively express a cic3a receptor (melillo et al., 2006) supporting the recruitment and activation of granular amebocytes and other effector cells in inflammation (c3ar positive circulating hyaline amebocytes identified by immunostaining could be granular amebocytes). inhibition experiments with the antibodies revealed that a cic3a-cic3ar binding is requested for exerting ci3a chemotactic activity on hyaline and granular amebocytes. a granular amebocyte population constitutively expresses cicd94-1 (homolog to vertebrate cd94 that marks nk cells), involved in the phagocytic activity, as a self-non-self recognition receptor with a c-type lectin domain exposed on the cell surface (zucchetti et al., 2008). after in vitro lps treatment 80% amebocytes express the cicd94 transcript. granulocytes characterized by the size and number of their granules they are large cells (ranging from 5.0 to 11.0 µm) with a cytoplasm, partially or almost entirely occupied by great granules containing materials of various density and refractile properties (rowley, 1981). the size and number of these granules can be very different (0.5-2.5 µm). granule number and size characterize granulocytes with many large granules, whereas other granulocytes contain a variable number of globules filled with material of low, moderate or high density. granules tend to fuse, and in the inflammatory reaction globular material appear to be released. an unique large granule occupies almost the whole cytoplasm and identify the unilocular refractile granulocytes. finally compartment and signet ring cells could be terminal hemocyte forms that release their content. the inflammatory response allows to distinguish the following hemocytes. granulocytes with large granules these granulocytes are abundant in the inflamed tunic, their inflammatory role is mainly indicated by phenoloxidase (po-2)-positive granules contained in the cytoplasm (parrinello et al., 2003). the enzyme was identified by dopa-mbth cytochemical reaction and anti-cipo-2 specific antibodies (immunoistochemistry) raised against a peptide designed from cloned and sequenced c. intestinalis po-2 (immesberger and burmester, 2004). the density of the po-positive granulocyte population as well as the enzyme activity increased in the tunic inflamed by lps inoculation, reaching the highest level within a few hours post injection (cammarata et al., 2008). in vitro assay of inflamed tissue and po assay of the tunic lysate supernatant supports that cells containing prophenoloxidase (propo) are activated. quinones, promptly (8 h) produced as an effect of cellular propo activation (proteolysis activated by lps), are distributed in the tunic matrix and presumably contribute to the inflammatory reaction. these granulocytes could have relationships with po-positive unilocular refractile granulocytes and morula cells. fine structure observations showed that numerous granulocytes with large granules undergo degranulation and granule contents (amorphous and granular) are released into the inflamed tunic matrix (de leo et al., 1992). unilocular refractile granulocytes (urgs) the cells present an unique large granule, filled with electrondense material, nearly occupies the whole cell and confines the nucleus at the periphery close to the cell membrane. this hemocyte-type has been identified in the hemolymph, the granule content appears to be refractile under phase contrast microscopy (rowley, 1981), provided with homogeneous fine granular content [electron microscopy (tem) observations] arranged in flocculent s61 structures or with loose or condensed fibrogranular material that forms masses aggregated into two or more foci often flowing together. the urgs are promptly involved in the tunic inflammatory reaction and are numerous in the inflamed tissue. the unique granule contains phenoloxidase (parrinello et al., 2003), cytochemical reaction with dopa-mbth shows a strong enzyme activity and the presence of cipo2 was revealed by specific antibodies. following lps injection (cammarata et al., 2008) numerous po-positive urgs occupy the tunic and presumably release products of po pathway. it is noteworthy that the circulating urgs exert cytotoxic activity, presumably cd94independent, when assayed in vitro with erythrocytes (parrinello et al., 1996). plaque forming cell assay demonstrated that cytotoxic molecules can be released following effector-target contacts (parrinello et al., 1996). an urg population from naïve ascidians expresses an antimicrobial peptide (cipap-a) that exerts a potent antimicrobial activity against a variety of bacteria and the yeast candida albicans (fedder and leippe, 2008). depending on the cell differentiation state, either the cytoplasm or the inclusion inside the large compartment contain cipap-a. although an assay on hemocytes from lps injected ascidians was not performed, it is reasonable that cipap-a can exert its antimicrobial activity in inflamed tissue injured by the injection procedure as well as by the inflammatory wound. multilocular granulocytes some large globular granules, tightly packed and close to the cell membrane, occupy the most cytoplasm. electron dense or electron transparent granule contents have also been used to distinguish two hemocyte types, morula cells and compartment cells respectively. morula cells they are spherical (8 16 µm), present variable number of tightly packed symmetrically arranged large globules (2 3 µm, max. 7). the nucleus, devoid of nucleolus, eccentrically located and compressed into an angular mass, is usually obscured by the globules. the globules are filled with high density homogeneous material, in some case the content appears electron-transparent with dense inclusion which may be granular or filamentous. it is peculiar that globules have a high refractive index (rowley, 1981). to one side of the nucleus there is a well-developed golgi apparatus containing irregularly shaped masses and an endoplasmic reticulum whose cisternae enclose dense granules (wright, 1981). cellular activity may be indicated by endoplasmic reticulum cisternae containing dense granules, and by irregularlyshaped masses in the golgi vesicles. when allowed to stand they assume a berry-like or morular appearance. presumably distinct morula cells populations could be distinguished. although phenoloxidase (dopa-mbth reaction and immunohistochemistry stain) was mainly found in granulocytes with numerous large granules and in urgs, it is noteworthy that, both in the hemolymph and in inflamed tissue (in vitro observations), some morula cells show globular granules with a faint po positivity. in addition these cells express (in situ hybridization) and contain (immunocytochemistry) citypeix-like (facit) collagen α-chain (parrinello et al., 2008). we cloned and sequenced a facit collagen (1α-chain) which is constitutively expressed in c. intestinalis tissues (vizzini et al., 2002). a prompt (4 h) expression of this collagen was shown by real-time pcr in the pharynx of lps inoculated ascidians as well as in circulating hemocytes challenged in vitro (flow cytometry analysis). collagens are major structural components of extracellular matrix in tissues of vertebrates and invertebrates, also involved in defence and reparative processes (singer and clark, 1999). in acute inflammatory reactions cellular, humoral, and molecular events are activated resulting in a regulated pattern of tissue repair with collagen fibres bundles organized during the remodelling (nwomeh et al., 1998). intermediate or fully developed hemocytes the possibility exists that compartment cells and signet ring cells may be intermediate or fully developed hemocytes. compartment cells spherical cells (8 12 µm) that present a variable number of large round globules containing electron transparent material with electron-dense granules of variable size on the inside. the nucleus contains no nucleolus and is centrally located, surrounded on one side by mitochondria and numerous profiles of dense rough endoplasmic reticulum and ribosomes (wright, 1981). rowley et al. (1984) report that compartment cells are usually far less common (5 % max.) than morula cells, moreover x-ray microanalysis results suggest interrelationship between these two cell types. we have found that, like morula cells they contain ci-facit collagen α-chain and are involved in tissue inflammatory response (vizzini et al., 2008). compartment cell-like cells releasing their globule content can be recognized in the inflamed tunic (de leo et al., 1992), suggesting they could be regarded as a fully developed hemocyte engaged in discharging inflammatory factors. in addition, they can express and contain inducible citnfα-chain in the cytoplasm lining the globules (parrinello et al., 2008). although both collagen chain and citnfα are constitutively expressed more numerous positive cells can be identified in the body wall after the lps challenge. the citnf enhanced time course expression is fast whereas that of the collagen increases after few days. finally, compartment cells express cic3a fragment (pinto et al., 2003) but not cic3a-receptor has been found (melillo et al., 2006). it is difficult to identify hemocyte types by using immunostaining method, and some cic3ar positive cells could be compartment cells. in additon, cic3ar positive granular amebocytes (presumably granulocytes with s62 table 1 features, activities and innate immunity genes expression of c. intestinalis inflammatory hemocytes observed in the inflamed tunic matrix or hemolymph after inoculation with lps. hemocyte drawings from light and electron microscopy observations hemoblast llc hematogenic tissue • steam cells circulating hemolymph • proliferation • cd34 positive • allograft reaction. liu et al., 2006; reddy et al., 1975; peddie et al., 1995 a gr an ul oc yt es circulantin hyaline amebocyte circulating hemolymph • express ci-tnf (lps) • fagocytosis fsrbc e. coli, saccharomices cerevisiae • necrosis/apoptosis. parrinello et al., 2008; rowley 1981; liu et al., 2006 granulocyte with small granule inflamed tunic • degranulation • cic3-1 expression • cic3a-r expression • cicd-94 expression circulating hemolymph • necrosis/apoptosis • phagocytosis: psicobacter immobilis, polystirene latex beads, fs rbc (not always), fix e. coli • degranulation following lps inoculation • cicd-94 expression. parrinello et al., 1990; liu et al., 2006; pinto et al., 2003; melillo et al., 2006; zucchetti et al., 2008; cammarata et al., 2008 granulocyte with large granules inflamed tunic • a few granules po-2 positive. cammarata et al., 2008 mc refractile globules inflamed tunic • same mcs contain po-2 positive globules • ci-facitα-chain collagen. vizzini et al., 2008 g ra nu lo cy te s urg unique refractile granulocyte inflamed tunic • po-positive unique large granule circulating hemolymph • po-positive unique large granule • cicd-94-independent cytotoxic activity (rbc, k562) • plaque forming cell • cipap-a expression against a variety of bacteria and candida albicans. zucchetti et al., 2008; parrinello et al., 1996; fedden and leippe, 2008; cammarata et al., 2008 compartment inflamed tunic (lps) • ci-facitα-chain collagen expression • ci-tnfα expression • ci-c3a • no ci-c3a-r. vizzini et al., 2008 parrinello et al., 2008 pinto et al., 2003 in te rm ed ia te a nd /o r te rm in al fo rm s signet ring cell inflamed tunic • encapsulation • release of electron transparent content. parrinello, 1981, 1990; de leo et al., 1992. s63 small granules) and some large round non ameboidic cells can be distinguished with difficulty from the negative cells identified as morula cells. signet ring cells these cells have been mainly described in the inflamed tunic (parrinello and patricolo, 1984; de leo et al., 1992), their frequency in the hemolymph may be very low (about 2 %), or they are absent. a large electron-transparent granule occupies almost entirely the cytoplasm and confines the nucleus at a peripheral site. electron microscopy observations show that these hemocytes (de leo et al., 1992) are very numerous in the inflamed tunic matrix where they discharge their granular or amorphous content as an effect of membrane dissolution. (parrinello 1981, 1990; de leo et al., 1992). the nature of the discharged granule materials is unknown, but they seem to contribute in encapsulation response. according to rowley (1981) presumably these cells represent the secretive state of challenged hemocytes. conclusions electron and light microscopy morphological features of c. intestinalis hemocytes allowed controversial classifications of the circulating cells. insights have been obtained by examining both cellular aspects of the inflammatory response and innate immunity genes expression (table 1). corpusculate and soluble inflammatory agents, bacteria and lps inoculation into the ascidian body wall, as well as in vitro challenge of circulating hemocytes, contribute to distinguish two main categories, agranulocytes (hemoblasts, lymphocytelike cells, hyaline amebocytes) and granulocytes (granulocytes with small granules, granulocytes with large granules, unilocular refractile granulocytes, morula cells). compartment cells and signet ring cells could be intermediate or fully developed hemocytes presumably involved in releasing inflammatory factors or tunic matrix components. gene expression studies, immunohistochemistry and immunocytochemistry assays disclose that hyaline amebocytes, mainly phagocytes, contain and presumably release citnfα-like cytokine, and show that granular amebocytes express cic3-1, cic3ar, cicd94 receptor and produce chemotactic cic3a supporting their inflammatory role. granulocytes with large granules show a weak po activity of a few granules, and could have relationships with po-positive unilocular refractile granulocytes and morula cells. urgs are the main components of the inflamed tissue where presumably release products of the po pathaway, whereas circulating urgs exert cytotoxic activity when assayed against erythrocytes and k562 tumor cell line. cytolysins cannot be related to the ca2+independent cicd94 nk-like receptor, they are released and exert ca2+-dependent activity. on the contrary, cicd94 nk-like receptor is involved in phagocytic activity. finally, an urg population from naïve ascidians expresses an antimicrobial peptide (cipap-a) with a potent antimicrobial activity. it is reasonable that cipap-a exerts its antimicrobial activity in inflamed tissue injured by the injection procedure and by the inflammatory wound. morula cells contain globules filled with high density homogeneous material showing high refractive index, and some of them are provided with faint po activity. in addition these cells express citypeix-like (facit) collagen α-chain following an lps challenge. some similarities suggest interrelationship between morula and compartment cells which contain a variable number of large round globules with electron transparent content. however, circulating compartment cells appear to be engaged in releasing inflammatory factors including citnfα-chain and cifacit α-chain, cic3a fragment. these hemocytes have been also found in inflamed tissues. finally signet ring cells could be a fully developed hemocytes discharging their granular or amorfous content as an effect of membrane dissolution, representing the secretive state of challenged hemocytes. we suggest that morpho-functional studies on challenged inflammatory hemocytes could contribute in establishing a more precise classification of the c. intestinalis hemocytes, taking in account that several populations of a same cell type can exert distinct roles. aknowledgments research on the c. intestinalis gene expression (citnf, cifacit collagen), po and cytotoxic activities of inflammatory hemocytes, as well as the present paper, have been supported by the princofin 2006 grant to prof n parrinello. references azumi k, de santis r, de tomaso a, rigoutsos i, yoshizaki f, pinto mr, et al. genomic analysis of immunity in a urochordate and the emergence of the vertebrate immune system: "waiting for godot”. immunogenetics 55: 570581, 2003. cammarata m, arizza v, cianciolo c, parrinello d, vazzana m, vizzini a, et al. the prophenoloxidase system is activated during the tunic inflammatory reaction of ciona intestinalis. cell tissue res. 333: 481-492, 2008. davidson b, swalla bj. a molecular analysis of ascidian metamorphosis reveals activation of an innate immune response. development 129: 4739-4751, 2002. de leo g, parrinello n, di bella ma. structural changes in granulocytes involved in the lysis of the tunic during inflammatory-like reaction induced in ciona intestinalis (tunicata, ascidiacea). eur. arch. biol. 103: 113–119, 1992. de leo g. ascidian hemocytes and their involvement in defense reactions. boll. zool. 59: 195–213, 1992. dehal p, satou y, campbell rk, chapman j, degnan ba, de tomaso b, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins science 298: 2157–2167, 2002. s64 delsuc f, brinkmann h, chourrot d, hervé p. tunicate and not cephalochordates are the clotest living relatives of vertebrates nature letter. 439: 965-968, 2006. di bella ma, and de leo g. hemocyte migration during inflammatory-like reaction of ciona intestinalis (tunicata, ascidiacea) j. invertebr. pathol. 76: 105-111, 2000. di bella ma, carbone mc, de leo g. aspects of cell production in mantle tissue of ciona intestinalis l. (tunicata, ascidiacea) micron 36: 477-481, 2005. du pasquier l. speculations on the origin of the vertebrate immune system. immunol. lett. 92: 3-9, 2004. ermak th. the hematogeneic tissues of tunicates. in: wright rk, cooper el (eds), phylogeny of thymus and bone marrow-bursa cells, elsevier/north-holland biomedical press, amsterdam, the netherlands, pp 45-55, 1976. ermak th. the renewing cell populations of ascidians. am. zool. 22: 795-805, 1982. fedders h, leippe ma. reverse search for antimicrobial peptides in ciona intestinalis: identification of a gene family expressed in hemocytes and evaluation of activity dev. comp. immunol. 32: 286-298, 2008. fujita t, endo y, nonaka m. the lectin-complement pathway its role in innate immunity and evolution. immunol. rev. 198: 185-202, 2004. fujita t. evolution of the lectin-complement pathway and its role in innate immunity. nature rev. immunol. 2: 346-353, 2002. fuke m, fukumoto m. correlative fine structural, behavioral, and histochemical analysis of ascidian blood cells. acta zool. 74: 61-71, 1993. furukawa, y. cell cycle regulation of the hematopoietic stem cells. hum. cell 11: 81-92, 1998. immesberger a, burmester t. putative phenoloxidases in the tunicate ciona intestinalis and the origin of the arthropod hemocyanin superfamily. j. comp. physiol. b 174: 169-80, 2004. kasahara m, suzuki t, du pasquier l. on the origins of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates. trend immunol. 25: 105-111, 2004. khalturin k, pancer z, cooper md, bosch tcg. recognition strategies in the innate immune system of ancestral chordates. mol. immunol. 41: 1077-1087, 2004. kimbrell da, beutler b. the evolution and genetics of innate immunity. nature rev. genet. 2: 256267, 2001. liu l, wu c, chen t, zhang x, li f, luo w, et al. effects of infection of egfp-expressing escherichia coli on haemocytes in ciona intestinalis j. exp. mar. biol. ecol. 332: 121134, 2006. marino r, kimura y, de santis r, lambris jd, pinto mr. complement in urochordates: cloning and characterization of two c3-like genes in the ascidian ciona intestinalis. immunogenetics 53: 1055-1064, 2002. melillo d, sfyroera g, de santis r, graziano r, marino r, lambris jd, et al. first identification of a chemotactic receptor in an invertebrate species: structural and functional characterization of ciona intestinalis c3a receptor. j. immunol. 177: 4132-4140, 2006. nonaka m, miyazawa s. evolution of the initiating enzymes of the complement system, genome biol. 3: 1-5, 2002. nwomeh bc, yager dr, cohen ik. physiology of the chronic wound. clin. plast. surg. 25: 341356, 1998. parrinello n, arizza v, chinnici c, parrinello d, cammarata m. phenoloxidases in ascidian hemocytes: characterization of the prophenoloxidase activating system comp. biochem. physiol. 135b: 583-591, 2003. parrinello n, arizza v, vazzana m, cammarata m, giaramita ft, di bella ml, et al. separated hemocyte populations from the ascidian ciona intestinalis contain and release in vitro opsonizing ca++-independent and β-galactoside specific lectins. inv. surv. j. 4: 55-64, 2007. parrinello n, cammarata m, arizza v. univacuolar refractile hemocytes from the tunicate ciona intestinalis are cytotoxic for mammalian erythrocytes in vitro. biol. bull. 190: 418-425, 1996. parrinello n, de leo g, di bella ma. fine structural observation of the granulocytes involved in the tunic inflammatory-like reaction of ciona intestinalis (tunicata). j. invert. pathol. 56: 181189, 1990. parrinello n, patricolo e, canicatti c. inflammatorylike reaction in the tunic of ciona intestinalis (tunicata). i. encapsulation and tissue injury. biol. bull. 167: 229-237, 1984. parrinello n, patricolo e, canicattì c. tunicate immunobiology. i. tunic reaction of ciona intestinalis l. to erythrocyte injection. boll. zool. 44, 373–381, 1977. parrinello n, patricolo e. inflammatory-like reaction in the tunic of ciona intestinalis (tunicata). ii. capsule components biol. bull. 167: 238-250, 1984. parrinello n, vizzini a, arizza v, salerno g, parrinello d, cammarata m, et al. enhanced expression of a cloned and sequenced ciona intestinalis tnfα-like (citnfα) gene during the lps-induced inflammatory response cell tissue res. 334: 305-17, 2008. parrinello n. the reaction of ciona intestinalis l. (tunicata) to subcuticular erythrocyte and protein injection dev. comp. immunol. 5: 105110, 1981. peddie cm, smith vj. lymphocyte-like cells in ascidians: precursors for vertebrate lymphocytes? fish shellfish immunol. 5: 613629, 1995. pinto rm, chinnici c, kimura y, melillo d, marino r, spruce la, et al. cic3-1a-mediated chemotaxis in the deuterostome invertebrate ciona intestinalis (urochordata). j. immunol. 171: 5521-5528, 2003. raftos da, cooper el. proliferation of lymphocytelike cells from the solitary tunicate, styela clava, in response to allogeneic stimuli. j. exp. zool. 260: 391-400, 1991. s65 raftos da, tait nn, briscoe da. cellular basis of allograft rejection in the solitary urochordate, styela plicata. dev, comp. immunol. 11: 713725, 1987. deuterostomes. syst. biol. 49: 52-64, 2000. terajima d, yamada s, uchino r, ikawa s, ikeda m, shida k, et al. identification and sequence of seventy-nine new transcripts expressed in hemocytes of ciona intestinalis, three of which may be involved in characteristic cell– cell communication, dna res.10: 203–212. 2003. roach jc, glusman g, rowen l, kaur a, purcell mk, smith kd, et al. the evolution of vertebrate toll-like receptors proc. natl. acad. sci. usa 102: 9577-9582, 2005. rowley, af. the blood cells of the sea squirt ciona rntestinalis. morphology, differential counts, and in vitro phagocytic activity. j. invertebr. pathol. 37: 91-100, 1981. vasselon t, detmers pa. toll receptors: a central element in innate immune responses. infect. immun. 70: 1033-1041, 2002. vizzini a, arizza v, cervello m, cammarata m, gambino r, parrinello n. cloning and expression of a type ix-like collagen in tissues of the ascidian ciona intestinalis. biochim. biophys. acta 1577: 38-44, 2002. rowley af. ultrastructure and cytochemical studies on the blood cells of the sea squirt ciona intestinalis. 1. stem cells and amoebocytes. cell tissue res. 223: 403-414, 1982. rowley af, rhodes cp, ratcliffe a. protochordate leucocytes: a review. zool. j. linn. sk 80: 283295, 1984. vizzini a, pergolizzi m, vazzana m, salerno g, di sano c, macaluso p, et al. facit collagen (1αchain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian ciona intestinalis. dev. comp. immunol. 32: 682-692, 2008. satoh n. the ascidian tadpole larva: comparative molecular development and genomics. nature rev. genet. 4: 285-295, 2003. satou y, yamada l, mochizuki y, takatori n, kawashima t, sasaki a, et al. a cdna resource from the basal chordate ciona intestinalis. genesis 33: 153-154, 2002. warr gw, decker jm, mandel te, de luca d, hudson r, marchalonis jj. lymphocyte-like cells of the tunicate, pyura stolonifera: binding of lectins, morphological and functional studies. aust. j. exp. biol. med. sci. 55: 151-164, 1977. shida k, terajima d, uchino r, ikawa s, ikeda m, asano k, et al. hemocytes of ciona intestinalis express multiple genes involved in innate immune host defense. biochem. biophys. res. commun. 302: 207-218, 2003. wright rk. urochordates in: ratcliff na, rowley af (eds), invertebrate blood cells, academic press, new york, vol 2, pp 565-626, 1981. zeng l, swalla bj. molecular phylogeny of the protochordates: chordate evolution. can. j. zool. 83: 24-33, 2005. singer aj, clark raf. mechanisms of desease: cutaneous wound healing. n. engl. j. med. 341: 738-743, 1999. zucchetti i, marino r, pinto mr, lambris jd, du pasquier l, de santis r. cicd94-1, an ascidian multipurpose c-type lectin-like receptor expressed in ciona intestinalis hemocytes and larval neural structures differentiation 76: 267-282, 2008. smith vj, peddie cm. cell cooperation during host defence in the solitary tunicate ciona intestinalis (l), biol. bull. 183: 211-219, 1992. swalla bj, cameron cb, corley ls, garey jr. urochordates are monophyletic within the s66 uninjured tissue under the carapace of intermoult crayfish consists of a low columnar epithelium immediately under the cuticle and underneath a layer of loose connective tissue forming large haemolymph sinuses isj 6: 125-137, 2009 issn 1824-307x review mechanisms of wound repair in crayfish x vafopoulou biology department, york university, 4700 keele street, toronto m3j 1p3, ontario, canada accepted september 8, 2009 abstract this review describes the complexity of events involved with repair to integumentary wounds and their regulation using the crayfish as a model system. injuries to integument precipitate a cascade of cellular events that lead to rapid healing of the wound, regeneration of damaged tissues and repair of the integument. the first step in this cascade is hemolymph clotting and subsequent melanization, events documented thoroughly elsewhere and not discussed here. wound healing and repair in crayfish involves the action of two physiological systems, the immune system and the neuroendocrine system regulating synthesis of the steroid molting hormones, ecdysteroids. injury promotes a swift rise in hemolymph ecdysteroids to a low, sustained plateau, followed by a premolt peak and molting. the plateau is essential for wound healing since its principal targets are the circulating cells of the immune system, the hemocytes, and healthy epidermal cells and fibrocytes. massive migration of these cells occurs under the wound and their concerted efforts under ecdysteroid control are paramount to wound healing and repair. these cells are likely engaged in physiological and biochemical activities that promote cell communication and cell to cell adhesion, removal of dead and harmful material and production of molecules essential to tissue regeneration. key words: crustacea; immune system; ecdysteroids; hemocytes; epidermis; regeneration introduction structural integrity in crustaceans is maintained by a rigid exoskeleton. the exoskeleton is also the main defense barrier against invasions by microbes and parasites. when this barrier is breached by mechanical injuries or erosion due to chemicals or bacteria, the animal becomes vulnerable to systemic infections. it is therefore vital for survival that injuries to integument are healed promptly and the damaged integument is repaired quickly. this review addresses events and mechanisms by which these responses are brought about. injury triggers a dramatic awakening of the machinery that directs restoration and repair of damaged tissues the details of which are discussed in the following section. wound healing is a complex and orderly process involving the coordinated behavior of different cell types. it relies heavily on rapid immune responses by the hemocytes which seal and protect the wound site and massive movements of cells which regenerate the damaged tissues. the initial event is coagulation of hemolymph components that ___________________________________________________________________________ corresponding author: xanthe vafopoulou biology department york university 4700 keele street, toronto m3j 1p3, ontario, canada e-mail: xanthev@yorku.ca seal off wounds temporarily and thus contain hemolymph loss and trap microbes (see for review lee and söderhäll, 2002; cerenius and söderhäll, 2004). ruptured hemocytes release clotting enzymes that coagulate hemolymph and enzymes that activate the pro-phenoloxidase (propo) cascade which leads to melanisation and ensures trapping of foreign and damaged cell material. additional immune responses are also triggered that include phagocytosis, cytotoxicity, nodule formation and encapsulation which kill invading microbes and remove cellular and matrix debris. concurrently with hemocyte recruitment, epidermis regeneration is triggered and when this process is completed the newly formed epidermal layer deposits a new cuticle that seals the wound permanently and restores the damaged integument to its original condition. the phenomena of wound healing and repair raise fundamental biological issues concerning the origin of cues that initiate the cascade of this process. cues may originate locally to act from one cell population to another or may be humoral in nature. for example the cues that trigger hemolymph coagulation are usually local. in the case of bacterial infection, local signals present on the surface of microbes initiate hemolymph clotting (see for review sritunyalucksana and söderhäll, 2000; theopold et al., 2002, 2004). by contrast, in 125 the case of physical wounding the cues are largely unknown. it is hypothesized that, as in vertebrates (e.g., gallucci and matzinger, 2001), unidentified “danger signals” are released from the damaged cells that trigger the clotting reaction. danger signals may include cellular components of damaged cells such as nucleotides, reactive oxygen intermediates or other intracellular proteins, which activate the hemolymph clotting cascade (review by theopold et al., 2002). another example of local signals derives from the study of limb regeneration in crabs following limb autotomy; regenerating limb buds release and respond to growth factor(s) that promote local growth (hopkins et al., 1979, 1999; hopkins, 2001). cues for the cascade of wound healing and repair may also be provided distantly, specifically from the central neuroendocrine system. the primary candidate for promoting tissue regeneration is the steroid molting hormones of crustaceans, ecdysteroids. for example, wound healing and repair depends on the presence of low levels of ecdysteroids following carapace injury in crabs (halcrow and steel, 1992) and crayfish (vafopoulou et al., 2007) or limb autotomy in crabs (mccarthy and skinner, 1977). freshwater crayfish provide an excellent model system to advance knowledge on the mechanisms of wound healing and repair. crayfish are of commercial and ecological importance. they are easy and inexpensive to rear in large numbers under laboratory conditions for experimentation in a simple aquatic environment. key components of the innate immunity system have been established in the crayfish (e.g., söderhäll et al., 1994; söderhäll and cerenius, 1998; wang et al., 2001a; lee and söderhäll, 2001, 2002) and much is known about the morphology and development of the animal (references in vogt, 2008). in the present review, we will discuss the current state of understanding of wound healing and tissue regeneration in the freshwater crayfish procambarus clarkii and the underlying mechanism of hormonal control that brings about these changes. wound healing and repair in crayfish we have found a direct physiological link between the immune system and the system that controls molting during wound healing and repair in the crayfish. specifically, we found in wounded animals immune responses affect the molting system and molting responses affect the immune system. interaction of these two systems is crucial in the ability and speed by which a crayfish heal wounds to the integument. the two physiological systems and their involvement in wound healing are described in details below. the first system to respond to injuries is the immune system. many aspects of the crustacean immune system have been elucidated in crayfish, making it a valuable animal to study the involvement of the immune system in wound healing. the immune system is centered on the circulating hemocytes and involves mechanisms to recognize and destroy non-self material and heal wounds. there are two components in innate immunity in crustaceans, the humoral and cellular components, both of which are activated upon immune challenges resulting from wounds to integument. cellular defenses include hemocyte-mediated responses such as phagocytosis, nodulation and encapsulation. humoral defences include antimicrobial peptides, coagulation and melanization of the hemolymph and production of reactive intermediates of oxygen and nitrite (see lee and söderhäll, 2002; lee et al., 2004). most of the bioactive peptides are produced by the hemocytes. among these bioactive compounds, the propoactivating system is the best studied and plays a pivotal role in innate immunity. in crayfish, propo and the enzymes responsible for its conversion to its active form phenoloxidase (po) reside as zymogens in the granules of granular and semigranular hemocytes, the contents of which are released by exocytosis into the hemolymph under challenges by pathogens or cell damage because of wounding (smith and söderhäll, 1991; wang et al., 2001a). this conversion ultimately results in the formation of melanin and the generation of a number of potent bioactive products, which assist phagocytosis, cell to cell adhesion and melanization (see söderhäll et al., 1994; söderhäll and cerenius, 1998; see for review lee and söderhäll, 2002; cerenius and söderhäll, 2004). propo has been cloned in crayfish (aspán et al., 1995). other important components of immune responses have also been extensively studied in crayfish. for example, components of the clotting and melanization system have been characterized such as a transglutaminase essential for hemolymph clotting (hall et al., 1999; wang et al., 2001b) and an inhibitory protein that halts melanization (söderhäll et al., 2009); antimicrobial peptides such as astacidin and crustin (jiravanichpaisal et al., 2007); hemocyanin which acts as a phenoloxidase in crayfish and a precursor to an antibacterial peptide (lee et al., 2004); peroxinectin, a multifunctional peptide critical for cell to cell adhesion (johansson and söderhäll, 1988), encapsulation (kobayashi et al., 1990) and degranulation (johansson and söderhäll, 1989); a pattern recognition peptide (lee and söderhäll, 2001; see for review sritunyalucksana and söderhäll, 2000; lee and söderhäll, 2002; cerenius and söderhäll, 2004). study of the cascade of events in wound healing depends on recognition of the morphological and functional characteristics of hemocytes. hemocytes are classified according to their morphology, cytochemistry and function (smith and söderhäll, 1983a; see for review johansson et al., 2000) and are recognized as hyaline, semigranular and granular hemocytes based on a classification system established by smith and söderhäll (1983a). based on this system, hemocytes have been characterized in several crayfish such as astacus astacus (smith and söderhäll, 1983a), procambarus zonangulus (cárdenas et al., 2000), pacifastacus leniusculus (wang et al., 2001a), procambarus clarkii (vafopoulou et al., 2007) and astacus leptodactylus (giulianini et al., 2007). in general, all three classes and subclasses of hemocytes, are recognized in all crustacean species (e.g., vázquez et al., 1997; johansson et al., 2000; sung and sun, 126 2002; battison et al., 2003; martin et al., 2003; liu et al., 2007; zhan et al., 2008). hemocytes have a defined division of labor in innate immunity. hyaline hemocytes are critical for hemolymph clotting (hall and söderhäll, 1994; wang et al., 2001a; reviews by theopold et al., 2002, 2004) and phagocytosis (smith and söderhäll, 1983b). the principal role of semigranular hemocytes is encapsulation (kobayashi et al., 1990) and cytotoxicity (söderhäll et al., 1985), whereas granular hemocytes are the primary source of the propo-activating system (johansson and söderhäll, 1985; see for review sritunyalucksana and söderhäll, 2000). the second system in crayfish that responds to injury is the neuroendocrine system that controls molting (vafopoulou et al., 2007). molting is the cyclical formation of a new exoskeleton and the shedding of the old one. growth and molting in crustaceans is regulated by ecdysteroids (such ecdysone and 20-hydroxyecdysone) the concentration of which displays a characteristic sequence of increases and decreases over a molt cycle (steel and vafopoulou, 1989). ecdysteroids are synthesized and secreted by the y-organs under the negative control of the neuropeptide molt-inhibiting hormone (mih). mih is synthesized by the x-organ in the brain and released from the sinus gland in the eyestalk (see for review webster, 1998). when present, it prevents the secretion of ecdysteroids from the y organ and holds crustaceans in intermolt (e.g., snyder and chang, 1986; see for review chang et al., 2001). new cuticle is secreted by the epidermis under the action of ecdysteroids; this period in the molt cycle is called ‘premolt’ and it culminates in shedding of the old exoskeleton (ecdysis) and hardening of the new one in the post-ecdysial period known as ‘postmolt’. during the remainder of a molt cycle, animals are devoid of ecdysteroids and not preparing to molt; this period is called ‘intermolt’. we have found that injuries to the crayfish integument during intermolt induce precocious production of ecdysteroids, measured by radioimmunoassay (ria), as shown in fig. 1 (details in vafopoulou et al., 2007). these injuries cause initially a small but significant elevation in the concentration of hemolymph ecdysteroids to a low, sustained plateau (10-12 days after injury) (fig. 1a, b; dark triangles). the plateau is then followed after about 2-3 weeks by a sharp increase to the peak values characteristic of premolt. ecdysis occurs at about 2 months later in crayfish. uninjured, healthy controls show only a slight increase in hemolymph ecdysteroid levels throughout the premolt period of wounded animals (fig. 1a, b; open triangles), and they never form a premolt peak. positive control animals which are subjected to removal of both eyestalks but no exocuticular injury exhibit no plateau phase but rather commence the steep premolt increase promptly after ablation (fig. 1a, b; open circles). bilateral eyestalk removal is known to precipitate an immediate premolt, because it removes the source of the neuropeptide moltinhibiting hormone (mih). therefore, both eyestalkless and wounded animals are induced to enter premolt but in wounded animals the premolt peak is delayed relative to eyestalkless animals by the duration of the plateau phase. fig. 1 induction of premolt and molt in intermolt crayfish determined by changes in hemolymph ecdysteroid titre using a ria. time zero indicates time of treatment. (a) bilateral eyestalk ablation (removal of molt inhibiting hormone) (positive control; open circles, orange line). premolt peak is reached at about 35 days and animals undergo ecdysis at about 50 days after treatment. dark triangles (light green line) represent wound to the integument. premolt peak is delayed compared to eyestalk ablated animals by about two weeks and animals undergo ecdysis at about 55 days after treatment. this delay is due to a plateau phase of low ecdysteroid level at days 10-12 after wounding. dark circles (brown line) represent integument wound plus bacteria infection. premolt peak is reached at about day 55 after treatment. a plateau phase (days 0-20) is further prolonged when compared to line b (dark triangles) by about a week. open triangles (dark green line) represent untreated, intact crayfish (negative control). these animals do not reach a premolt peak during the experiment and do not undergo ecdysis. (b) enlarged view of the plateau phase of ecdysteroid titre (days 0-15 underlined with a light blue line on the x-axis of panel (a). (modified from vafopoulou et al., 2007). 127 fig. 2 digital images of cells under the wound using confocal laser scanning microscopy and fluorescent immunohistochemistry on whole tissue mounts. tissues were stained with an antibody against ecr. ecdysteroidresponsive cells are revealed by ecr fluorescence which appears as yellow-green in the nuclei. cellular material and fibres of coagulated hemolymph without ecr are shown as olive green or red. optical sections are 1 μm thick. image a was taken immediately after wounding. images b-i were taken at 2 h after wounding. (a) hyaline hemocytes (short arrows) and strands of coagulated hemolymph fibres (long arrow). (b) aggregation of large number of ecdysone-responsive hyaline hemocytes (stack of 9 optical sections from a z-series). (c) granular hemocytes (arrows) interspersed among hyaline hemocytes (stack of 12 optical sections from a z-series). red, spherical cytoplasmic inclusions represent granules. (d-f) enlarged views of the three types of hemocytes in crayfish: (d) hyaline hemocytes; (e) semigranular hemocytes; arrow shows small size cytoplasmic granules; (f) granular hemocytes; arrow shows large size cytoplasmic granules. (g-i) high magnification of a single nucleus from hyaline hemocyte double-labelled with anti-ecr (g, green) and a fluorescent nucleic acid dye, propidium iodide (i, red) showing co-localization of ecr with chromatin fibres in the nucleus (h, shows merged image of g and f; co-localization is shown as yellow-green). this configuration of distribution of ecr fluorescence in the nuclei of ecdysone-responsive cells is suggested to represent the active state of ecr engaged in gene transcription (vafopoulou et al., 2005; vafopoulou and steel, 2006). bar = 10 μm. the plateau phase of ecdysteroids following injury is necessary for wound healing, epidermal regeneration and initiation of the process of repair of the wound on the integument. this plateau represents the main difference in the premolt processes between eyestalk ablated animals and wounded animals. successful restoration of the damaged integument during molting for wounded crayfish depends on previous regeneration of the damaged epidermis layer; a fully restored epidermis is required to secrete a new cuticle. during the plateau phase, we found the ecdysteroid receptor (ecr) localized in the nuclei of various types of cells involved in wound healing and repair using fluorescent antibodies to ecr, immunohistochemistry (ihc) and confocal laser scanning microscopy (fig. 2) (details in vafopoulou et al., 2007). cellular responses to ecdysteroids are mediated by ecr. ecr belongs to the nuclear receptor superfamily that includes receptors of hormones like estrogen, progesterone, thyroid hormone, vitamin d and others. ecr acts as ligand 128 fig. 3 confocal images of ecr-positive hemocytes with fillopodia (arrows) under the wound at 4 days after wounding. colors as in fig. 2. (a) hyaline hemocytes (modified from vafopoulou et al., 2007). (b) semigranular hemocytes. bar = 10 μm. inducible transcription factor (henrich, 2005). in crayfish, the presence of ecr was restricted in nuclei in association with chromatin (figs 2g-i). this property of ecr identifies these cells as specific targets of ecdysteroid action engaged in ecdysteroid-directed activities (vafopoulou et al., 2005, 2007). the morphology of uninjured tissue under the cuticle of intermolt crayfish reveals a single layer of columnar epithelium immediately under the cuticle and underneath a layer of loose connective tissue forming large hemolymph sinuses. fibrocytes and their long processes form these sinuses which are sparsely populated by small mostly hyaline hemocytes. these processes appear to provide a scaffolding structure the function of which is at the present unknown but it may be involved as a facilitator of cell movement and structural support under the integument. crayfish hyaline hemocytes are recognized as small, oval-shaped cells (about 10 μm in diameter) with a high nuclear to cytoplasmic (n:c) ratio and few, if any, small granules in the cytoplasm (fig. 2d). an immediate response to injury is coagulation of hemolymph under the damaged cuticle (fig. 2a, long arrow). the wound area is quickly populated by fibres of clotted hemolymph which protects and seals the wounded area probably by activation of the resident hemocytes (fig. 2a, short arrows). following an injury to integument, the morphology of the underlying tissue undergoes swift changes including rapid movements of various cell types. the integrated efforts of three classes of cells i.e., hemocytes, epidermal cells and fibrocytes, are required for proper wound healing and repair. by day 2 after wounding, some unknown cue (probably the release of a chemotactic factor from damaged cells?) triggers migration of large number of hemocytes into the wounded area. cell migration begins while the ecdysteroid level is still rising towards its plateau level. hemocytes rapidly infiltrate the area under the wound from the surrounded intact areas. masses of hyaline hemocytes form initially large aggregates and later a continuous sheath, embedded in a now thick disorganized matrix of strand-like coagulated hemolymph (fig. 2b). the rapid aggregation of hemocytes under the wound is to be expected since hyaline hemocytes are the primary cause of hemolymph clotting. the establishment of a hemocyte sheath now seals and protects the wound. a few semigranular and granular hemocytes also move under the wound and become dispersed among the hyaline hematocytes in the hemocyte sheath (fig. 2c, arrows). semigranular hemocytes are cells of about 20 μm in diameter and are characterized by a low n:c ratio. they carry a few small granules in their cytoplasm and their nucleus is slightly eccentrically located. (fig. 2e). granular hemocytes on the other hand are large cells (about 30 μm in diameter), have a slightly irregular shape and an eccentrically located nucleus. they are characterized by a low n:c ratio and the presence of numerous, large cytoplasmic granules (fig. 2f). fillopodia project from the cell bodies of various hemocytes at this time (fig. 3), presumably to facilitate movement under the wound. hemocyte movement under the wound coincides with the appearance of ecr in the nuclei of all hemocyte types (figs 2, 3) suggesting that hemocytes respond swiftly to slight elevation 129 fig. 4 confocal images of cells at 4 days after wounding. ecr-positive epidermal cells are shown in (a) and (c-f). colors as in fig. 2. (a) migrating epidermal cells under the wound originating from the border of the wound (modified from vafopoulou et al., 2007). (b) injured epidermis under the wound with clumped nuclear material (arrows) characteristic of apoptosis. these cells do not stain with anti-ecr. (c) epidermal cells begin to form a new epidermal sheath. (d) enlarged view of epidermal cells forming an epidermal layer. (e) migrating fibrocytes with elongated nuclei (modified from vafopoulou et al., 2007). bar = 10 μm. in ecdysteroid titre after injury and are therefore principal targets of ecdysteroid action. at this day after injury, the compacted cells of epidermis under the wound still have a normal appearance and they do not appear to carry ecr. around day 4 (beginning of ecdysteroid plateau) the epidermal cells under the wound have already become apoptotic as indicated by the presence of highly condensed chromatin in the nuclei (fig. 4b). these cells do not display ecr. a scab has also formed by this time above the sheath of hemocytes. the scab becomes quickly melanized, probably entrapping necrotic tissue. healthy epidermal cells around the periphery of the wound become aligned into rows pointing towards the wound site (fig. 4a). this arrangement of epidermal cells is indicative of cell migration from the edge of the wound towards its centre. as the ecdysteroid titre reaches the top of its plateau, the uninjured epidermal cells display abundant ecr in their nuclei (figs 4a, c, d), showing that they respond to ecdysteroid elevation. therefore, epidermal cells around the wound respond to ecdysteroids but only a few days later than the hemocytes and at relatively higher hormone concentrations. thus, injured and uninjured epidermal cells of the same animal respond differentially to ecdysteroids. these findings indicate that a primary response to the low ecdysone levels induced by injury is to activate epidermal cells to migrate across the injured area to restore the continuous sheath of epidermal cells. the migrating healthy epidermal cells make cell to cell contacts and are aligned in a single-cell layer (figs 4c, d), which becomes a complete sheath under the wound a few days later (see below). therefore, one of the early events in wound healing is cell to cell adhesion. if cell proliferation of epidermal cells takes 130 fig. 5 confocal images of granulocytes at 6 days after wounding with large cytoplasmic inclusions showing aggregation of these cells. these cells do not exhibit immunoreactivity with the ecr antiserum. (a) granulocyte containing enlarged granules and large cytoplasmic inclusions shown in red. (b) granulocyte with giant cytoplasmic inclusions (red). (c) aggregation of granulocytes with giant cytoplasmic inclusions. (d) a stack of 16 optical sections from a z-series shows a layer of hyaline hemocytes (small, round green nuclei) and underneath a layer of granulocytes with giant cytoplasmic inclusions (red). bar = 10 μm. place (and we suspect it does), it occurs not immediately under the wound but in healthy undamaged regions in the neighbourhood of the wound to produce the migrating sheets of epidermal cells that populate the wound area. along with the epidermal cells, fibrocytes, which are characterized by elongated nuclei, also migrate from the wound margin under the wound, probably to restore the scaffolding seen in intact, uninjured areas (fig. 4f). fibrocytes also develop ecr in their nuclei at this time indicating that they are also targets of ecdysteroid action (fig. 4f). at day 6 after injury, the area under the wound possesses abundant semigranular and granular hemocytes all of which display abundant nuclear ecr. many semigranular hemocytes possess conspicuous fillopodia. by this day, the aggregated hyaline hemocytes have formed a multilayered sheath. the appearance within 6 days following injury of ecr in the nuclei of cell types essential to wound healing and repair, such as epidermal cells, fibrocytes and all three types of hemocytes, shows unequivocally that all these cell types are targets of ecdysteroid action. however, the response to ecdysteroids is apparently elicited in each cell type by different hormone concentration. the gathering of granulocytes under the wound site is of particular interest because it indicates that copious amounts of po are probably needed locally for melanisation. indeed, concomitant to the rise of ecdysteroids to the plateau phase, the hemolymph po activity increases greatly (fig. 6, orange bar), and remains significantly higher during the plateau phase compared to those of intact animals (fig. 6, brown bar) and eyestalk-ablated crayfish (fig. 6, green bar). therefore wounding provoked the synthesis of high levels of po activity. the close correlation between the time of appearance of ecr in the hemocytes and the time of increase in po activity is suggestive of ecdysteroid control. this hypothesis is supported by the finding that ecdysone response elements are present in the flanking regions of the 131 fig. 6 changes in hemolymph po activity in crayfish during the plateau phase of the ecdysteroid titre shown in fig. 1. measurements were taken at time 0 (time of treatment) and then at days 2, 4, 6 and 10 after treatment. bars indicate measurements from 10 animals per treatment ± sem. brown bars show control, untreated animals. orange bars show animals with an integumentary wound. green bars show animals with bilateral eyestalk ablations. highly significant (p<0.01) increases in po activity were observed at days 2, 4 6 and 10 when animals with integumentary wounds were compared to controls. likewise, significant (p<0.05) increases in po activity were also measured at days 2, 4 and 6 between wounded and eyestalkless animals. enzyme responsible for catalyzing conversion of propo to po in the insect manduca sexta (zou et al., 2005) indicating direct control by ecdysteroids. further, injection of 20-hydroxyecdysone increased expression of propo mrna in hemocytes of honey bees (zufelato et al., 2004) and in a hemocyte cell line from mosquitoes (ahmed et al., 1999; müller et al., 1999). the phenomenon of increase of po activity following wounding in crayfish is also in agreement with observations in other crustaceans (mucklow and ebert, 2003; mucklow et al., 2004) and insects (nayar and knight, 1995). propo was found immunohistochemically to be distributed widely in the wound site in an insect suggesting that it acts locally and thus assists in the healing process (lai et al., 2002). therefore, it is plausible that the increase in po activity in injured crayfish indicates local participation of po essential for wound healing and repair. the close correlation between the time of appearance of ecr in the hemocytes and the time of increase in po activity suggests that this hemocyte function is probably directly influenced by ecdysteroids. at day 10 (end of plateau and beginning of premolt rise of ecdysteroids), a sheath of flattened and highly condensed granulocytes has been established between the necrotic tissue and the newly forming epidermis (fig. 5d) underneath the multilayer sheath of hyaline hemocytes (fig. 5d). the distinction between semigranular and granular hemocytes becomes unclear at this time because the cells become swollen with giant inclusions probably containing phagocytized and/or encapsulated material (figs 5a, b). ecr is present in the nuclei of all cell types, showing that they still respond to ecdysteroids. at this time, a pale ring around the melanized scab at the edges of the wound is seen under the dissecting microscope when the uppermost layer of the wound surface is peeled away, suggesting that deposition of repair cuticle had already started, as also reported in other crustaceans (dillaman and roer, 1980). around day 10, ecr ceases to be present in the nuclei of cells during the period of study of wound healing and repair. by day 15, after termination of the plateau phase, the titre commences its premolt rise and the epidermal cells have already become organised into a continuous sheet resulting from the coalescence of migrating epidermal cells which fully seals off the wound. at day 20, as the ecdysteroid titre continues to rise sharply to its premolt peak, the deposition of repair cuticle has become completed on the injured site. repair cuticle lacks epicuticle the characteristic outermost layer of arthropod cuticle; this is a phenomenon common to all crustaceans studied so far (halcrow, 1988). the requirement of a low level ecdysteroid plateau for wound healing and repair was highlighted in experiments where integumentary injury was combined with simultaneous injection of bacteria through 132 fig. 7 diagram showing the cascade of events of wound healing and repair in crayfish during the plateau phase of ecdysteroid titre, as summarised in the conclusion section of the text. red arrows indicate pathways where the triggering factors are known. white arrows indicate pathways where the triggering factors are speculated. the wound (vafopoulou et al., 2007). crayfish exposed to both injury and bacteria exhibited an extended ecdysteroid plateau and the consequent delay of the onset of the premolt peak when compared to injured animals (figs 1a, b). the presence of bacteria clearly taxes the resources of the immune system to a much greater extent and results in long delays in wound healing. therefore, the length of the hormonal plateau appears to be dependent on the extent of the immune challenges. thus, it is likely that the immune system functions as a modulator of the neuroendocrine system responsible for ecdysteroid synthesis. the dependence of tissue regeneration during 133 wound healing and repair on low levels of ecdysteroids can be generalized for decapod crustacean and probably for other arthropods. earlier studies in crabs suffering limb autotomy demonstrated that limb regeneration depends on low ecdysteroid levels (see for review skinner, 1985; hopkins, 2001) and results in ecr expression in whole limb regenerates (durica et al., 1996; 1999; 2002; chung et al., 1998). in insects, regeneration of imaginal discs also requires low ecdysteroid level and is inhibited by high (madhaven and schneiderman, 1969; kunieda et al., 1997). why are ecdysteroids needed for wound healing? the work with crayfish shows that hemocytes are principal targets of ecdysteroids and wounding results in elevation of po activity. these findings suggest that the nature of hemocyte responsiveness to ecdysteroids is activation of immune responses. this conclusion is supported by the fact that expression of several immune parameters in other decapod crustaceans is correlated with molt stage (cheng et al., 2003; liu et al., 2004, 2006; kuballa and elizur, 2008; ho et al., 2009; yeh et al., 2009) or with changes in ecdysteroid level following eyestalk ablation (sainzhernández et al., 2008). all this reinforces the hypothesis that immune responses in crustacea are regulated by ecdysteroids during wound healing. even though the area of ecdysteroid control of immune responses in crustaceans is largely unexplored, there is considerable evidence from insect systems supporting this hypothesis. hemocytes failed to respond to bacterial challenge in the absence of ecdysteroids (ahmed et al., 1999; müller et al., 1999). molt-dependent variations in immune parameters (yamamoto et al., 2001; zufelato et al., 2004; meylaers et al., 2006; eleftherianos et al., 2008) and positive correlations between ecdysteroids and immune responses have been observed in insects (meister and richards, 1996; dimarcq et al., 1997; dimopoulos et al., 1997; lee et al., 2002; sorrentino et al., 2002; korayem et al., 2004; aye et al., 2008; flatt et al., 2008). most interesting is the finding that ecdysteroid induction of antimicrobial peptides in the fruit fly drosophila melanogaster required the presence of ecr (flatt et al., 2008). therefore, it appears that the situation of ecdysteroid control of immune responses unites arthropods with vertebrates in which steroid hormones, their nuclear receptors and other members of the nuclear receptor family regulate adaptive and innate immunity (see for review flatt et al., 2008). another cellular aspect of ecdysteroid influence in wound healing in crayfish appears to be the massive migration of various cell types under a wound, notably hemocytes, epidermal cells and fibrocytes. again convincing supporting evidence derives from insect studies where it has been shown that cell migration during morphogenesis is regulated by ecdysteroids (hackney et al., 2007; jang et al., 2009; see for review montell, 2001). a less understood aspect of wound healing process is the cue(s) that activate the crayfish neuroendocrine system to synthesize and release ecdysteroids following an integumentary injury. we speculate that these cues may be supplied by the wound itself as is the case with limb regeneration in crabs. an inhibitory factor similar to molt-inhibiting hormone is released by the regenerating limb bud that inhibits proecdysial growth of limb buds and delays molting by acting on the central neuroendocrine system (see for review mykles, 2001; yu et al., 2002). conclusions in summary, integumentary wounds damage the underlying tissues and set in motion a cascade of events that lead to regeneration and repair of the damaged area. in the event of a wound, both the immune and neuroendocrine systems (ecdysteroid synthesis) are activated to repair the wound rapidly and prevent hemolymph and tissue loss and infection. a schematic summary of the events involved is shown in fig. 7. several triggering factors in this cascade are known (red arrows) and have been described above in detail, whereas the existence of many others is speculative (white arrows) and invites further research. in this scheme, a wound damages cells that lie under the cuticle and these cells leak a variety of cellular components which may trigger several reactions. they may act chemotactically and trigger cell migration towards and under the wound from neighboring healthy areas, e.g., hemocytes, epidermis and fibrocytes, and they may also activate synthesis of specific proteins by these cell types. or they may act on the neuroendocrine system and induce synthesis of ecdysteroids. likewise, the act of wounding itself may activate the neuroendocrine system by some unknown mechanism to initiate synthesis of ecdysteroids. factors released from damaged hemocytes are known to stimulate hemolymph clotting. migration of cells may also be triggered by ecdysteroids. ecdysteroids released into the hemolymph target primarily the immune system, e.g., the hemocytes, thereby appearing to regulate immune responses. conversely, the immune system may also transmit feedback signals to the molting system that modulates ecdysteroid synthesis. ecdysteroids act also upon epidermal cells and fibrocytes to synthesize the necessary building blocks that restore the damaged area. acknowledgements we are grateful to cch steel for his constructive comments in the writing of this review. supported by natural science and engineering research council of canada discovery grant 6069. references ahmed a, martin d, manetti ago, han s-j, lee wj, mathiopoulos kd, et al. genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector anopheles gambiae. proc. natl. acad. sci. usa 96: 14795-14800, 1999. aspán a, huang t, cerenius l, söderhäll k. cdna cloning of prophenoloxidase from the freshwater crayfish pacifastacus leniusculus and its activation. proc. natl. acad. sci. usa 92: 939-943, 1995. 134 aye tt, shim jk, rhee ik, lee ky. upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the indian meal moth, plodia interpunctella. j. insect physiol. 54: 1301-1305, 2008. battison a, cawthorn r, horney b. classification of homarus americanus hemocytes and the use of differential counts infected with aerococcus viridans var homari (gaffkemia). j. invert. pathol. 84: 177-197, 2003. cárdenas w, jenkins ja, dankert jr. a flow cytometric approach to the study of crustacean cellular immunity. j. invert. pathol. 76: 112-119, 2000. cerenius l, söderhäll k. the prophenoloxidaseactivating system in invertebrates. immunol. rev. 198: 116-126, 2004. chang es, chang sa, mulder ep. hormones in the lives of crustaceans: an overview. amer. zool. 41: 1090-1097, 2001. cheng wt, juang fm, li jt, lin mc, liu ch, chen jc. the immune response of the giant freshwater prawn macrobrachium rosenbergii and its susceptibility to lactococcus garvieae in relation to the molt stage. aquaculture 218: 3345, 2003. chung ac-k, durica ds, clifton sw, roe ba, hopkins pm. cloning of crustacean ecdysteroid receptor and retinoid-x receptor gene homologs and elevation of retinoid-x receptor mrna by retinoic acid. mol. cell. endocrinol. 139: 209227, 1998. dillaman md, roer rd. carapace repair in the green crab, carcinus maenas (l.). j. morphol. 163: 135-155, 1980. dimarcq j-l, imler j-l, lanot r, ezekowitz rab, hoffmann ja, janeway ca, et al. treatment of l(2)mbn drosophila tumorous blood cells with the steroid hormone ecdysone amplifies the inducibility of antimicrobial peptide gene expression. insect biochem. molec. biol. 27: 877-886, 1997. dimopoulos g, richman a, müller hm, kafatos fc. molecular immune responses of the mosquito anopheles gambiae to bacteria and malaria parasites. proc. natl. acad. sci. usa 94: 11508-11513, 1997. durica ds, hopkins pm. expression of the genes encoding the ecdysteroid and retinoid receptors in regenerating limb tissues from the fiddler crab, uca pugilator. gene 171: 237-241, 1996. durica ds, chung a c–k, hopkins pm. characterization of ecr and rxr gene homologs and receptor expression during the molt cycle in the crab, uca pugilator. amer. zool. 39: 758-773, 1999. durica ds, wu x, anilkumar g, hopkins pm, chung ac-k. characterization of crab ecr and rxr homologs and expression during limb regeneration and oocyte maturation. mol. cell. endocrinol. 189: 59-76, 2002. eleftherianos i, baldwin h, french-constant rh, reynolds se. developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of manduca sexta to infection by photorhabdus. j. insect physiol. 54: 309-318, 2008. flatt t, heyland a, rus f, porpiglia e, sherlock c, yamamoto r et al. hormonal regulation of the humoral innate immune response in drosophila melanogaster. j. exp. biol. 211: 2712-2724, 2008. gallucci s, matzinger p. danger signals: sos to the immune system. curr. opin. immunol. 13: 114119, 2001. giulianini pg, biert m, lorenzon s, battistella s, ferrero ea. ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge. micron 38: 49-57, 2007. hackney jf, pucci c, naes e, dobens l. ras signalling modulates activity of the ecdysone receptor ecr during cell migration in the drosophila ovary. dev. dynamics 236: 12131226, 2007. halcrow k. 1988. absence of epicuticle from the repair cuticle produced by four malacostracan crustaceans. j. crust. biol. 8: 346-354, 1988. halcrow k, steel cgh. cuticular repair in the mature male snowcrab, chionocetes opilio: relation to ecdysteroids. can. j. zool. 70: 314319, 1992. hall m, söderhäll k. crayfish α-macroglobulin as a substrate for transglutaminases. comp. biochem. physiol. 108b: 65-72, 1994. hall m, wang r, antwerpen rk, sottrup-jensen l, söderhäll k. the crayfish plasma clotting: a vitellogenin-related protein responsible for clot formation in crustacean blood. proc. natl. acad. sci. usa 96: 1965-1970, 1999. henrich v. the ecdysteroid receptor (ecr). in: gilbert li, iatrou k, gill s (eds), comprehensive molecular insect science, elsevier science, oxford, uk, vol. 3, pp 243-285, 2005. ho py, cheng ch, cheng wt. identification and cloning of the alpha 2-macroglobulin of giant freshwater prawn macrobrachium rosenbergii and its expression in relation with the molt stage and bacteria infection. fish shellfish immunol. 26: 459-466, 2009. hopkins pm. limb regeneration in the fiddler crab, uca pugilator: hormonal and growth factor control. amer. zool. 41: 389-398, 2001. hopkins pm, bliss de, sheehan sw, boyer jr. limb growth-controlling factors in the crab gecarcinus lateralis, with special reference to the limb growth-inhibiting factor. gen. comp. endocrinol. 39: 192-207, 1979. hopkins pm, chung c-k, durica ds. limb regeneration in the fiddler crab, uca pugilator: histological, physiological and molecular considerations. amer. zool. 39: 513-617, 1999. jang acc, chang yc, bai jw, montell c. bordercell migration requires integration and temporal signals by the btb protein abrupt. nat. cell biol. 11: 569-u100, 2009. jiravanichpaisal p, lee sy, kim ya, andren t, söderhäll i. antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish pacifastacus leniusculus: characterization and expression pattern. dev. comp. immunol. 31: 441-455, 2007. 135 johansson mw, söderhäll k. isolation and purification of a cell adhesion factor from crayfish blood cells. j. cell biol. 106: 17951803, 1988. johansson, m.w., söderhäll k. a cell adhesion factor from crayfish hemocytes has degranulating activity towards crayfish granular cells. insect biochem. 19: 183-190, 1989. johansson mw, söderhäll k. exocytosis of the phenoloxidase activating system from crayfish hemocytes. j. comp. physiol. 156b: 175-191, 1985. johansson mw, keyser p, sritunyalucksana k, söderhäll k. crustacean hemocytes and hematopoiesis. aquaculture 191: 45-52, 2000. kobayashi m, johansson mw, söderhäll k. the 76 kd cell-adhesion factor from crayfish hemocytes promotes encapsulation in vitro. cell tissue res. 260: 13-18, 1990. korayem am, fabbri m, takahashi k, scherfer c, lindgren m, schmidt o, et al. a drosophila salivary gland mucin is also expressed in immune cells: evidence for a function in coagulation and the entrapment of bacteria. insect biochem. mol. boil. 34: 1297-1304, 2004. kuballa av, elizur a. differential expression profiling of components associated with exoskeletal hardening in crustaceans. bmc genomics 9: 575, 2008. kunieda t, kurat s, natori s. regeneration of sarcophaga imaginal discs in vitro: implication of 20-hydroxyecdysone. dev. biol. 183: 86-94, 1997. lai sc, chen cc, hou rf. immunolocalization of prophenoloxidase in the process of wound healing in the mosquito armigeres subalbatus (diptera: culicidae). j. med. entomol. 39: 266274, 2002. lee k-y, horodyski fm, valaitis ap, denlinger dl. molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth, lymantria dispar. insect biochem. mol. boil. 32: 1457-1467, 2002. lee sy, söderhäll k. characterization of a pattern recognition protein, a masquerade-like protein, in the freshwater crayfish, pacifastacus leniusculus. j. immunol. 166: 7319-7326, 2001. lee sy, söderhäll k. early events in crustacean innate immunity. fish shellfish immunol. 12: 421-437, 2002. lee sy, lee bl, söderhäll k. processing of crayfish hemocyanin subunits into phenoloxidase. biochem. biophys. res. commun. 322: 490496, 2004. liu yc, li fh, wang b, dong b, zhang ql, luan w, et al. a transglutaminase from chinese shrimp (fenneropenaeus chinensis), full-length cdna cloning, tissue localization and expression profiles after challenge. fish shellfish immunol. 22: 576-588, 2007. liu c-h, tseng d-y, lai c-y, cheng w, kuo c-m. molecular cloning and characterization of prophenoloxidase cdna from hemocytes of the giant freshwater prawn, macrobrachium rosenbergii, and its transcription in relation with the molt stage. fish shellfish immunol. 21, 6069, 2006. liu ch, yeh st, cheng sy, chen jc. the immune response of the white shrimp litopenaeus vannamei and its susceptibility to vibrio infection in relation with the molt cycle. fish shellfish immunol. 16: 151-161, 2004. madhavan k, schneiderman ha. hormonal control of imaginal disc regeneration in galleria mellonella (lepidoptera). biol. bull. 137: 321331, 1969. martin gg, castro c, moy n, rubin n. n-acetyl-dglucosamine in crustacean hemocytes; possible functions and usefulness in hemocyte classification. invert. biol. 122: 265-270, 2003. meister m, richards g. ecdysone and insect immunity: the maturation of the inducibility of the diptericin gene in drosophila larvae. insect biochem. mol. biol. 26: 155-160, 1966. mccarthy jf, skinner dm. proecdysial changes in serum ecdysone titers, gastrolith formation and limb regeneration following molt induction by limb autotomy and /or eyestalk removal in the land crab, gecarcinus lateralis. gen. comp. endocrinol. 33: 278-292, 1977. meylaers k, freitak d, schoofs l. immunocompetence of galleria mellonella: sexand stage-specific differences and the physiological cost of mounting an immune response during metamorphosis. j. insect physiol. 53: 146-156, 2007. montell dj. command and control: regulatory pathways controlling invasive behaviour of the border cells. mech. dev. 105: 19-25, 2001. mucklow pt, ebert d. physiology of immunity in the water flea daphnia magna: environmental and genetic aspects of phenoloxidase activity. physiol. biochem. zool. 76: 836-842, 2003. mucklow tp, vizoso db, jensen kh, refardt d, ebert d. variation in phenoloxidase activity and its relation to parasite resistance within and between populations of daphnia magna. proc. r. soc. london b 271: 1175-1183, 2004. müller hm, dimopoulos g, blass c, kafatos fc. a hemocyte-like cell line established from the malaria vector anopheles gambiae expresses six prophenoloxidase genes. j. biol. chem. 274: 11727-11735, 1999. mykles dl. interactions between limb regeneration and molting in decapod crustaceans. amer. zool. 41: 399-406, 2001. nayar jk, knight jw. wounding increases intracellular encapsulation (melanization) of developing brugia malayi (nematoda: filarioidea) larvae in thoracic muscles of anopheles quadrimaculatus. comp. biochem. physiol. 112a: 553-557, 1995. sainz-hernández jc, racotta is, dumas s, hernández-lópez j. effect of unilateral and bilateral eyestalk ablation in litopenaeus vannamei male and female on several metabolic and immunologic variables. aquaculture 283: 188-193, 2008. skinner dm. molting and regeneration. in: bliss de, mantel lh (eds), the biology of crustacea, academic press, new york, usa, vol 9, pp 43146, 1985. 136 smith vj söderhäll k. β-1,3 glucan activation of crustacean hemocytes in vitro and in vivo. biol. bull. 164: 299-314, 1983a. smith vj söderhäll k. induction of degranulation and lysis of hemocytes in the freshwater crayfish, astacus astacus by components of the prophenoloxidase activating system in vitro. cell tissue res. 233: 295-303, 1983b. smith vj söderhäll k. a comparison of phenoloxidase activity in the blood of marineinvertebrates. dev. comp. immunol. 15: 251261, 1991. snyder mj, chang es. effects of sinus gland extracts on larval molting and ecdysteroid titers of the american lobster, homarus americanus. biol. bull. 170: 244-254, 1986. söderhäll k, cerenius l. role of the prophenoloxidase-activating system in invertebrate immunity. curr. opin. immunol. 10: 23-28, 1998. söderhäll k, cerenius l, johansson mw. the prophenoloxidase activating system and its role in invertebrate defence. annals ny acad. sci. 712, 155-161, 1994. söderhäll k, wingren a, johansson mw, bertheussen k. the cytotoxic reaction of hemocytes from the freshwater crayfish, astacus astacus. cell immunol. 94, 326-332, 1985. söderhäll i, wu c, novotny m, lee bl, söderhäll k. a novel protein acts as a negative regulator of prophenoloxidase activation and melanisation in the freshwater crayfish pacifastacus leniusculus. j. biol. chem. 284: 6301-6310, 2009. sorrentino rp, carton y, govind s. cellular immune response to parasite infection in the drosophila lymph gland is developmentally regulated. dev. biol. 243: 65-80, 2002. sritunyalucksana k, söderhäll k. the propo and clotting system in crustaceans. aquaculture 191: 53-69, 2000. steel cgh, vafopoulou x. ecdysone titre profiles during growth and development of arthropods. in: koolman j (ed), ecdysone: from chemistry to mode of action, georg thieme verlag, stuttgart, germany, pp 221-231, 1989. sung hh, sun r. use of monoclonal antibodies to classify hemocyte subpopulation of tiger shrimp (penaeus monodon). j. crustacean biol. 22: 337-344, 2002. theopold u, li d, fabbri m, scherfer c, schmidt o. the coagulation of insect haemolymph. cell. mol. life sci. 59: 363-372, 2002. theopold u, schmidt o, söderhäll k, dushay ms. coagulation in arthropods: defence, wound closure and healing. trends immunol. 25: 289294, 2004. vafopoulou x, laufer h, steel cgh. spatial and temporal distribution of the ecdysteroid receptor (ecr) in haemocytes and epidermal cells during wound healing in the crayfish, procambarus clarkii. gen. comp. endocrinol. 152: 359-370, 2007. vafopoulou x, steel cgh. ecdysteroid hormone nuclear receptor (ecr) exhibits circadian cycling in certain tissues, but not others, during development in rhodnius prolixus (hemiptera). cell tissue res. 323: 443-455, 2006. vafopoulou x, steel cgh, terry kl. ecdysteroid receptor (ecr) shows marked differences in temporal patterns between tissues during larval-adult development in rhodnius prolixus: correlations with haemolymph ecdysteroid titres. j. insect physiol. 51: 27-38, 2005. vázquez l, pérez a, millán d, agundis c, martin g, cooper rl, et al. morphology of hemocytes from the freshwater prawn macrobrachium rosenbergii. j. morphol. 234: 147-153, 1997. vogt g. the marbled crayfish: a new model organism for research on development, epigenetics and evolutionary biology. j. zool. 276: 1-13, 2008. wang r, lee sy, cerenius l, söderhäll k. properties of the prophenoloxidase activating enzyme of the freshwater crayfish, pacifastacus leniusculus. eur. j. biochem. 268: 895-902, 2001a. wang r, liang z, hall m, söderhäll k. a transglutaminase involved in the coagulation system of the freshwater crayfish, pacifastacus leniusculus. tissue localisation and cdna cloning. fish shellfish immunol. 11: 623-637, 2001b. webster sg. neuropeptides inhibiting growth and reproduction in crustaceans. in: coast gm, webster sg (ed), recent advances in arthropod endocrinology, cambridge university press, cambridge, uk, pp 33-52, 1998. yamamoto k, fujii h, kusakabe t, koga k, aso y, ishiguro m. change in phenoloxidase and its precursor during silkworm (a80 strain) development. j. fac. agric. kyushu univ. 45: 487-493, 2001. yeh ms, lai cy, liu ch, kuo cm, cheng w. a second propo present in white shrimp litopenaeus vannamei and expression of the propos during a vibrio alginolyticus injection, molt stage. fish shellfish immunol. 26: 49-55, 2009. yu x, chang es, mykles dl. characterization of limb autotomy factor-proecdysis (lafpro), isolated from limb regenerates, that suspends molting in the land crab gecarcinus lateralis. biol. bull. 202: 204-212, 2002. zhan wb, wei xm, xing j, zhang zd. characterization of monoclonal antibodies to haemocyte types of the shrimp, fenneropenaeus chinensis. crustaceana 81: 931-942, 2008. zou z, wang y, jiang h. manduca sexta prophenoloxidase activating proteinase-1 (pap1) gene: organization, expression, and regulation by immune and hormonal signals. insect biochem. mol. biol. 35: 627-636, 2005. zufelato ms, lourenco ap, simŏes zlo, jorge ja, bitondi mmg. phenoloxidase activity in apis mellifera honey bee pupae, and ecdysteroiddependent expression of the prophenoloxidase mrna. insect biochem. mol. biol. 34: 12571268, 2004. 137 39 isj 15: 39-51, 2018 issn 1824-307x research report the effects of cyanobacterial blooms on the immune system of elliptio complanata in urban and agricultural areas in the yamaska river watershed f gagné1*, m gélinas1, m fortier2, m fournier2 1acquatic contaminants research division, environment and climate change canada, 105 mcgill street, montreal, quebec, canada, h2y 2e7 2inrs – institut armand-frappier, 531 boulevard des prairies, laval, quebec, canada, h7v 1b7 accepted february 6, 2018 abstract the cumulative effects of cyanobacterial blooms at sites impacted by urban or agricultural activity, could be detrimental to local freshwater mussels. the purpose of this study was to examine and compare the resulting toxicity using an upstream–downstream approach at four sites in the yamaska river. mussels were caged and placed at a site where cyanobacterial blooms were present, a site that receives municipal effluent from a small city of 75,000 inhabitants, and a site that drains a large agricultural area—plus a reference site in lake saint-pierre (quebec, canada) for four months spanning the summer and fall (june to october). effects on immunocompetence were monitored by testing for hemocyte counts, viability, phagocytosis, production of nitric oxide (no), lysozyme (ly), reactive oxygen species (ros), thiol contents, and inflammation induced by cyclooxygenase (cox) activity and glutathione s-transferase (gst) activity. cyanobacterial blooms occurred at the target site only, reaching levels of 2.1 million cyanobacteria/l and 3 g/l of microcystin-lr in surface waters. although most of the immune parameters were affected at the urban, agricultural and cyanobacteria sites, ros and ly were the most responsive to cyanobacterial bloom, with a significantly greater response than the agriculture, urban and reference sites. the results also suggest that the effects of cyanobacterial blooms are spatially localized; they are not found at the downstream urban and agriculture sites in the yamaska river. key words: cyanobacteria, microcystins, hemocytes, immunocompetence, lysozymes, oxidative stress introduction the yamaska river is located in southeastern quebec (canada). it is 177 km long, covers a watershed of 4,784 km2 and originates from lake brome. the river ends in lake saint-pierre, which is part of the st. lawrence river. the watershed consists mainly of small townships (<75000 inhabitants) and supports intense agricultural activity. urban and agricultural pollutants including nutrients (total phosphorus and nitrogen) are released into the river, degrading its water quality. eutrophic conditions in aquatic environments can lead to blooms of pelagic and benthic cyanobacteria. for example, blooms of unicellular planktonic microcystis aeruginosa or filamentous anabaena, aphanizomenon or planktothrix can develop in lakes enriched with high nutrient loads. ___________________________________________________________________________ corresponding author: francois gagné acquatic contaminants research division environment and climate change canada 105 mcgill street, montreal, quebec, canada e-mail: francois.gagne@canada.ca m. aeruginosa produce toxic secondary metabolites called microcystins (mycst). these toxins are potent inhibitors of protein phosphatase 1 and 2a and cause liver damage and death to animals (jochimsen et al., 1998). the production of toxins is also associated with other unfavourable environmental conditions (e.g., hypoxia and high concentrations of suspended solids, organic matter and other associated pollutants), and the combined or cumulative effects can have profound effects on aquatic organisms. in freshwater environments, harmful algal blooms often occur in locales where bivalve molluscs are found. as sedentary filter-feeder organisms, mussels accumulate (cyano)bacteria, fungi, algae and suspended matter as sources of nourishment, thereby ingesting an important variety of environmental contaminants. thus, they may filter and accumulate large quantities of possibly toxic cyanobacteria. in a previous study, freshwater mussels (elliptio complanata) fed with anabeana flos-aqua in the laboratory for five days showed toxic effects, although the cyanobacteria were not 40 fig. 1 site location. the lake saint-pierre (lsp) site is part of the st. lawrence river and is located downstream from the other sites. the yam site is located 0.5 km from the junction of the yamaska river and lake saintpierre. lake boivin (boi) is within the municipality of granby. the choinière (cho) basin is northwest of granby. actively producing microcystins (gélinas et al., 2013). indeed, acetylcholinesterase activity and lipid peroxidation were higher in mussels fed with the cyanobacteria. although cyanobacteria are considered gram-negative, they contain unusual surface mucopeptides thick enough to react weakly with the gram stain (hoiczyk and hansel 2000). in addition, exposure of fish for 90 days to m. aeruginosa stimulated the immune system, including lysozyme activity, which is involved in the degradation of cell walls of gram-positive bacteria (das et al., 2013). in clams exposed to 100 µg/l microcystin-lr for 10 days, an increase in enzymes involved in oxidative stress, such as superoxide dismutase and catalase, was observed (pham et al., 2016). these responses were observed at tissue microcystin levels of 12.7 ± 2.4 µg/g dry weight, indicating that corbicula leana clams are relatively tolerant to these toxins. decreased glutathione stransferase (gst) activity was observed in dreissena polymorpha exposed to 10 µg/l and 50 µg/l microcystin-lr for 24 h and 7 days (burmester et al., 2012). the long-term and cumulative impacts of urban, agricultural and cyanobacteria-related pollution on organisms exposed to multiple contaminants are currently not well understood. for example, decreased gst activity in organisms exposed to cyanobacterial blooms could limit biotransformation and elimination of other organic pollutants to which they are exposed concurrently. increased levels of cyanobacteria in mussel digestive glands could have major impacts on the immune system. as a mussel filters its food, these harmful algae interact directly with the gills, digestive gland and other tissues during ingestion, allowing diffusion to the hemolymph throughout the bivalve’s open circulatory system. the internal defences against invading foreign bodies are mainly based upon non-specific innate immune system responses, such as phagocytosis and the production of humoral factors such as cytokines (auffret, 2005). hemocytes are responsible for phagocytosis and the production of humoral factors (cheng et al., 1996). these cells are not only found in the circulatory system, but also nest in interstitial tissues waiting to return to circulation following a stress signal. the release of hemocytes following an invasion by foreign bodies is a first response; phagocytosis and lysozyme (ly) production usually follow. hemocyte viability could be also affected following exposure to cyanobacteria extracts in crassostrea gasar oysters (queiroga et al., 2017). ly 41 is an enzyme involved in the degradation of cell walls of most gram-positive bacteria and cyanobacteria (which are gram-indeterminate), leading to lysis. this process is supported by phagocytocis, a further attempt to eradicate the pathogens/toxins in which the ingested bodies in phagosomes are destroyed by oxidative burst involving the production of nitric oxide (no) and hydrogen peroxide (peroxynitrite) as the main form of reactive oxygen species (ros). increased cyclooxygenase (cox) activity is associated with the production of pro-inflammatory precursors to assist the immune response (ottaviani 2004; gagné et al., 2005). recently, ros formation and antioxidant enzyme responses have been reported in organisms exposed to purified microcystins or microcystis extracts (wiegand and pflugmacher 2005; pietro et al., 2006; amado et al., 2010). ros, when improperly inactivated, can inflict damage on a wide range of cellular components (dna, proteins and lipids), and it is linked with the development of many pathological states. the observed resistance of mussels against cyanotoxins could also be attributed to increased biotransformation through gst activity and gsh, a main source of reduced thiols in cells (fernandes et al., 2009). the increase in gst activity coincided with peak levels of microcystin-lr in tissues, followed by a phase of decreasing activity after 8-day and 12-day depuration periods in toxin-free water. given that toxic marine algae could impair the immune system, we investigated whether blooms of m. aeruginosa could affect the immune system of a freshwater mussel, e. complanata, already multiply stressed by agricultural and urban activities in the yamaska river. this mussel is typical of the native fauna of the yamaska watershed and is representative of a sessile species that would endure blooms of m. aeruginosa in addition to other pollution inputs from agricultural and urban activities. the aim of the study was therefore to examine the effects of m. aeruginosa blooms on the immune system in caged mussels during the summer/fall months. we included sites contaminated by urban activities (municipal discharges) and agriculture during the same period to assess the contribution of other stressors to the health status of caged mussels. materials and methods study area sites were selected using an upstreamdownstream approach: the sites polluted by urban activity and algal blooms are located upstream in the yamaska river which discharges on the south shore of lake saint-pierre (lsp), which is part of the st. lawrence river (figure 1). three sites were located in the yamaska watershed, which supports agricultural activity and an urban area (granby; population circa 75,000). two sites were located upstream from granby: lake boivin (boi) and the choinière reservoir (cho). cho, the site located farthest upstream, is a 4.7 km2 reservoir built in 1972 that is prone to frequent cyanobacterial blooms (rolland et al., 2005). the boi site, which is a little farther downstream, was also formed by dams in the early 19th century. it receives the municipal effluents from the city of granby (aeration lagoons) and is also subject to cyanobacterial blooms. however, in this study, a cyanobacterial bloom occurred only at the cho site. the third site (yam) was in the yamaska river 3 km upstream from where it flows into lake saint-pierre, which drains more than 50 km of intensely farmed land. the fourth site was the reference site, lsp, located in lake saint-pierre near île plate which does not receive input from the yamaska river. mussel collection and caging exposure wild freshwater elliptio complanata mussels were collected in the first week of june 2011 in lake achigan (quebec, canada), which is not subjected to any direct sources of pollution. the animals were maintained in an aquarium at 15 °c under constant aeration, with a 16h-light/8h-dark photoperiod. they were fed daily with concentrates of phytoplankton (phytoplex®) and laboratory-cultured pseudokirchneriella subcapitata algae. mussels were then placed in two cylindrical nets (65×30 cm), with 30 mussels in each net (which includes extra mussels in case of unexpected mortality), and immersed at the four sites (lsp, yam, boi and cho) on june 20 and 21. the cages were secured with a cable and 10-kg cinderblocks. mussels were collected (10 randomly collected mussels per time) for analysis after one month (on july 30) and three months (september 30), placed in coolers at 4 ℃ and brought back to the laboratory for immediate immunocompetence assessments, as described below. water sampling and analysis water sampling was carried out monthly (in july, august, september and october). temperature, oxygen concentration, conductivity and ph were measured at 1 m depth in the water column with a ysi multiprobe. water samples were transferred into two 1 l polypropylene containers: a dark one for chlorophylla a (chl a) analysis and a clear one for the measurement of total phosphorus (tp), total nitrogen (tn), dissolved organic carbon (doc), ammonia (nh3) and nitrite-nitrate (no2-no3) (environment canada 2005). in the laboratory, chla water samples were filtered on gf/f whatman filters (0.7 µm pore size) that were kept frozen until analysis. chla pigments were extracted in cold 95% ethanol for 24 h and measured before and after acidification at 665 nm and 750 nm (spectronic genesys 5 spectrophotometer) (rice et al., 2012). tp and total dissolved phosphorus (tdp) were determined by acid digestion followed by colorimetry with ammonium molybdate (rice et al., 2012). tn was analyzed with a lachat continuous flow quick-chem 8000. no2-no3 was measured by reducing nitrate to nitrite in a cadmium column prior to colorimetry; nh3 was analyzed by colorimetry after the addition of sodium nitroprusside and phenate sodium. doc was oxidized to carbon dioxide by the addition of persulfate prior to infra-red detection (shimadzu tco-5000) (rice et al., 2012). the levels of mc-lr toxins in the water were determined using a commercial competitive immunoassay kit (enzo life sciences, usa). 42 hemolymph collection mussel hemolymph of 10 individuals per site and time of collection (july and end of september) was drawn (between 750 l and 1,000 l) from the posterior adductor muscle with a 3 ml syringe and a 23g needle. a portion of the hemolymph was set aside to measure hemocyte concentration and viability, reactive oxygen species (ros), nitric oxide production (no) and reduced thiols by flow cytometry. about 800 l was kept to measure lysozyme (ly), cyclooxygenase (cox) activities and nitric oxide (no) concentration. for ly, the hemolymph was centrifuged at 1,000 × g at 4 °c for 10 min, 100 µl of supernatant (plasma) was withdrawn for activity measurements, and the remaining pellet was re-suspended in 150 µl of phosphate buffered saline (pbs; pre-diluted 1/3 in water) for cox activity. total proteins in the cell-free hemolymph and hemocytes were determined by the protein-dye binding principle (bradford, 1976). flow cytometric analysis hemocyte counts and viability were evaluated by flow cytometry using a 3-colour guava easycyte plus cytometer with a laser emitting at 488 nm, using the supplied viacount kit (guava technologies, hayward, ca, usa). an aliquot of 20 μl of hemolymph was mixed with 80 µl of viacount solution after 10 min, in accordance with the supplier’s recommended procedure. the flow rate was set at 0.6 µl/sec, and 5,000 events were counted. the data were expressed as the number of cells/ml and percentage of live cells for hemocyte concentration and viability respectively. for phagocytosis activity, hemocytes were incubated with fluorescently labelled beads based on the protocol of brousseau et al., 2000. fluorescent latex beads (polysciences, pa, usa) were added to the cell suspensions at a 30:1 (beads:cell) ratio and incubated for 18 h at 15 oc in a humidified incubator. after the incubation period, the hemocyte suspension was re-suspended in prediluted pbs and layered over 4 ml of rpmi cell culture media (diluted 1/4 in water) supplemented with 3% bovine serum albumin (bsa) (sigma, ontario, canada). elimination of free or loosely bound beads was followed by centrifugation at 150 × g for 8 min at 4 °c. the cell pellet was then suspended in 0.5 ml of 0.5% formaldehyde and 0.2% sodium azide (sigma chemicals, canada) diluted in pbs (becton dickinson, ca, usa). cells were analyzed using flow cytometry, and at least 10,000 events were recorded for analysis. hemocyte populations were defined on the basis of their forward and right-angle scatter properties (fsc and ssc, respectively). results were analyzed with the cell quest pro software (becton dickinson). the percentage of cells (hemocytes) that engulfed at least one bead and at least three beads was measured in order to assess phagocytosis activity and efficiency, respectively (farcy et al., 2011). intracellular ros and reduced thiol were measured as previously described (brousseau et al., 2000). the probe dihydrochlorofluorescein diacetate (h2dcfda; cas 4091-99-0) was used to measure ros levels in hemocytes (collén and davison 1997). a 2 µm final concentration of h2dcfda was prepared and mixed with hemocytes and allowed to stand for 45 min in the dark. the presence of intracellular ros was determined by oxidation of h2dcfda dye and the dye is retained in viable cells by non-specific esterases (deacetylation of the diacetates into carboxylates). fluorescence was measured for 3,000 events using flow cytometry. for reduced thiol, the 5-chloromethylfluorescein diacetate probe (cmfda) was used. as the first step, non-specific thiol fluorescence background was blocked using n-ethylmaleimide (nem) as a negative control: hemocytes were treated with 100 µm nem for 10 min and washed in pre-diluted pbs. afterward, all hemocytes were stained with 5-µm cmfda and a total of 2,500 events were acquired by flow cytometry. immunological biomarkers the production of no was estimated by measuring the levels of nitrite concentration in the plasma (verdon et al., 1995). because no reacts readily with oxygen to produce nitrites and nitrates, nitrate reductase was added to convert nitrates into nitrites. following a 30 min pre-incubation step of 50 µl of hemolymph with 25 µl of nitrate reductase (80 units/l) and 25 µl of nadph (3 mm), the concentration of no was measured by adding 100 µl of griess reagent and reading the absorbance at 450 nm after 30 min. results are expressed as µmol of no/mg proteins. the activity of cox in hemocytes was measured by oxidation of the 2,7-dichlorofluorescein in the presence of arachidonic acid (gagné, 2014). a volume of 50 µl of the suspension (hemocytes and 1/4 pbs) was mixed with 150 µl of tris-hcl buffer 50 mm, ph 8.0, containing 0.05% tween 20, 0.1 µg/ml horseradish peroxidase, 25 µm arachidonic acid and 2 µm 2,7-dichlorofluorescein. fluorescence readings were taken at 485 nm for excitation and 520 nm for emission every 10 min for 30 min at 30 °c. a standard solution of fluorescein in pbs at 1 µm was used for instrument calibration. cox activity was expressed as fluorescein units/min/mg proteins. total protein concentration was determined using bovine serum albumin as standard, via the proteindye binding principle (bradford, 1976), with absorption at 595 nm measured by a microplate reader (powerwave, biotek). statistical analysis the biomarker responses were determined in n=10 mussels for each site and time of collection (season). the biomarkers were normalized to the lsp reference site for each season. the data was tested for normal distribution and homogeneity of variance using the shapiro–wilk and bartlett tests respectively. two-way factorial analysis of variance (anova) was used to test for the effects of sites (boi, choi, lsp, yam), seasons (summer and fall) and their interaction. intersite and between time comparisons were done using the least square difference test as the post-hoc test. correlation was examined using the pearson-moment procedure. significance was set at p<0.05. all statistical analyses were performed using statistica version 8. 43 table 1 change in physico-chemical characteristics of surface waters parameter turbidity oxygen (mg/l) chl a (mg/ml) blue-green algae (cells/ml) total n/nh3/no2 (mg/l) doc1 (mg/l) conductivity/ph s.cm-1 dissolved and total phosphates (mg/l) lsp j a s o 4.4 3.3 2.5 6.1 8.9 8.7 9.0 10.3 0.62 2.7 1.9 2.8 nd nd nd nd 0.545/0.04/0.18 0.475/0.04/0.17 0.54/0.05/0.19 0.58/0.04/0.22 3.5 3.5 3.4 3.4 260/8.5 276/8.6 272/8.3 271/8.2 0.18/0.01 0.18/0.01 0.02/0.01 0.03/0.01 yam j a s o 15.8 7.8 59.1 27.7 10.1 9.2 8.5 9.7 1.13 7,7 6.3 4.3 nd nd nd nd 0.48/0.02/0.05 0.69/0.02/0.03 0.54/0.05/0.2 1.9/0.05/1.3 3.2 3.3 7.5 7.5 280/8.3 286/8.7 289/8.1 332/8.3 0.03/0.008 0.03/0.01 0.19/0.07 0.14/0.06 cho j a s o 10.2 8.2 18.8 261 13.1 9.6 8.6 7.8 9.9 15.3 17.6 18 nd 613 2424 20900 0.06/0.001/0.02 0.68/0.02/0.02 0.75/0.08/1.2 0.75/0.01/0.02 5.2 5.4 6.8 6.8 129/9.2 129/8.6 122/7.5 130/6.9 0.02/0.01 0.03/0.01 0.04/0.01 0.04/0.01 boi j a s o 3.7 4.2 3.3 1.5 6.0 6.3 6.8 9.0 48 29.7 8.6 9 60 nd nd nd 0.1/0.02/0.02 1.1/0.03/0.02 0.84/0.02/0.02 0.84/0.02/0.03 9.5 9.5 11 11 156/7.4 161/7.8 180/7.2 218/8.0 0.22/0.1 0.17/0.1 0.11/0.09 0.11/0.09 1. dissolved organic carbon. results surface water chemistry during the first four weeks of the summer period, water temperature was stable at around 24 ℃. during the fall period, the mean water temperature was 17 °c and varied between 20 °c and 14 ℃. chl a measurement was higher at the boi site in the first summer month (july) but did not contain blue-green algae. chl a was higher at the cho site in fall, during the m. aeruginosa bloom (table 1). the number of blue-green algae increased considerably during the fall at cho site exclusively (table 1). cyanobacteria toxins were measured on every sampling date at all sites. no toxins were detected at any site in the summer, but during just one month in september at cho site. microcystin-lr equivalents were detected in filtered water (0.45 µm) taken from the water column (not the algae) at concentrations between 1.5 µg/l and 3 µg/l. immunocompetence responses for hemocyte density, two-way factorial anova revealed a positive interaction (f=32; p<0.001) between site and season (figure 2a). hemocyte density was significantly higher in the summer than in the fall: the mean cell density at the reference site, lsp, was 960 000 and 580 000 cells/ml in summer and fall respectively. in the summer, hemocyte density did not change significantly between sites. in the fall, hemocyte counts were significantly higher at the cho (5-fold) and boi (3.5-fold) sites than at the lsp and yam sites. the cho and boi levels were also significantly different from each other. hemocyte density was significantly correlated with ammonia (r=0.52; p<0.05), nitrates (r=-0.53; p<0.05) and ph (r=0.54; p<0.05). in respect to hemocyte metabolic activity (viability), based on general esterase activity, two-way factorial anova confirmed a significant interaction (f=15.7, p<0.001) between site and season (figure 2b). the initial metabolic activity (viability) index was 82% and 50% at the lsp site in summer and fall respectively. in the summer, hemocyte metabolic activity was significantly lower at the cho site (0.9-fold) compared to the lsp site. in the fall, hemocyte activity was significantly higher at the yam (1.2fold), boi (1.5-fold) and cho (1.45-fold) sites relative to the lsp site. hemocyte density was not significantly different between the cho and boi sites. hemocyte activity was significantly correlated with hemocyte density (r=0.39, p<0.05). phagocytosis activity and efficiency were also examined in mussels (figure 3). two-way factorial anova revealed a significant interaction (f=18.3, p<0.001) with site and season for phagocytosis activity. phagocytosis activity was significantly higher in the summer than in the fall (figure 3a). the initial activity was 41% and 14% at the lsp site for summer and fall respectively. in the summer, phagocytosis activity was significantly higher at the boi site (1.4-fold) compared to the lsp site. in the fall, phagocytosis activity at cho (2.3-fold), boi (2.1-fold) and yam (0.6-fold) were significantly 44 lsp yam boi cho sites 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 h e m o c y te d e n s it y (n o rm a liz e d t o r e fe re n c e s it e ) summer autumn a,b,c a,c a lsp yam boi cho sites 0,7 0,8 0,9 1,0 1,1 1,2 1,3 1,4 1,5 1,6 1,7 h e m o c y te v ia b il ty (n o rm a li z e d t o r e fe re n c e l s p s it e ) summer autumn a,c a,c a a b fig. 2 change in hemocyte density and esterase activity. mussels were caged at the sites in june and removed at the end of july and september. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no bluegreen algae). the letter c indicates a significant difference from yam. different than at lsp. phagocytosis activity was significantly correlated with hemocyte density (r=0.55; p<0.001), viability (r=0.43; p<0.001), nitrates (r=-0.67; p<0.01), chl a (r=0.61; p=0.01) and water conductivity (r=-0.53; p<0.01). phagocytosis efficiency responded similarly to phagocytosis activity with a significant interaction (f=22.8, p<0.001) between site and time (figure 3b). the efficiency values were 27% and 5% at the lsp site in summer and fall respectively. in the case of ly activity, two-way factorial anova revealed a significant interaction (f=7.0, p<0.001) between site and season (figure 3c). ly activity was at 3.9 and 1.5 absorbance change/min/mg proteins at the lsp site in the summer and fall respectively. in the summer season, ly activity remained unchanged between sites. however, a significant increase in ly activity was observed at boi (6-fold) and cho (10-fold) compared to the lsp. there was also a significant difference in ly activity between the cho site (rural cyanobacterial bloom) and the boi (urban) site. correlation analysis revealed that ly activity was significantly correlated with blue-green cyanobacteria counts (r=0.50; p<0.05), ammonia (r=-0.52; p<0.05) and conductivity (r=-0.60; p=0.01). multiple regression analysis revealed a correlation 45 lsp yam boi cho sites 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 p h a g o c y to s is a c ti v it y (n o rm a liz e d t o r e fe re n c e l s p s it e ) summer autumn a a,c a,c a,c a lsp yam boi cho sites -0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 p h a g o c y to s is e ffi c ie n c y (n o rm a liz e d to r e fe re n c e l s p s ite ) summer autumn b a,b,c a,b,c a lsp yam boi cho sites -2 0 2 4 6 8 10 12 14 l y s o z y m e a c tiv ity (n o rm a liz e d to r e fe re n c e l s p s ite ) summer autumn a,b,c a,c c fig. 3 change in phagocytosis and lysozyme activity in mussels exposed to multiple sources of pollution. mussels were caged at four sites from june to october. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no blue-green algae). the letter c indicates a significant difference from yam. between lysozyme activity (dependent variable) and blue-green cell counts (β=0.50) with conductivity (β=-0.44) (r=0.80; p=0.002), suggesting that hemolymph ly activity increases with blue-green cell levels in water that has low conductivity. ros production following phagocytosis was measured, as well as the production of total nitrites in hemocytes (figure 4). for ros production, twoway factorial anova showed a significant interaction (f=12.6, p<0.001) between season and site (figure 4a). mean ros levels were 2,941 and 36 fluorescence units at the lsp site during the summer and fall seasons, respectively. in the summer, ros production did not differ significantly between sites. in the fall, ros production was significantly higher at the cho site only (20-fold) than at the yam and lsp sites. although the ros levels were high (5-fold) at boi, the difference was not significant owing to the variability. ros levels between cho and boi were significantly different. correlation analysis revealed that ros production was significantly correlated with hemocyte density (r=0.32; p=0.001), viability (r=0.42; p<0.001) and dissolved p/total p ratio (r=-0.68; p<0.01). for no production, two-way factorial anova revealed a significant interaction (f=4.6, p<0.01) between season and site (figure 4b). the mean no levels were 3.3 µg/mg and 0.1 µg/mg protein at the lsp site in the summer and fall respectively. in the summer, no production showed no significant difference between sites. in the fall, no production was significantly higher at cho (8-fold) compared to the lsp site. correlation analysis revealed that no level was significantly correlated with hemocyte density (r=0.37; p<0.001), phagocytosis efficiency (r=0.25; p=0.05) and dissolved p/total p ratio (r=0.49; p=0.05). cox activity was measured in hemocytes to assess inflammation in freshwater mussels (figure 4c). two-way factorial anova revealed a significant interaction (f=4.5, p<0.01) between site and season. cox activity was at 8,700 and 7,300 fluorescein units/min/mg proteins at the lsp site in the summer and fall respectively. in the summer, cox activity did not differ significantly by 46 lsp yam boi cho sites -5 0 5 10 15 20 25 30 h e m o c y te r o s le v e ls (n o rm a liz e d to l s p r e fe re n c e s ite ) summer autumn a,b,c a lsp yam boi cho sites -4 -2 0 2 4 6 8 10 12 n o p ro d u c ti o n (n o rm a li z e d t o r e fe re n c e l s p s it e ) summer autumn a,b,c b lsp yam boi cho sites 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 c o x a c ti v it y (n o rm a li z e d t o l s p r e fe re n c e s it e ) summer autumn a,cc c fig. 4 oxidative burst and inflammation in freshwater mussel hemolymph. mussels were caged at four sites during the summer and fall. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no blue-green algae). the letter c indicates a significant difference from yam. 47 site. in the fall, cox activity was significantly lower at cho (0.6-fold) and boi (0.5-fold) compared to the reference site, lsp. correlation analysis revealed that cox activity was significantly correlated with hemocyte density (r=0.56; p<0.01), gst (r=0.26; p<0.01), thiol content (r=0.83; p<0.001) and lysozyme activity (r=-0.67; p<0.01). biotransformation activity was followed by reduced thiol content (for metals and ros inactivation) and gst (for organic pollutants) activity. for hemocyte thiol content, two-way factorial anova revealed a significant interaction (f=10.9, p<0.001) between site and season. the mean thiol content was 109 and 121 fluorescence units at the lsp site in the summer and fall respectively. in the summer, thiol content was significantly higher at cho (1.8 fold) and lower at yam (0.3-fold) compared to lsp (figure 5a). thiol content was significantly higher at the cho site than at the boi site. in the fall, thiol content was significantly lower at cho (0.2-fold) and boi (0.18fold) compared to yam and lsp. there was no significant difference between the boi and cho sites. thiol content was significantly correlated with ph (r=0.61; p=0.01) and oxygen content (r=0.51; p<0.05). lsp yam boi cho sites -0,5 0,0 0,5 1,0 1,5 2,0 2,5 h e m o c y te t h io l c o n te n t (n o rm a liz e d t o l s p r e fe re n c e s it e ) summer autumn a a,b,c c a a,b,ca,c lsp y am boi cho sites -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 g s t a c ti v it y (n o rm a liz e d t o l s p r e fe re n c e s it e ) summer autumn b a,b,c a,b,c a,c a,c fig. 5 biotransformation activity in hemocytes. mussels were caged at four sites in the yamaska river during the summer and fall. biotransformation activity was determined by total thiol content and gst activity. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no blue-green algae). the letter c indicates a significant difference from yam. 48 for gst activity, two-way factorial anova revealed a significant difference for sites only (f=29.5, p<0.001) i.e. no seasonal effects and no interaction between season and sites. gst activity was globally similar in the summer and in the fall (figure 5b). the mean gst activity was 0.11 and 0.06 absorbance change/min/mg proteins at the lsp site in the summer and the fall. in the summer, gst activity significantly decreased at boi (0.45-fold) and cho (0.25-fold) compared to lsp. in the fall, the same pattern was observed but more intensely than in the summer, with 0.18-fold and 0.2-fold decreases at boi and cho respectively. there was no significant difference between the boi and cho sites by season. correlation analysis revealed that gst activity was significantly correlated with hemocyte density (r=0.23; p<0.05), phagocytosis efficiency (r=0.23; p<0.05), no production (r=0.42; p<0.001), doc (r=-0.66; p<0.01), conductivity (r=0.61; p=0.01) and dissolved p/total p ratio (r=-0.56; p<0.05). we examined the immunocompetence data to attempt to determine whether cyanobacterial blooms could produce specific, discernible effects in mussels. in respect to cyanobacterial blooms that occurred at the cho site only in the fall, we sought biomarkers that responded to the cho site and differed significantly from the urban pollution (boi), agriculture (yam) and reference (lsp) sites. we found that hemocyte density, phag efficiency, ros, no and ly activity satisfied the above criteria based on two-way factorial anova (table 2). moreover, ly activity was significantly correlated (r=0.67; p<0.01) with the levels of blue-green algae (cyanobacteria) in surface waters. multiple regression revealed that lysozyme activity was predicted (r=0.85) by blue-green algae (β =0.54; p<0.05), no3/total n (β=0.32; p<0.05) and conductivity (β=-0.42; p<0.05). table 2 identification of effects in caged freshwater mussels associated with cyanobacterial blooms biomarker difference before and after blooms at cho site difference from reference site lsp difference from urban pollution (boi) difference from agricultural pollution relationship with water parameters hemocyte density yes (+) yes(+) yes(+) yes(+) ph (r=0.5) hemocyte viability yes(+) yes(+) no yes(+) nil phagocytosis activity yes (-) yes (+) no yes (+) turbidity (r=-0.51) chl a (r=0.61) cond (r=-0.59) phagocytosis efficiency yes (+) yes (+) yes(+) yes (+) chl a (r=0.61) cond (r=-0.53) lysozyme yes (+) yes(+) yes(+) yes(+) cyanobacteria (r=0.66) cond (r=-0.63) ph (r=-0.60) ros yes(+) yes(+) yes(+) yes(+) pdissol/total p (r=-0.68) no yes(+) yes(+) yes(+) yes(+) cod (r=-0.58) pdissol/total p (r=-0.57) thiol content yes (-) yes (-) no yes (-) o2 (r=0.50) ph (r=0.61) gst activity no yes (-) no yes (-) cod (r=-0.66) cond (r=0.61) ph (r=0.5) pdissol/total p (r=-0.56) cox activity yes (-) yes (-) no yes (-) ph (r=0.67) the shaded rows indicate biomarkers that responded more strongly to cyanobacterial blooms than to urban and agricultural pollution. 49 discussion in nutrient (n and p)-rich environments, exponential growth (blooms) of cyanobacteria develop annually during summer and fall in lakes, reservoirs and slow-flowing rivers in temperate latitudes. however, it was found that total p levels alone were not always able to predict the presence of algal blooms (shimoda et al., 2016). multiple regression analysis showed that the occurrence of blue-green algae was significantly correlated with acidification of surface waters (ph, r=-0.52; p<0.05) but not with total phosphate and nitrogen levels. conditions in the yamaska river watershed are known to favour cyanobacterial blooms, which usually occur in late summer and fall (mddefp: http://www.mddelcc.gouv.qc.ca/eau/inter.htm). our two upstream sites, cho and boi, were located within the urban and agricultural area and one of them, cho, exhibited cyanobacterial bloom during the fall period, as confirmed by the presence of blue-green algae and microcystin-lr in surface waters and visual inspection (a thick green mat covered the water). given that mussel populations also thrive in the yamaska watershed, which drains both urban and agricultural areas, freshwater mussels are exposed to the cumulative input of urban and agricultural pollution and cyanobacterial blooms. the health consequences of this exposure are currently not well understood. gst activity in hemocytes was significantly depressed in both summer and fall at both the cho (algal blooms) and boi (municipal effluent) sites relative to lsp (the reference site), suggesting that not only cyanobacterial blooms but also other factors were at play in decreasing the activity. the decrease in gst activity could reflect a depletion of gsh stores from oxidative stress and biotransformation in mussels exposed long-term to pollution. this was corroborated by reduced thiol content in hemocytes in the fall (figure 3). interestingly, thiol content levels were significantly higher in the summer, which suggests that thiol stores were not fully depleted at this time. in a study with the freshwater clam diplodon chilensis patagonicus, clams were fed with a toxic strain of m. aeruginosa for 6 weeks, then oxidative stress and biotransformation activity were determined (sabati et al., 2011). antioxidant enzymes (superoxide dismutase and catalase), reduced gsh and gst activity were all significantly upregulated after 6 weeks. the reverse was found with decreased thiol content and gst in mussels caged at the cho site in the fall during cyanobacterial blooms. moreover, the ros increase was much greater than the increase in no production and was weakly correlated (r=0.19; p<0.05) with no, suggesting that oxidative stress involved other mechanisms than oxidative burst following phagocytosis. shrimps exposed for 70 days to cyanobacterial blooms had decreased hemocyte density and increased phagocytosis activity (gao et al., 2017). phagocytosis activity was increased at both the urban (boi) and cyanobacterial bloom (cho) sites, and the increase was stronger in the fall. however, both cho (cyanobacterial blooms) and boi (urban) showed increased phagocytosis activity and efficiency, although phagocytosis efficiency differed significantly between the cho and boi sites during the cyanobacterial bloom in the fall. nevertheless, the increase in phagocytosis activity and hemocyte density in mussels exposed to municipal effluents was reported elsewhere (farcy et al., 2011). interestingly, ly activity was not significantly increased by the effluents, indicating less sensitivity to human-generated sources of bacteria than to cyanobacterial blooms. microcystins from algal blooms could be toxic to clams (pham et al., 2016). exposure to crude extracts of cyanobacteria containing 400 µg/l of microcystin-l in clams for 10 days resulted in increased gst, superoxide dismutase and catalase in the gills and mantle, and inhibition of catalase and gst activities in the foot and other tissues. gst activity was also decreased at cho site, which contained cyanobacteria and microcystins at 3 µg/l but exposure was for much longer times than 10 days. gst activity was also reduced at boi, which receives urban discharges from the city of granby. no signs of cyanobacterial blooms were found there, indicating that other contaminants could also reduce the activity. in another study, freshwater clams (d. chilensis) were fed a toxic strain of m. aeruginosa for 6 weeks (sabatini et al., 2011). although neither protein nor lipid damage were observed, significant increases in gsh (thiol), superoxide dismutase, catalase and gst were observed, suggesting that antioxidant enzymes and cofactors were able to contain oxidative stress. this is in keeping with decreased inflammation (cox activity) in mussels caged at boi and cho. however, total thiol content was decreased at cho and boi indicating that other factors could have contributed to reduced thiol levels in the hemolymph. another possible explanation for the observed decreased in gst activity and the tolerance of bivalves to toxins is compensation mechanisms involving a multi-xenobiotic resistant p-glycoprotein (p-gp) pump (contardo-jara et al., 2008). the activity of the p-gp pump showed a significant increase up to 72 h in clams exposed to 100 µg/l microcystin-lr, indicating enhanced excretion of the toxin. this was accompanied by decreased gst and catalase activities after 72 h exposure times. hemocyte metabolic activity was greater when hemolymph was exposed in vitro to m. aeruginosa than when exposed to lyngbia wollei extracts (gélinas et al., 2014). this is consistent with increased hemocyte metabolic activity and density observed at the cho site, which was undergoing cyanobacterial blooms. however, hemocytes exposed to m. aeruginosa extracts had elevated ros levels and cox activity. a possible explanation is that protein thiol content was depleted by sustained exposure to contaminants including cyanotoxins and oxidative stress in mussels after four months. lower water temperatures in the fall might also have reduced the turnover rate of thiols and proteins in the hemolymph. no production, ly activity and ros levels appeared to be the most responsive and selective endpoints to cyanobacterial blooms when compared to urban and agriculture sites. indeed, ly activity http://www.mddelcc.gouv.qc.ca/eau/inter.htm 50 was maximal at the cyanobacterial bloom (cho) site, differing significantly from the urban (boi) and agriculture (yam) sites (table 2). ly activity was elevated at boi, but significantly less than at cho, compared to the reference lsp site, which makes sense given the occurrence of microorganisms in municipal effluents. increased hemolymph ly activity was also observed in hyriopsis cumingii mussels exposed to m. aeruginosa (hu et al., 2015). moreover, hypoxia was found to dampen the increase in ly activity. although oxygen content at cho (7.8 mg/l) was lower than at boi (9 mg/l), there was no significant correlation between ly activity and o2 levels in surface waters, within the range of o2 measured in the present study (6 mg/l to 13.1 mg/l). in conclusion, exposure to cyanobacterial blooms could contribute to contamination from urban and agricultural pollution in caged freshwater mussels. ros production and ly activity were the most responsive biomarkers for cyanobacterial blooms at the cho site and responded more strongly there than at the boi site, which receives municipal effluents from the city of granby. ros levels were not correlated with phagocytosis activity and were correlated only weakly with no levels, which suggests that there are other sources of ros from the immune response (oxidative burst), perhaps from mitochondria activity and biotransformation. the results support the hypothesis that cyanobacterial blooms exert additional stress on freshwater mussels by contributing to oxidative stress and to enhanced immunocompetence, as with other contaminants. acknowledgments this study was supported by the st. lawrence action plan of environment and climate change canada. the authors thank chantale andré and sophie trépanier for their assistance with mussel collection and cage preparation. references amado ll, monserrat jm. oxidative stress generation by microcystins in aquatic animals: why and how. environ. internat. 36: 226-235, 2010. auffret m. bivalves as models for marine immunotoxicology. in: tryphonas h, fournier m, blakley br, smits jeg., brousseau p. (eds) investigative immunotoxicology. taylor and francis, new york, pp29-48, 2005. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254, 1976. brousseau p, pellerin j, morin y, cyr d, blakey b, boermans h, et al. flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam mya arenaria hemocytes following in vitro exposure to heavy metals. toxicol. lett. 142: 145-165, 2000. burmester v, nimptsch j, wiegand c. adaptation of freshwater mussels to cyanobacterial toxins: response of the biotransformation and antioxidant enzymes. ecotoxicol. environ. saf. 78: 296-309, 2012. cheng tc. hemocytes: forms and functions. the eastern oyster crassostrea virginica. maryland sea grant book, college park, pp. 299-333, 1996. collé j, davison ir. in vivo measurement of active oxygen production in the brown alga fucus evanescens using 2′,7′-dichlorohydrofluorescein diacetate. j. phycology 33: 643-648, 1997. contardo-jara v, pflugmacher s, wiegand c. multixenobiotic-resistance a possible explanation for the insensitivity of bivalves towards cyanobacterial toxins. toxicon 52: 936-943, 2008. das bk, pradhan j, sahu s, marhual np, mishram bk, eknath aa. microcystis aeruginosa (kütz) incorporated diets increase immunity and survival of indian major carp labeo rohita (ham.) against aeromonas hydrophila infection. aquaculture res. 44: 918-927, 2013. farcy e, gagné f, martel l, fortier m, trépanier s, brousseau p, et al. short-term physiological effects of a xenobiotic mixture on the freshwater mussel elliptio complanata exposed to municipal effluents. environ res. 111: 10961106, 2011. fernandes s, welker m, vasconcelos vm. changes in the gst activity of the mussel mytilus galloprovincialis during exposure and depuration of microcystins. j. exp. zool. a ecol. genet. physiol. 311: 226-230, 2009. gagné f, bérubé e, fournier m, blaise c. inflammatory properties of municipal effluents to elliptio complanata mussels: lack of effects from anti-inflammatory drugs. comp biochem physiol c toxicol pharmacol. 141: 332-337, 2005. gagné f. biomarkers of infection and diseases. chapter 11 in: biochemical ecotoxicology: principle and methods, 1st edition. new york: elsevier inc. pp197-207, 2014. gao j, zuo h, yang l, he jh, niu s, weng s, et al. long-term influence of cyanobacterial bloom on the immune system of litopenaeus vannamei. fish shellfish immunol. 61: 79-85, 2017. gélinas m, fortier m, lajeunesse a, fournier m, gagnon c, gagné f. energy status and immune system alterations in elliptio complanata after ingestion of cyanobacteria anabaena flos-aquae. ecotoxicology 22:457468, 2013. gélinas m, fortier m, lajeunesse a, fournier m, gagnon c, barnabé s, et al. responses of freshwater mussel (elliptio complanata) hemocytes exposed in vitro to crude extracts of microcystis aeruginosa and lyngbya wollei. ecotoxicology 23: 260-266, 2014. hoiczyk e., hansel a. 2000. cyanobacterial cell walls: news from an unusual prokaryotic envelope. j bacteriol. 182: 1191-1199, 2000. hu m, wu f, yuan m, li q, gu y, wang y, et al. antioxidant responses of triangle sail mussel hyriopsis cumingii exposed to harmful algae microcystis aeruginosa and hypoxia. chemosphere. 139: 541-549, 2015. jochimsen em, carmichael ww, an js, cardo dm, cookson st, holmes ce, et al. liver failure and death after exposure to microcystins at a https://www.ncbi.nlm.nih.gov/pubmed/?term=burmester%20v%5bauthor%5d&cauthor=true&cauthor_uid=22172521 https://www.ncbi.nlm.nih.gov/pubmed/?term=nimptsch%20j%5bauthor%5d&cauthor=true&cauthor_uid=22172521 https://www.ncbi.nlm.nih.gov/pubmed/?term=wiegand%20c%5bauthor%5d&cauthor=true&cauthor_uid=22172521 https://www.ncbi.nlm.nih.gov/pubmed/22172521 https://www.ncbi.nlm.nih.gov/pubmed/?term=contardo-jara%20v%5bauthor%5d&cauthor=true&cauthor_uid=18930753 https://www.ncbi.nlm.nih.gov/pubmed/?term=pflugmacher%20s%5bauthor%5d&cauthor=true&cauthor_uid=18930753 https://www.ncbi.nlm.nih.gov/pubmed/?term=wiegand%20c%5bauthor%5d&cauthor=true&cauthor_uid=18930753 https://www.ncbi.nlm.nih.gov/pubmed/?term=farcy%20e%5bauthor%5d&cauthor=true&cauthor_uid=21831370 https://www.ncbi.nlm.nih.gov/pubmed/?term=gagn%c3%a9%20f%5bauthor%5d&cauthor=true&cauthor_uid=21831370 https://www.ncbi.nlm.nih.gov/pubmed/?term=martel%20l%5bauthor%5d&cauthor=true&cauthor_uid=21831370 https://www.ncbi.nlm.nih.gov/pubmed/?term=fortier%20m%5bauthor%5d&cauthor=true&cauthor_uid=21831370 https://www.ncbi.nlm.nih.gov/pubmed/?term=tr%c3%a9panier%20s%5bauthor%5d&cauthor=true&cauthor_uid=21831370 https://www.ncbi.nlm.nih.gov/pubmed/?term=brousseau%20p%5bauthor%5d&cauthor=true&cauthor_uid=21831370 https://www.ncbi.nlm.nih.gov/pubmed/21831370 https://www.ncbi.nlm.nih.gov/pubmed/?term=fernandes%20s%5bauthor%5d&cauthor=true&cauthor_uid=19189307 https://www.ncbi.nlm.nih.gov/pubmed/?term=welker%20m%5bauthor%5d&cauthor=true&cauthor_uid=19189307 https://www.ncbi.nlm.nih.gov/pubmed/?term=vasconcelos%20vm%5bauthor%5d&cauthor=true&cauthor_uid=19189307 https://www.ncbi.nlm.nih.gov/pubmed/19189307 https://www.ncbi.nlm.nih.gov/pubmed/19189307 https://www.ncbi.nlm.nih.gov/pubmed/16126009 https://www.ncbi.nlm.nih.gov/pubmed/16126009 https://www.ncbi.nlm.nih.gov/pubmed/16126009 https://www.ncbi.nlm.nih.gov/pubmed/?term=gao%20j%5bauthor%5d&cauthor=true&cauthor_uid=27986602 https://www.ncbi.nlm.nih.gov/pubmed/?term=zuo%20h%5bauthor%5d&cauthor=true&cauthor_uid=27986602 https://www.ncbi.nlm.nih.gov/pubmed/?term=yang%20l%5bauthor%5d&cauthor=true&cauthor_uid=27986602 https://www.ncbi.nlm.nih.gov/pubmed/?term=he%20jh%5bauthor%5d&cauthor=true&cauthor_uid=27986602 https://www.ncbi.nlm.nih.gov/pubmed/?term=niu%20s%5bauthor%5d&cauthor=true&cauthor_uid=27986602 https://www.ncbi.nlm.nih.gov/pubmed/?term=weng%20s%5bauthor%5d&cauthor=true&cauthor_uid=27986602 https://www.ncbi.nlm.nih.gov/pubmed/27986602 https://www.ncbi.nlm.nih.gov/pubmed/?term=g%c3%a9linas%20m%5bauthor%5d&cauthor=true&cauthor_uid=24395509 https://www.ncbi.nlm.nih.gov/pubmed/?term=fortier%20m%5bauthor%5d&cauthor=true&cauthor_uid=24395509 https://www.ncbi.nlm.nih.gov/pubmed/?term=lajeunesse%20a%5bauthor%5d&cauthor=true&cauthor_uid=24395509 https://www.ncbi.nlm.nih.gov/pubmed/?term=fournier%20m%5bauthor%5d&cauthor=true&cauthor_uid=24395509 https://www.ncbi.nlm.nih.gov/pubmed/?term=gagnon%20c%5bauthor%5d&cauthor=true&cauthor_uid=24395509 https://www.ncbi.nlm.nih.gov/pubmed/?term=barnab%c3%a9%20s%5bauthor%5d&cauthor=true&cauthor_uid=24395509 https://www.ncbi.nlm.nih.gov/pubmed/?term=gagn%c3%a9%20f%5bauthor%5d&cauthor=true&cauthor_uid=24395509 https://www.ncbi.nlm.nih.gov/pubmed/?term=hoiczyk%20e%5bauthor%5d&cauthor=true&cauthor_uid=10671437 https://www.ncbi.nlm.nih.gov/pubmed/?term=hu%20m%5bauthor%5d&cauthor=true&cauthor_uid=26318116 https://www.ncbi.nlm.nih.gov/pubmed/?term=wu%20f%5bauthor%5d&cauthor=true&cauthor_uid=26318116 https://www.ncbi.nlm.nih.gov/pubmed/?term=yuan%20m%5bauthor%5d&cauthor=true&cauthor_uid=26318116 https://www.ncbi.nlm.nih.gov/pubmed/?term=li%20q%5bauthor%5d&cauthor=true&cauthor_uid=26318116 https://www.ncbi.nlm.nih.gov/pubmed/?term=gu%20y%5bauthor%5d&cauthor=true&cauthor_uid=26318116 https://www.ncbi.nlm.nih.gov/pubmed/?term=wang%20y%5bauthor%5d&cauthor=true&cauthor_uid=26318116 https://www.ncbi.nlm.nih.gov/pubmed/?term=liu%20q%5bauthor%5d&cauthor=true&cauthor_uid=26318116 https://www.ncbi.nlm.nih.gov/pubmed/26318116 https://www.ncbi.nlm.nih.gov/pubmed/?term=jochimsen%20em%5bauthor%5d&cauthor=true&cauthor_uid=9516222 https://www.ncbi.nlm.nih.gov/pubmed/?term=carmichael%20ww%5bauthor%5d&cauthor=true&cauthor_uid=9516222 https://www.ncbi.nlm.nih.gov/pubmed/?term=an%20js%5bauthor%5d&cauthor=true&cauthor_uid=9516222 https://www.ncbi.nlm.nih.gov/pubmed/?term=cardo%20dm%5bauthor%5d&cauthor=true&cauthor_uid=9516222 https://www.ncbi.nlm.nih.gov/pubmed/?term=cookson%20st%5bauthor%5d&cauthor=true&cauthor_uid=9516222 https://www.ncbi.nlm.nih.gov/pubmed/?term=holmes%20ce%5bauthor%5d&cauthor=true&cauthor_uid=9516222 51 hemodialysis center in brazil. n. engl. j. med. 338: 873-878, 1998. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. pham tl, shimizu k, kanazawa a, gao y, dao ts, utsumi m. microcystin accumulation and biochemical responses in the edible clam corbiculaleana p. exposed to cyanobacterial crude extract. j. environ. sci. 44: 120-130, 2016. prieto ai, jos a, pichardo s, moreno i, cameán a.m. differential oxidative stress responses to microcystins lr and rr in intraperitoneally exposed tilapia fish (oreochromis sp.). aquat toxicol. 77: 314-321, 2006. queiroga fr, marques-santos lf, hégaret h, sassi r, farias nd, santana ln, et al. effects of cyanobacteria synechocystis spp. in the hostparasite model crassostrea gasar-perkinsus marinus. aquat. toxicol. 187: 100-107, 2017. rice ew, baird rb, eaton ad, clesceri ls. standard methods for the examination of water and wastewater, 22nd edition. american public health association, american water works association, water environment federation, 2012. sabatini se, brena bm, luquet cm, san julián m, pirez m, carmen ríos de molina md. microcystin accumulation and antioxidant responses in the freshwater clam diplodon chilensis patagonicus upon subchronic exposure to toxic microcystis aeruginosa. ecotoxicol. environ. saf. 74: 1188-94, 2011. shimoda y, watson sb, palmer me, koops ma, mugalingam s, morley a, et al. delineation of the role of nutrient variability and dreissenids (mollusca, bivalvia) on phytoplankton dynamics in the bay of quinte, ontario, canada. harmful algae 55: 121-136, 2016. verdon cp, burton ba, prior rl. sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by nadp+ when the griess reaction is used to assay for nitrite. anal biochem 224: 502-508, 1995. wiegand c, pflugmacher s. ecotoxicological effects of selected cyanobacterial secondary metabolites: a short review. toxicol appl pharmacol. 203: 201-218, 2005. https://www.ncbi.nlm.nih.gov/pubmed/9516222 https://www.ncbi.nlm.nih.gov/pubmed/14979662 https://www.ncbi.nlm.nih.gov/pubmed/14979662 https://www.ncbi.nlm.nih.gov/pubmed/14979662 https://www.ncbi.nlm.nih.gov/pubmed/?term=pham%20tl%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/pubmed/?term=shimizu%20k%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/pubmed/?term=kanazawa%20a%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/pubmed/?term=gao%20y%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/pubmed/?term=dao%20ts%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/pubmed/?term=utsumi%20m%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/m/pubmed/?term=prieto%20ai%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=jos%20a%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=pichardo%20s%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=moreno%20i%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=came%c3%a1n%20am%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=came%c3%a1n%20am%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/pubmed/?term=queiroga%20fr%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=marques-santos%20lf%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=h%c3%a9garet%20h%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=sassi%20r%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=sassi%20r%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=farias%20nd%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=santana%20ln%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=da%20silva%20pm%5bauthor%5d&cauthor=true&cauthor_uid=28407513 https://www.ncbi.nlm.nih.gov/pubmed/?term=hegaret+cyanobacteria https://www.ncbi.nlm.nih.gov/pubmed/?term=sabatini%20se%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=brena%20bm%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=luquet%20cm%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=san%20juli%c3%a1n%20m%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=pirez%20m%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=carmen%20r%c3%ados%20de%20molina%20md%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=shimoda%20y%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=watson%20sb%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=palmer%20me%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=koops%20ma%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=mugalingam%20s%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=morley%20a%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/28073525 https://www.ncbi.nlm.nih.gov/pubmed/28073525 http://www.sciencedirect.com/science/article/pii/s0003269785710792#! http://www.sciencedirect.com/science/article/pii/s0003269785710792#! http://www.sciencedirect.com/science/article/pii/s0003269785710792#! http://www.sciencedirect.com/science/journal/00032697 http://www.sciencedirect.com/science/journal/00032697/224/2 https://www.ncbi.nlm.nih.gov/pubmed/15737675 https://www.ncbi.nlm.nih.gov/pubmed/15737675 https://www.ncbi.nlm.nih.gov/pubmed/15737675 transplant rejection in m0lluscs isj 8: 15-20, 2011 issn 1824-307x minireview transplant rejection in terrestrial molluscs e furuta, k yamaguchi the research institute for comparative immunology, 1250-9-401, hasunuma, minuma-ku, saitama 337-0015, japan accepted december 20, 2010 abstract to know whether or not molluscs are capable of recognizing tissue allo-antigens, dorsal skin-allografts were exchanged between adult terrestrial slug, incilaria fruhstorferi. we succeeded for the first time in orthotopic transplantation of allografts and observed chronic rejection of allografts. cellular changes in the rejection process continued over for 40 days. two functional types of “effector” cells were recognized at the rejection site, but they were observed to be macrophages possessing perforin granules and phagocytosing damaged cells of the allograft. three days after transplantation, the perforin-positive cells were recognized only in the recipient tissue surrounding the allograft. five days after transplantation, these cells started to appear in the graft, while they disappeared from the host tissue. however, tunel-positive cells (apoptotic cells) were not observed throughout the graft-rejection process. electron microscopic examination of the graft tissue revealed autophagic degeneration of epithelial cells, mucous cells, pigment cells, fibroblasts, and muscle cells. these observations suggest that the slugs have the capability to recognize differences in cell-surface molecules between the allogeneic and recipient tissue, and that an allograft is chronically rejected due to a type of immunocyte (macrophage) that can induce perforin-dependent cell death. key words: allogeneic rejection; molluscs; orthotopic transplantation; autophagic cell death; perforin indroduction all metazoan animals need an internal defense system to protect themselves against foreign materials that succeed in getting past the external defense. it is well known that the internal defense mechanism in vertebrate consists of non-specific humoral and cellular factors, and specific cellular and humoral ones. vertebrates are able to respond against invading microorganisms first by non-specific element, such as lysozyme, complements, interferon, lysine, transferin and then by typical immune reactions of a clonal nature. moreover, macrophages are involved in part or all of those sequential defense processes both as phagocytes and antigen-presenting cells in some way and t and b-lymphocytes are engaged in only the later stage of specific defense reactions. however, invertebrates including the molluscs do not possess classical immune recognition molecules of vertebrates such as immunoglobulins, t-cells, mhc ___________________________________________________________________________ corresponding author: emiko furuta the research institute for comparative immunology 1250-9-401, hasunuma, minuma-ku saitama 337-0015, japan e-mail: furutaemk@nifty.com or antigen receptors. but they still manage to normally keep their internal body fluids sterile. they possess the capacity to distinguish not only between self and non-self, but also among non-self materials which differ in chemical properties (bayne, 1990; cooper et al., 1992). phagocytosis is considered to be the primary clearance mechanism in molluscs. incilaria fruhstorferi, the largest slug native of japan, possesses only macrophage, a kind of hemocyte, being able to recognize and phagocytose biotic and abiotic non-self materials (furuta et al., 1987; yamaguchi et al., 1988; furuta et al., 1990). the macrophage performs intracellular digestion of non-self materials. the destruction of tissue allografts in mammals is thought to be mediated primarily by cellular immune mechanisms, an interpretation supported by histological observations of the dense infiltration of recipient cells into the organs to be rejected. detailed and precise studies of the nature and the function of such cells are essential in order to clarify the mechanisms of transplant rejection. in mammals, the principal target of the immune response to allografts is the major histocompatibility complex (mhc, h-2 in mice). however, mhc molecules have not previously 15 fig. 1 procedure of allotransplantation of dorsal skin graft in the slug, incilaria fruhstorferi. a small piece of dorsal skin (2.5x4.5 mm in size and 0.1-0.2 mm in depth) was peeled off and placed on the recipient graft bed of other individual. been reported in invertebrates, and invertebrates possess neither antigen-specific lymphocytes nor any form of secreted immunoglobulins. yet, despite the lack of a highly developed specific immune response, those animals apparently have thrived for hundreds million years in a world full of pathogens. invertebrates possess several types of hemocytes and at least one of these functions as a phagocyte. phagocytes are known to play a crucial role in the internal defense system in invertebrates. in addition, a body of evidences suggests that invertebrate animals, especially molluscs, may possess the capability to recognize and reject allogeneic transplants. this is controversial. when digestive glands of helisoma duryinormale, the head/foot and digestive gland tissues, and the heart of biomphalaria glabrata were implanted into cephalopedal sinus of the other individuals, these heterotopic allografts had been encapsulated by hemocytes (cheng and galloway, 1970; jourdane and cheng, 1987; sullivan et al., 1992). on the other hand, when hemocyte producing organ was implanted into the hemocel, this kind of allograft not only survived without being encapsulated but hemopoietic activity was also still retained (sullivan, 1990). in lymnaea stagnalis, when vasa deferentia was implanted into the cephalopedal blood sinus, hemocytes had aggregated transiently at the cut surface at 24 h after implantation, and thereafter small groups of hemocytes were contact with the graft (sminia et al., 1974). sullivan et al. (1998) had found no conclusive evidence for chronic allograft rejection in b. glabrata regardless of tissue that was transplanted. the above results show that some grafts can often survive in a recipient for many months, whereas, others undergo varying degrees of hemocytic encapsulation and degenerative changes. these contradictory results may be due to the site, i.e., the heterotopic position. only two reports have been described so far on orthotopic transplantation in molluscs. according to röegener and renwrantz (1984), all small pieces of skin of the head-foot (autografts) of helix pomatia were destroyed within 6-9 days. in this case, the skin grafts may have been deficient in size for survival. however, alloand congeneric xeno-grafts of cerebral ganglia were tolerated in the mesocerebrum which were removed from helix aspersa (gomot and gomot, 1996). since results from mammals seem to indicate the presence of a barrier, considering the brain as privileged site for transplantation is still under discussion and this may apply in even to molluscs. in order to unravel these confused results mentioned above, orthotopic transplantation would be required. obstacles in performing orthotopic transplantation seem to lie in the technical difficulty of holding the donor tissue at the appropriate site in the recipient. we overcome this difficulty by a method recorded elsewhere (yamaguchi et al., 1999). we attempted to elucidate in our examination cytological change in both grafted and recipient tissue in response to orthotopic transplants of allogeneic skin using the terrestrial slug, i. fruhstorferi. 16 m acrophage n um bers /field 0 20 40 60 80 100 120 4 8 20 weeks after transplantation (a) autotransplantation 0 20 40 60 80 100 120 4 (b) allotransplantation 140 8 20 * * ; graft ; graft bed fig. 2 changes of macrophage numbers in autoand allografts, and their beds after transplantation. the number of macrophages phagocytosed cell debris in grafts and their beds were counted out at 10 visual fields randomly selected for each sample. in autotransplantation, the numbers at 20 weeks after transplantation significantly decreased in grafts and their beds comparing with them at 4 or 8 weeks. mean ± sem (n = 10). *p < 0.001 (student’s t-test) (yamaguchi et al., 1999). histology of the epithelial cells of the dorsal surface skin external surface of the dorsal skin epidermis is composed of a single layer of microvillous columnar cells which hold in place the covering of mucus and underneath epidermal cell layer, numerous pigment cells with many dendritic processes exist. the epidermis is supported by a mat of connective tissue through which run muscle fibers. five main cell types are distinguished in the epidermis: (1) microvillous cells, (2) round mucous cells (3) tubular mucous cells (4) channel cells and (5) ciliated cells. the epidermal cells closely cling to one another by zonula adherens in the apical region of cells. the presence of unicellular mucous glands is well established in the slug and the cells typically possess cell bodies located in the subepidermal connective tissue and secretary processes extending to the surface of the epidermis. macrophages are hardly observed in subepidermal connective tissue. furuta and shimozawa (1983), in an in vitro experiment, found that fibroblast of i. fruhstorferi differentiated into macrophage after injection of foreign materials, and the main macrophage producing site is in the cells that line the hemocel wall, which are derived from fibroblasts (furuta et al., 1994). tissue transplantation the terrestrial slug was selected as the donor and recipient for implantation, because it is easy to use, being not aquatic and possessing no shell. these features are favorable because the graft is not readily peeled off from the recipient body mechanically, as by a water stream or contact with a shell. transplants were made orthotopically: a piece of the dorsal skin (2.5x4.5 mm in size, 0.1~0.2 mm in depth) from a donor animal was placed on the cut surface at the corresponding site of the dorsal skin in the recipient (fig. 1). epidermis of the dorsal skin is arranged as simple columnar epithelial cells. although the body surface of the terrestrial slug is covered with only simple epithelium, the surface is prevented from evaporation and is protected from mechanical 17 fig. 3 perforin immunohistochemistry of an allografted recipient site of dorsal skin, 1 day (a) and 3 days (b) after transplantation. perforin-positive cells (arrowheads) are evident in the recipient connective tissue (ct) surrounding the graft 3 days after transplantation, but not 1day after transplantation as well as controlled immunohistochemistry. ep, epithelial cell; mu, mucous cell; pi, pigment cell. bar = 10 µm (furuta et al., 2006). injuries. the reason is that the surface skin of the slug is covered a large amount of mucus which is secreted by two mucous cell types whose necks reach the apical portion of the dorsal surface skin; these cells were present among the epithelial cells (yamaguchi et al.,1999). as the mucus rendered the physical attachment of the graft to the host graft bed difficult, secretion was inhibited by anesthetizing slugs on ice. on other hand, bacterial infection of the graft appears to be inhibited after transplantation by lectins in the mucus (furuta et al., 1995; yuasa et al., 1998). fate of autografts in transplant experiment of slugs, mucous cells, pigment cells and muscle fibers were provided as convenient criteria of graft viability. two weeks after transplantation, grafts had connected to host beds and various macrophages congregated around, particularly, underneath the graft sites and infiltrated into the graft matrix. numerous macrophages had already phagocytosed cells damaged by mechanical trauma. as a result of it, pigment cells, mucous cells and muscle fibers decreased in number in grafts and their beds. this phenomenon is seemed to heal wounds. until eight weeks after transplantation many macrophages observed in the graft site, whereas at four weeks after transplantation, grafted tissues such as muscle fibers and mucous cells began to regenerate slowly and the regeneration of these cells had been over twenty weeks after transplantation. at twenty weeks after transplantation, the macrophage numbers in autografts and their beds decreased significantly comparing with the numbers at four and eight weeks (p < 0.001, student’s t-test). by contrast, numerous macrophages were still present in allografts and their beds at eight and twenty weeks and revealed active phagocytosis (fig. 2). thus, the dorsal skin of host slug was completely repaired in autograft transplantation. fate of allografts the rejection of allografts in mammals is mainly mediated by cytotoxic t-lymphocytes, whereas no comparable immune cells have been described in invertebrates. the examination was undertaken to determine whether similar cytotoxic effector cells are present when allograft rejection occurs in the terrestrial slug i. fruhstorferi (yamaguchi et al., 1999; furuta et al., 2006). immunohistochemistry for perforin, detection of apoptosis by the tunel (tdt-mediated dutp-biotin nick-end labeling) method and electron microscopy were performed using both donor and recipient tissues. one day after transplantation, the grafted skin appeared normal, and both perforin-positive and tunel-positive cells were not recognized in the graft tissues (fig. 3a). three days after transplantation, perforin-positive cells (macrophages) were evident in the recipient tissue surrounding the graft (fig. 3b), but not present in the grafted tissue. five days after transplantation, perforin-positive cells were observed in the graft, but no these cells were seen any longer in the recipient tissue surrounding graft. beneath the epithelial layer, the cells composing the connective tissue of the graft (fibroblasts, muscle cells, pigment cells, nerve cells, etc.) began to appear shrunken, and chromatin-condensed nuclei (ccn, pro-ccn) were scattered throughout the tissue. autophagic 18 fig. 4 electron micrographs of the tissue of a dorsal skin allograft, 5 days after transplantation. columnar epithelial cells (ep) of the graft have become simple-squarmous type. autophagic or early degenerating cells are seen in the connective tissue of the graft. a) ep decreases in height and becomes simple-squarmous type. the cells composing the connective tissue of the graft begin to appear shrunken and contain chromatin-condensed nuclei (pro-ccn; p-ccn), and macrophages containing perforin-like granules (arrowheads) have infiltrated into the graft. bar = 5 µm. b) a mucous cell containing granules (mg) irregular in shape becomes autophagic and is surrounded by a macrophage (mp) containing several perforin-like granules (arrowheads). bar = 2 µm (furuta et al., 2006). vacuoles were observed in the cytoplasm of these cells (fig.4a, b). macrophages containing small perforin-like granules often appeared surrounding the cells with pccn (fig. 4b). the remnants of autophagic cell death were phagocytosed by macrophages that infiltrated into the grafted connective tissue. twelve days after transplantation, the constitutive cells of the connective tissue of the graft disappeared through phagocytosis by infiltrating macrophages. in the graft tissue, cell elements were gradually lost and cell-free space was formed at the site of cell disappearance. autophagic cell death was found to play an important role in tissue destruction. one-hundred forty days after transplantation, the grafted tissues were completely displaced by recipient tissues. from the above data, we concluded that terrestrial slugs (molluscs) have the capability of recognizing and rejecting allogeneic tissue transplants and that they possess perforin-like molecules that lead to a cytotoxic reaction for autophagic cell death in the rejection of allografts. the presence of a putative perforin in molluscs suggests that perforin may be an immune defense mechanism conserved across phylogenetic lineages. the existence of a perforin in terrestrial slugs implies a much earlier evolutionary origin of this molecule than has been previously thought. references bayne cj. phagocytosis and non-self recognition in invertebrates. bioscience, 40: 723-731, 1990. cheng tc, galloway pc. transplantation immunity in mollusks: the histocompatibility of helisoma duryinormale with allografts and xenografts. j. invertebr. pathol. 15: 177-192, 1970. cooper el, rinkevich g, valembois p. invertebrate immunity: another viewpoint. scand. j. immunol. 35: 247-266, 1992. furuta e, shimozawa a. primary culture of cells from the foot and mantle of the slug, incilaria fruhstorferi collinge. zool. mag. 92: 280-296, 1983. furuta e, yamaguchi k, shimozawa a. hemolymph cells and the platelet-like structures of the land slug, incilaria bilineata (gastropoda: pulmonata) anat. anz. 170: 99-109, 1990. furuta e, seo n, yamaguchi k. perforin-dependent cell death in skin allograft rejection of the terrestrial slug, incilaria fruhstorferi. zool. sci. 23: 1093-1100, 2006. 19 furuta e, takagi t, yamaguchi k, shimozawa a. incilaria mucus agglutinated human erythrocytes. j. exp. zool. 271: 340-347, 1995. furuta e, yamaguchi k, shimozawa a. blood cell-producing site in the land slug, incilaria fruhstorferi. acta. anat. nipponica 69: 751-764, 1994. furuta e, yamaguchi k, shimozawa a. phagocytosis by hemolymph cells of the land slug, incilaria fruhstorferi collinge (gastropoda: pulmonata). anat. anz. 163: 82-99, 1987. gomot a, gomot l. allogeneic and xenogeneic grafts in pumonate gastropod mollusks: fates of neural transplants. dev. comp. immunol. 20: 193-205, 1986. jourdane j, cheng tc. the two-phase recognition process allografts in a brazillian strain of biomphalaria glabrata. j. invertebr. pathol. 49: 145-158, 1987. rögener w, renwrantz l. destruction of autografts and wound healing in helix pomatia. zool. jb. physiol. 88: 515-527, 1984. sminia t, borghart-reinders te, van de linde aw. encapsulation of foreign materials experimentally introduced into the freshwater snail limnaea stagnalis. an electron microscopic and autoradiographic study. cell tissue res. 153: 307-326, 1974. sullivan jt, galvan ag, lares rr. comparison of several types of allografts in biomphalaria glabrata (mollusca: pulmonata). j. invertebr. pathol. 71: 1-8, 1998. sullivan jt. long-term survival of heterotopic allografts of amoebocyte producing organ in biomphalaria glabrata (mollusca: pulmonata). trans. am. microsc. soc. 109: 52-60, 1990. sullivan jt, andrew ja, currie rt. heterotopic heart transplantats in biomphalaria glabrata (mollusca: pulmonata): fate of allografts. trans. am. microsc. soc. 111: 1-15, 1992. yamaguchi k, furuta e, nakamura h. chronic skin allograft rejection in terrestrial slugs. zool. sci. 16: 485-495, 1999. yamaguchi k, furuta e, shimozawa a. morphological and functional studies on hemolymph cells of land slug, incilaria bilineata, in vitro and in vivo. in kuroda y, kurstak e, maramorosch k. (eds), invertebrate and fish tissue culture, springer-verlag, berlin, germany, pp 247-250, 1988. yuasa hj, furuta e, nakamura a, takagi t: (1998) cloning and sequencing of three c-type lectins from body surface mucus of the land slug, incilaria fruhstorferi. comp. biochem. physiol. 119b: 479-484, 1998. 20 research report isj 7: 89-106, 2010 issn 1824-307x research report the effect of oxidative stress on phagocytosis and apoptosis in the earthworm eisenia hortensis sl fuller-espie, t nacarelli, el blake, fm bearoff science department, cabrini college, 610 king of prussia road, radnor, pennsylvania 19087-3698, usa accepted february 17, 2010 abstract the effect of exogenous hydrogen peroxide (h202) on phagocytic function and apoptosis in coelomocytes from eisenia hortensis was investigated. treating coelomocytes with h202 (0.26 to 8.4 mm) evoked a significant increase in phagocytosis for one or more of the concentrations of h202 employed in 67 % of cases. using annexin v-fitc we show that h202 induced apoptosis of coelomocytes in vitro. we found that 100 % of viable coelomocyte populations exhibited significant increases in phosphatidylserine translocation for one or more of the concentrations of h202 tested (8.4 to 67.6 mm). using a fluorescent inhibitor of caspases, we revealed the presence of activated caspases observing increased caspase activity in 67 % of viable coelomocyte populations treated with 33.8mm h202, and in 100 % of cases treated with 67.6 mm h202. agarose gel electrophoresis and the tunel assay showed dna fragmentation in samples treated with 16.9 and 33.8 mm h202. in addition, endogenous h202 production during phagocytosis by hyaline amoebocytes was detected using a fluorogenic substrate. thus, free radicals not only appear to facilitate phagocytosis and are produced during phagocytosis, but they also promote an oxidative-stress-induced apoptosis that may play an important function in regulating innate immune responses in e. hortensis. key words: phagocytosis; hydrogen peroxide; annexin v; dna fragmentation; caspase, tunel assay introduction when the production of reactive oxygen species (ros) in microsomes, peroxisomes, mitochondria and the cytosol overwhelms a cell’s ability to either neutralize reactive intermediates or repair toxic effects from ros, a state of oxidative stress is initiated. toxic effects from ros include damage to nucleic acids (mutagenesis) and carbohydrates, lipid peroxidation of cellular membranes, and enzyme inactivation (imlay, 2003). oxidative stress is also linked to the aging process (larsen, 1993; helfand and rogina, 2003; rattan, 2006; csiszar et al., 2007). ros encompass a wide array of oxidants including hydrogen peroxide (h202, the focus of this study), superoxide anions, hydroxyl radicals, peroxyl radicals and organic hydroperoxides (dunford, 1987; coffey et al., 1995; panasenko et al., 2002). these oxidants are produced through various means including radiation, uncomplexed metals such as iron and copper, organic compounds such as quinones, uric acid and ___________________________________________________________________________ corresponding author: sl fuller-espie science department cabrini college 610 king of prussia road, radnor, pa 19087-3698, usa email: sfuller-espie@cabrini.edu homocysteines, certain classes of xenobiotics such as polycyclic aromatic hydrocarbons (pah) going through redox cycling, and thermal stress (diguilioi et al., 1989; livingstone et al., 1990; sundaram et al., 1990; livingstone et al., 1995; abele et al., 2001; tyagi et al., 2005; valko et al., 2006; strazzullo and puig, 2007; fato et al., 2008; pichaud et al., 2008; kell, 2009). they are also generated by intracellular enzymes including nadph oxidase, xanthine oxidase and cytochrome p450 (lewis, 2002; bedard and krause, 2007; jankov et al., 2008). paradoxically, ros not only exert damaging effects in cells, but they also afford protecive effects, for example during immune defense for phagocytosis where ros are toxic to phagocytized pathogens. another benefit of ros is their aility to participate in redox signaling (thannickal and fanburg, 2000; forman and torres, 2002; wang, 2009). because of the detrimental effects of ros on cellular components, it is imperative that organisms possess cellular antioxidant defense mechanisms for the detoxification of ros, often measured as the total oxyradical scavenging capacity (tosc), an important biomarker of oxidative stress (regoli, 2000; gorbi and regoli, 2003; dovzhenko et al., 2005). ros are neutralized by a variety of   89 mailto:sfuller-espie@cabrini.edu antioxidant processes aimed at stabilizing free radicals, terminating free radical reactions, and preventing the transfer of electrons from oxygen to organic molecules. one mechanism relies upon enzymatic detoxification of ros by catalase, glutathione peroxidase, superoxide dismutase, thioredoxin reductase, peroxiredoxins and sulfiredoxin (raes et al., 1994; nordberg and arnér, 2001; flohé et al., 2003; findlay et al., 2005). nonenzymatic antioxidants also provide antioxidant defenses and include a diverse array of molecules including antioxidant quenchers comprising cellular proteins (e.g. transferrin, ferritin, metallothionein, ceruplasmin and others) that chelate pro-oxidant minerals (cairo et al., 1995; kang et al., 2001; yamaji et al., 2004; laukens et al., 2009). in addition, glutathione, selenium, phytochemicals, vitamin e, vitamin c and provitamin a compounds (e.g. beta carotene) also provide protective antioxidant defenses (sies et al., 1992; loo, 2003; brenneisen et al., 2005; ghezzi, 2005). the primary goal of this study was to investigate the in vitro effects of oxidative stress on cellular activities in the immune cells (coelomocytes) of the earthworm eisenia hortensis (also known as the european nightcrawler) which reside in the coelomic cavity. investigations of innate immunity in earthworms have identified three distinct subpopulations of coelomocytes (leukocyte equivalents): hyaline amoebocytes (large coelomocytes), granular amoebocytes (small coelomocytes) and chloragocytes (eleocytes), most likely diverging developmentally from a common progenitor cell (prohemocyte), as suggested by hartenstein (2006). the immune functions of coelomocytes can be studied in vitro after harvesting the coelomic fluid, which is rich in coelomocytes, by extruding the coelomocytes through the dorsal pores of the body wall from experimentally-induced earthworms. the hyaline amoebocytes are the major phagocytic cells, the granular amoebocytes constitute the subpopulation exhibiting nk-like activity, and the eleocytes contain chloragosomes and do not participate in either phagocytic or nk-like activities, but they do secrete lytic substances (cooper, 1996; cossarizza et al., 1996; adamowicz and wojtaszek, 2001; engelmann et al., 2002; engelmann et al., 2005). differences in granularity and size between coelomocytes permits amoebocytes (hyaline and granular) and eleocytes to be distinguished using flow cytometry methodology employing forward light scatter (fsc) and side light scatter (ssc) measurements (cossarizza et al., 1996, 2005; engelmann et al., 2004; patel et al., 2007; fuller-espie et al., 2008). selective analysis of subpopulations is facilitated by specifying regions to identify particular subpopulations, and then gating on assigned regions, permitting the investigator to include only desired subpopulations and exclude irrelevant subpopulations from final analyses. light and fluorescent microscopy have been used by researchers to study immune functions in earthworms (adamowicz and wojtaszek, 2001; kalaç et al., 2002), however, these methods are more subjective than flow cytometry, and they impose restrictions on the number of cells included in analyses owing to time constraints. in contrast, flow cytometry is an objective, quantitative methodology that analyzes thousands of cells per second with the option of restricting analyses to predetermined subpopulations. this investigation focused specifically on phagocytic function and the induction of apoptosis in the amoebocytes of e. hortensis following exposure to exogenous h202. we evaluated the phagocytic uptake of escherichia coli using flow cytometry and found that in vitro exposure to h202 (0.26 8.4 mm) enhanced phagocytosis. using flow cytometric and agarose gel electrophoresis methodologies we also examined the effect of h202 on three events associated with apoptosis: 1) translocation of phosphatidylserine (ps) to the extracellular face of the plasma membrane using annexin-v binding; 2) caspase activation using a fluorescein-conjugated inhibitor of caspase activation (flica); and 3) dna fragmentation. we present data supporting an apoptotic-like cell death in coelomocytes of e. hortensis resulting from in vitro exposure to exogenous h202 (8.4 67.6 mm). finally, using the fluorogenic substrate dhr 123, a probe widely used to measure intracellular h202, we also show that h202 is generated in hyaline amoebocytes during phagocytosis of bacillus megaterium and pseudomonas stutzeri. materials and methods cell culture supplies and chemical reagents tissue culture plasticware was purchased from fisher scientific. phosphate buffered saline (pbs) was purchased from invitrogen. dulbecco’s modified eagle medium (dmem, invitrogen) was supplemented with either 10 % heat-inactivated fetal calf serum (invitrogen) or serum supreme (lonza biowhittaker), plus 100 μg ml-1 ampicillin (shelton scientific), 10 μg ml-1 kanamycin (shelton scientific), 10 μg ml-1 tetracycline, 5 μg ml-1 chloramphenicol (fluka biochemika), 1× penicillin, streptomycin and amphotericin b, 1× nonessential amino acids (invitrogen) and 1× l-glutamine (invitrogen) to comprise super dmem (sdmem). sdmem supplemented with serum supreme was used for all exogenous h202 assays while sdmem supplemented with fetal calf serum was used for endogenous h202 assays. earthworm husbandry eisenia hortensis (european nightcrawlers) was purchased from vermitechnology unlimited, orange lake, florida, usa, who imports e. hortensis from star food, holland, scherpenzeelseweg 95, 3772me barneveld, the netherlands. species identity was determined by the united states department of agriculture, usda permit #52262 (vermitechnology, personal communication). shortterm colonies were maintained at rt in the dark on moistened autoclaved pine woodchips sprinkled with single grain rice cereal or rice with bananas cereal (gerber) and covered with autoclaved, shredded and moistened paper towels. habitats were changed twice weekly. animals were euthanized by freezing at -20 oc.   90 extrusion of coelomocytes prior to experimentation, earthworms were first washed with distilled water on paper towels using a water bottle to remove wood chip fragments or food particles. they were then placed overnight on paper towels moistened with 2.5 μg ml-1 fungizone (fisher scientific) in 100 mm petri dishes to minimize fecal contamination during the extrusion process, and remove further any surface contaminants. to collect coelomocytes, earthworms were placed in either 100 mm petri dishes or in multichannel pipette reservoirs containing 3 ml bd facsflow sheath fluid (bd biosciences). the earthworms extruded their coelomocytes through their dorsal pores in response to this external stimulus without the need to use the alcohol extrusion method reported by others (engelmann et al., 2005). the coelomocytes were then transferred to 0.5 ml accumax (innovative cell technology) in 15 ml conical test tubes for a 5 min incubation period at rt to reduce aggregation of cells. finally, 5 ml pbs was added and the samples were centrifuged immediately at 150 x g, 5 min at 4 oc. after decanting the supernatant, the coelomocyte pellet was gently mixed by flicking the bottom of the centrifuge tube, and coelomocytes were resuspended in 0.5 ml sdmem. enumeration was carried out using a hemacytometer. only hyaline amoebocytes (large coelomocytes) and hyaline granulocytes (small coelomocytes) were included in the cell count; eleocytes were not counted but did factor into a quality score. samples with large numbers of eleocytes compared to large and small coelomocytes were not used in phagocytosis assays. samples were adjusted to 3.8 x 105 or 5 x 105 (phagocytosis and annexin v assays, see below) or 1 x 106 (caspase assays) coelomocytes ml-1 in sdmem. bacteria for phagocytosis assays e. coli/gfp: escherichia coli hb101 transformed with pglo (biorad) and expressing green fluorescent protein (gfp) were grown on tryptic soy agar containing 100 μg ml-1 ampicillin and 0.2 % (w/v) arabinose at 32 °c for 24 h. after washing the cells once in pbs, they were fixed chemically with 4 % (v/v) paraformaldehyde in pbs, 1 h at rt with periodic mixing, followed by three pbs washes. centrifugation was carried out at 3273xg for 5 min at 4 oc. the final cell pellet was resuspended in pbs, bacteria were enumerated using a hemacytometer, and then stored in the dark at 4 °c. bacillus megaterium and pseudomonas stutzeri (presque isle cultures) were grown overnight in tryptic soy broth at 37 oc in a shaking incubator. absorbance was measured using a spectrophotometer (600 nm) and compared to a standard curve to determine concentration. standard curves were generated by correlating absorbance with cell count using a hemacytometer. all bacteria were diluted in sdmem to obtain the desired multiplicity of infection (m.o.i.). phagocytosis: exogenous h202 pretreatment phagocytosis assays were carried out in sdmem. coelomocytes (50,000 per well) were pretreated with or without h202 (0 8.4 mm final concentration) 5 % co2, at 25 oc in 96-well, roundbottom plates in 200 μl sdmem. duplicate samples were used in every assay. following h202 pretreatment, cells were centrifuged (150xg) and washed once with pbs. finally, 200 μl e. coli/gfp was added to each well at a multiplicity of infection of 1000 bacteria:1 coelomocyte and incubated for 3 h at 30 °c. to control for non-specific binding of e. coli/gfp to the external surface of coelomocytes, 50 μm cytochalasin b (sigma aldrich) [an antibiotic that interferes with microfilament activity and thereby inhibits phagocytosis (axline and reaven, 1974)] was added to control wells 45 min before the addition of e.coli/gfp. following e. coli/gfp uptake, trypan blue (biowhittaker) was used at a final concentration of 0.02 % (w/v) for 30 min at rt in the dark, for quenching purposes to reduce background fluorescence (mosiman et al., 1997). the cells were transferred to flow cytometry tubes containing 100 μl facs flow buffer (bd biosciences), placed on ice in the dark, and run immediately on the flow cytometer. annexin v-fitc/pi assay recombinant human annexin v-fitc (invitrogen, annexinv01)) and propidium iodide (pi) (invitrogen, p3566) were used to detect ps translocation and to enable exclusion of dead cells from analyses. using a 96-well, round-bottom plate, 5 x 104 (assay 1) or 3.8 x 104 (assay 2) coelomocytes in 50 μl sdmem were added to appropriate experimental (h202-treated) and control (double negative autofluorescent background; single positive fitc; single positive pi; double positive annexin v/pi background) wells in triplicate. experimental wells received 50 μl of h202 (final concentrations of 67.6, 33.8, 16.9, and 8.45 mm). single positive fitc controls received 50 μl of h202 (270mm final). single positive pi controls received 50 μl saponin (0.01 % final). the plate was incubated 6 h, 25 oc, 5 % co2 before adding 100 μl pbs and centrifuging (5 min, 4 oc, 150xg). after removing the supernatant fraction, the wells were washed with 200 μl well-1 of pbs, and centrifuged again. the supernatant fraction was removed and the cells were resuspended in 200 μl sdmem containing 1× binding buffer (0.01m hepes, 0.14 mm nacl, 2.5mm cacl2) with or without annexin vfitc (3.75 μl well-1) and/or pi (0.5 μl well-1). the autofluorescent background control did not receive annexin v-fitc or pi. the single positive pi control did not receive annexin v-fitc. the single positive fitc control did not receive pi. all other samples received both annexin v-fitc and pi. samples were incubated for 5 min at rt and transferred to flow cytometry tubes containing 150 μl of facs flow sheath buffer containing 1× binding buffer. samples were kept on ice protected from light and analyzed immediately by flow cytometry. caspase assay caspase activation was measured using a vybrant® fam caspases assay kit (fitc) (invitrogen/molecular probes) according to the manufacturer’s instructions. this assay utilized a fluorescent inhibitor of caspases known as flica™   91 which detects activation of caspase enzymes in cells undergoing apoptosis. using a 96-well, vbottom plate, 1 x 105 coelomocytes in 0.1 ml sdmem were added to appropriate experimental (h202-treated) and control [untreated (0 mm) double negative autofluorescent background; single positive fitc; single positive pi; double positive caspase background] wells in duplicate. experimental wells received 50 μl h202 (final concentrations of 33.8 mm for ew f1-f3; 16.9 mm, 33.8 mm and 67.9 mm for ew f4-f6); single positive pi control wells received 50 μl 0.03 % saponin in sdmem; and single positive fitc control wells received 50 μl sdmem. all control and experimental samples were incubated for 6 h, 25 °c, 5 % co2. after centrifugation at 150xg (5 min, 4 oc), the supernatant fraction was removed and the wells were washed with 200 μl pbs. again the plate was centrifuged and the supernatant fraction was removed. untreated double-negative, autofluorescent control samples (fitc negative, pi negative) and single positive pi controls were resuspended in 100 μl of sdmem. untreated samples (caspase background) and h202treated samples were resuspended in 90 μl sdmem plus 10 μl of 10x flica reagent. single positive fitc control wells were resuspended in 50 μl sdmem, 10 μl of 10x flica reagent, and 40 μl of 10 % formaldehyde. the plate was incubated in the dark, 1 h, 25 °c, 5 % co2, with gentle mixing every 20 min before adding 100 μl of 1× washing buffer and centrifuging as above. after removing the supernatant fraction, the cells were washed twice with 200 μl well-1 of 1× wash buffer. following the last centrifugation and removal of the supernatant fraction, 200 μl well-1 of 1× wash buffer with or without pi was added to each well; double-negative and single positive fitc controls did not receive pi, all other samples received pi. samples were placed on ice protected from light and analyzed immediately by flow cytometry. cell volume measurements ew f4-f6 used in the caspase assay were also subjected to cell volume analysis using flow cytometry. forward scatter measurements of pi negative large coelomocytes treated in duplicate with 0, 16.9, 33.8 and 67.6 mm h202 were averaged and analyzed by student’s t test to determine if differences observed in forward light scatter measurements were statistically significant compared to controls. flow cytometry fluorescence was measured using fl-1 (fitc, gfp and rhodamine 123) and fl-2 (pi) detectors of a facscalibur flow cytometer (bd biosciences). autofluorescent controls were used to set voltages for forward scatter (fsc), side scatter (ssc), fl-1 and fl-2 during instrument set-up. single positive fitc and pi controls were used to adjust compensation settings (spectral overlap removal) for annexin v and caspase assays. listmode data was acquired and analyzed using cell quest (bd biosciences) and winlist5.0 (verity software house) software. only coelomocytes corresponding to the large coelomocyte population (phagocytosis assays) or large and small coelomocyte populations combined (annexin v and caspase assays), as determined by appropriate granularity and size, were gated for further analyses. dna fragmentation assay dna purification was carried out according to hermann et al. (1994) with some modifications. briefly, coelomocytes from ten individual earthworms were extruded and plated at 1.5 x 105 coelomocytes well -1 in 200 μl. for each treatment, 10 wells were used (1.5 x 106 coelomocytes per treatment from 10 individual earthworms), one well for each earthworm extruded, and each earthworm exposed to all treatments of the assay. h202 was added at 0, 8.4, 16.9 or 33.8 mm and then incubated at 30 oc, 5 % co2, 6 h. after the incubation period, the plate was centrifuged (150xg, 10 min, 4 oc), the supernatant was removed, and the cells were gently resuspended by vortexing. then 40 μl of lysis buffer (1% np-40, 20 mm edta, 50 mm tris-hcl, ph 7.5) was added to each well before pooling the 10 wells (10 individual earthworms) for each treatment group into a single microcentrifuge tube and centrifuging the lysate at 1600xg, 5 min to pellet debris. the supernatant was transferred to a new tube and the pellet was reextracted with 40 μl lysis buffer and respun. supernatants were combined for each treatment group, and adjusted to 1 % sds, 5 μg ml-1 rnase (fermentas life sciences) and incubated 2 h at 56oc before adding proteinase k (fisher, bp170050) (2.5 μg ml-1) and incubating 2 h at 37 oc. then 0.5 volume 7.5 mm ammonium acetate and 2.5 volume of absolute ethanol was added to precipitate the dna. dna pellets were collected by centrifugation (14,000xg), rinsed with ice cold 70 % ethanol and air dried. pellets were resuspended in 21 μl te (10 mm tris-cl, 1 mm edta, ph 8,0) at 37 oc, 5 min before adding 4 μl loading dye (6x blue/orange loading dye, promega, g1881) containing 1:100 sybr® safe gel stain (10,000x concentration in dmso, invitrogen). molecular weight markers (10 μl well-1) (exactgene, fisher bioreagents, bp257110) containing sybr® safe gel stain (2 μl well-1) and dna samples from each treatment group were electrophoresed in a 1.5 % agarose gel containing 1:10,000 sybr® safe gel stain in 1 x tbe (89 mm tris base, 89 mm boric acid, 2 mm edta, ph 8.3), 120v until first dye front was ~ 2 cm from bottom of gel. gels were photographed using a gel-documentation system (bio-rad). tunel assay a flow tacs apoptosis detection kit (trevigen, inc.) was used according to the manufacturer’s instruction except 150,000 cell ml-1 were used in 0.5 volume recommended, and optional pi was not included. coelomocytes were incubated in 0.2 ml sdmem for 12 h at 25oc, 5% co2 with or without h2o2 (33.8 mm) before washing and fixing the cells. flow cytometry measured fl-1 signals from gated amoebocytes. samples were run in duplicate and subjected to statistical analysis by student’s t test.   92 phagocytosis: endogenous h202 production values were statistically significant as exhibited as a p-value less than or equal to 0.05. all data is based on averages of either triplicate (exogenous h202/phagocytosis and annexin v assays) or duplicate (endogenous h202/phagocytosis, caspase and tunel assays) samples. bacteria were introduced to coelomocytes (50,000 per well in 96 v-bottom plate in duplicate) at a m.o.i. ranging from 10:1 to 1000:1. following 90 min incubation at 30 oc, dhr 123 (invitrogen, d632, 1 mm stock in dmso) was added (1 μm final, 1:1000). after 10 min incubation at rt, samples were placed on ice, protected from light, and run on the flow cytometer immediately. negative controls (no dhr 123) were incubated with 1:1000 dmso to control for carrier effect of dmso. fluorescence was measured using the fl-1 detector. results flow cytometry: exogenous phagocytosis assay figure 1 illustrates how our data was collected and analyzed to determine specific phagocytosis by hyaline amoebocytes from e. hortensis when using exogenous h202. the left panel of the top row is a dot plot representing a typical coelomocyte profile obtained on the flow cytometer when analyzing forward scatter (fsc) (abscissa) versus side scatter (ssc) (ordinate) properties of earthworm statistical analysis data analysis and graphs were generated using microsoft excel 2007. the student’s t-test assuming unequal variance was utilized with a 95 % confidence interval to determine if the experimental fig. 1 representative flow cytometry profile from phagocytosis assays. top row: left panel shows a typical coelomocyte profile of fsc (size) (abscissa) versus ssc (granularity) (ordinate) of extruded earthworm coelomocytes where r1 = hyaline amoebocytes, r2 = granular amoebocytes, and r3 = eleocytes; right panel shows fsc (abscissa) versus fl-1 (relative fluorescence intensity) (ordinate) of r1-gated, untreated coelomocytes cultured without e. coli/gfp. bottom row: fsc (abscissa) versus fl-1 (ordinate) of r1-gated coelomocytes cultured with e. coli/gfp without pretreatment (left panel) and with pretreatment (right panel) of h202. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of gfp; ur = upper right; lr = lower right.   93 fig. 2 phagocytosis is enhanced by pretreating earthworm coelomocytes with h202. asterisks indicate p ≤ 0.05. top row: earthworms ew-p1 – ew-p4 were pretreated with 0, 1.1, 2.1, 4.2 and 8.4 mm h202. middle and bottom rows: earthworms ew-p5 – ew-p12 were pretreated with 0, 0.26, 0.53, 1.1, 2,1, 4.2 and 8.4 mm h202. coelomocytes. r1 depicts large coelomocytes (hyaline amoebocytes), r2 depicts small coelomocytes (granular amoebocytes), and r3 depicts chloragocytes (eleocytes). for analysis purposes and the determination of percent specific phagocytosis, fsc (abscissa) versus fl-1 (ordinate) dot plots were gated on hyaline amoebocytes (r1), the phagocytic cell population. the fsc versus fl-1 dot plots were partitioned into quadrants moving the horizontal bar (left to right) such that all of the events fell within the two right quadrants. the vertical bar (up and down) for fl-1 was established based on the negative control population (i.e., autofluorescence). relative fluorescence intensity values for fl-1 delineate positive events (upper right quadrant ur) and negative events (lower right quadrant lr). the right panel of the top row shows fsc versus fl-1 for an untreated sample in the absence of e. coli/gfp, the negative control population. note that the vertical bar was placed above the majority of events. the left panel of the bottom row shows an untreated sample in the presence of e. coli/gfp, while the right panel of the bottom row shows an h202-treated sample in the presence of e. coli/gfp. note the shift of the coelomocyte population from the lr (negative events) to the ur (positive events) quadrants between these two dot plots as fluorescence intensity increases due to phagocytic uptake of e. coli/gfp when pretreated with h202. in this example percent positive events in ur increases from 7.54 to 13.23 % when coelomocytes were pretreated with h202. effects of exogenous h202 on phagocytosis having established the data analysis protocol, we studied the effect of h202 at concentrations ranging from 0.26 8.4 mm on the phagocytosis of e. coli/gfp by earthworm coelomocytes. percent specific phagocytosis of e. coli/gfp was determined by subtracting the percent positive events (ur) of negative controls (absence of e. coli/gfp) in fl-1 from each of the experimental samples (presence of e. coli/gfp with or without h202). the average of duplicates of controls (0 mm h202) versus h202treated samples were plotted and statistical significance was determined using the student’s t test. figure 2 displays the results obtained for   94 a b c d e fig. 3 representative flow cytometry profile of annexin v-fitc/pi assay using ew-a7 as an example for data collection. coelomocytes were pretreated with 0 (spontaneous apoptosis) (a), 8.4 (b), 16.9 (c), 33.8 (d) and 67.6 (e) mm h202. left hand column: fsc (abscissa) versus ssc (ordinate) of total, ungated coelomocytes population. region 1 (r1) depicts the amoebocytes population (hyaline and granular amoebocytes). middle column: fsc (abscissa) versus fl-2 (pi) (ordinate) of r1 gated amoebocytes (excluding eleocytes). region 6 (r6) depicts pinegative (fl-2 negative), viable amoebocyte population. right hand column: fl-1 (abscissa) versus cell number (ordinate) of amoebocytes gated on r1 and r6 (i.e. only viable amoebocytes that have not taken up pi). region 7 (r7) corresponds to annexin v negative amoebocytes while region 8 (r8) corresponds to annexin v positive (early apoptotic) amoebocytes. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of fitc, fl-2 = relative fluorescence intensity of pi.   95 table 1 percent early apoptotic cells binding annexin v-fitc in untreated and h202-treated samples. only cells residing in r1 and r6 were gated for annexin v-fitc analysis. r1 included hyaline and granular amoebocytes (not eleocytes) and r6 included pi-negative cells. background autofluorescence of untreated samples not receiving annexin v-fitc or pi was subtracted from all values for each indicated earthworm sample before averaging triplicate data and performing statistical analyses. results indicate data obtained from two assays performed by two independent researchers. percent positive values for annexin v-fitc (± sd) are shown for spontaneous apoptosis (0 mm h202) and two-fold serial dilutions from 67.6 to 8.4 mm h202. statistically significant values above spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by student’s t test. earthworm sample spontaneous apoptosis 0 mm 8.4 mm 16.9 mm 33.8 mm 67.6 mm ew-a1 46.23 (±2.42) 31.75 (±1.33) 42.27 (±0.95) 62.81 (±2.39)*** 82.86 (±1.48)*** ew-a2 54.68 (±2.07) 52.82 (±0.74) 64.91 (±2.17)** 66.70 (±1.10)** 81.72 (±1.40)*** ew-a3 31.76 (±1.67) 31.88 (±0.49) 39.51 (±1.13)** 49.99 (±1.26)*** 62.03 (±1.45)*** a ss ay 1 ew-a4 47.01 (±0.57) 36.99 (±1.22) 44.93 (±1.00) 49.78 (±5.04) 63.24 (±1.76)** ew-a5 63.11 (±4.80) 50.40 (±0.96) 58.90 (±1.22) 79.79 (±2.35)* 85.99 (±0.67)* ew-a6 72.68 (±1.68) 69.51 (±0.63) 70.75 (±1.30) 71.56 (±2.65) 80.43 (±2.40)** ew-a7 31.95 (±0.51) 59.35 (±1.48)*** 62.06 (±1.45)*** 67.56 (±2.45)** 75.95 (±2.04)*** a ss ay 2 ew-a8 38.75 (±2.25) 27.83 (±0.08) 27.25 (±1.23) 37.60 (±0.59) 46.88 (±1.12)* coelomocytes from 12 earthworms pretreated in vitro with h202 prior to phagocytosis. earthworms 1, 2, 3 and 4 (ew-p1-p4) were pretreated in the range of 1.1 8.4 mm h202, while ew-p5-p12 were pretreated in the range of 0.26 8.4 mm h202. eight of the 12 earthworms tested (67 %) exhibited statistically significant enhancement of phagocytosis to at least one of the concentrations employed. at doses above 8.4 mm, inhibitory effects on phagocytosis were observed (data not shown). flow cytometry detection of early apoptosis for the next two experiments, which were aimed at investigating the effects of h202 on ps translocation and caspase activation in amoebocytes of e. hortensis, it was important to be able to discriminate between necrotic/late apoptotic amoebocytes and amoebocytes undergoing early apoptosis to ensure that analyses were restricted to amoebocytes with intact plasma membranes. to do this, we utilized pi in addition to the fluoresceintagged reporters of ps translocation and caspase activation. pi exhibits a sufficiently large stokes shift compared to the fluorescein permitting simultaneous detection of fluorescein-labelled moieties and nuclear dna providing the appropriate optical filters and compensation adjustments for spectral overlap are utilized. in our case, we used a facscalibur flow cytometer which employes fl-1 for fluorescein detection and fl-2 for pi detection. pi is membrane impermeant and is thus excluded from viable cells, making this fluorescent counterstain an ideal marker for identifying necrotic/late apoptotic cells in a population when used together with a second fluorescent dye which has minimal spectral emission overlap. figure 3a illustrates the gating strategy used for the analysis of both the ps translocation and caspase activation experiments. first the coelomocytes were analyzed for fsc versus ssc (fig. 3a left panel) and region (r1) was set around the amoebocytes. next a dot plot of fsc versus fl2 gated on the r1 population was created and quadrants were established according to procedure described in fig. 1 (fig. 3a middle panel) positioning the vertical bar at a location that clearly delineated live from dead cells based on the saponin-treated, pi-stained single-positive control employed for compensation purposes. the quadrants corresponded to regions 2-5 (r2-r5). next another region (r6) was drawn around the pinegative cell population, serving to identify viable cells (this would include non-apoptotic, non-necrotic and early-apoptotic cells whose membranes were still intact). finally, a single parameter histogram measuring fluorescein (fl-1) was generated and gated on r1 (amoebocytes) and r6 (pi-negative cells). therefore, only events that satisfied both prerequisites of belonging to the amoebocytes pool (hyaline and granular) as well as being viable were quantified in the r1/r6 gated fl-1 histogram (fig. 3a right panel). this strategy permitted the exclusion of necrotic/late apoptotic cells from the final analysis.   96 table 2 relative fluorescence intensity (rfi) of early apoptotic, annexin v-fitc-positive cells in untreated and h202-treated samples. only cells residing in r1 and r6 were gated for annexin v analysis. r1 included hyaline and granular amoebocytes (not eleocytes) and r6 included pi-negative cells. triplicate data was averaged and subjected to statistical analyses. results indicate data obtained from two assays performed by two independent researchers. rfi values (geometric mean) above background detected by fl-1 (± sd) are shown for spontaneous apoptosis (0 mm h202) and two-fold serial dilutions from 67.6 to 8.4 mm h202. statistically significant values exceeding spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by student’s t test. earthworm sample spontaneous apoptosis 0 mm 8.4 mm 16.9 mm 33.8 mm 67.6 mm ew-a1 14.75 (±0.59) 10.65 (±0.42) 11.22 (±0.08) 14.03 (±0.66) 19.69 (±0.94)** ew-a2 11.99 (±0.16) 12.56 (±0.44) 12.58 (±0.02)* 13.91 (±0.21)*** 21.66 (±1.50)** ew-a3 15.12 (±0.57) 10.72 (±0.48) 11.70 (±0.47) 13.53 (±0.57) 14.90 (±0.41) a ss ay 1 ew-a4 12.79 (±0.12) 10.19 (±0.06) 11.20 (±0.12) 13.33 (±0.53) 18.02 (±0.39)** ew-a5 29.88 (±1.92) 17.81 (±1.46) 18.40 (±0.19) 30.45 (±3.50) 81.52 (±5.72)** ew-a6 25.55 (±2.74) 19.13 (±0.40) 22.40 (±0.70) 28.61 (±0.28) 45.16 (±1.65)** ew-a7 12.23 (±1.61) 14.46 (±0.24) 23.22 (±0.40)** 26.48 (±0.82)*** 31.84 (±0.65)*** a ss ay 2 ew-a8 10.31 (±0.18) 10.35 (±0.35) 11.72 (±0.75)* 13.58 (±0.56)** 19.37 (±2.04)* ps translocation: annexin v-fitc binding cells undergoing early apoptosis can be easily identified using annexin v, an anticoagulant protein that exhibits a high degree of specificity for ps, and when conjugated to a reporter molecule, such as fluorescein isothiocyanate (fitc), can be used an indicator of early apoptosis. ps is a phospholipid that is normally retained on the inner leaflet of the plasma membrane, however, in cells undergoing apoptosis, it is translocated to the outer leaflet and is exposed on the surface of the cell. once exposed on the surface, it is accessible to annexin v binding. early apoptotic cells maintain the integrity of their plasma membrane and are thus impermeable to pi. we conducted experiments to determine the effect of h202 on ps translocation. we treated coelomocytes with h202 in the range of 0 to 67.6 mm and after washing incubated them annexin vfitc and pi. figure 3 illustrates a representative set of flow cytometry obtained for earthworm 7 (ewa7). as the concentration of h202 increased, the degree of ps translocation also increased (right panels). the left hand panels illustrate that h202 affected the morphology of the coelomocytes; as h202 concentration increased, granularity (ssc) decreased (note tightening of ssc signal on ordinate). the middle panels shows that h202 induced cell necrosis/late apoptosis at 33.8 mm and above. in two separate assays performed by two independent researchers on separate days, eight earthworms were analyzed. tables 1-3 report the overall findings of these experiments. table 1 shows % annexin v-fitc binding of viable amoebocytes; 100 % (8/8), 63 % (5/8), 38 % (3/8) and 12.5 % (1/8) exhibited statistically significant ps translocation when exposed to 67.6, 33.8, 16.9 and 8.4 mm h202, respectively. note that untreated amoebocytes exhibit signs of spontaneous apoptosis, perhaps attributed to a stimulus delivered during isolation and/or in vitro culturing. table 2 illustrates the increase in relative fluorescence intensity (rfi) of annexin v-fitc compared to baseline controls; 88 % (7/8), 38 % (3/8), and 38 % (3/8) revealed statistically significant increases in annexin v-fitc levels on the cell surface of viable amoebocytes following exposure to 67.6, 33.8 and 16.9 mm h202, respectively. there was no significant increase in rfi in any of the earthworms when exposed to 8.4 mm h202. table 3 reveals that necrosis/late apoptosis correlates with increasing concentration of h202 but only at the highest concentrations used; 100 % (8/8) and 63 % (5/8) of samples had statistically significant increases of necrosis/cell death compared to controls when exposed to 67.6 and 33.8 mm h202, respectively. no significant difference in necrosis/cell death was observed at concentrations of 16.9 and 8.4 mm h202. caspase activation in coelomocytes to detect the events associated with signal transduction in cells undergoing apoptosis, we measured the activation of caspases in coelomocytes exposed to h202 by using a reporter reagent called fam-vad-fmk flica where 1) fam is the carboxyfluorescein group which fluoresces and is detectable in the fl-1 detector of the flow cyometer, 2) vad is the three amino acid (valine, alanine, aspartic acid) generic probe that binds to most caspases (including caspases -1, -3, -4, -5, -6, -7, -8, and -9) and 3) fmk is the fluoromethyl ketone moiety which anchors the fam-vad-fmk reagent   97 table 3 percent late apoptotic/necrotic cells in untreated and h202-treated samples. only cells residing in r1 were gated for pi analysis. r1 included hyaline and granular amoebocytes (not eleocytes). triplicate data was averaged and subjected to statistical analyses. results indicate data obtained from two assays performed by two independent researchers. percent pi-positive cells detected by fl-2 (± sd) are shown for spontaneous apoptosis (0 mm h202) and two-fold serial dilutions from 67.6 to 8.4 mm h202. statistically significant pi values exceeding spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by student’s t test. earthworm sample spontaneous apoptosis 0 mm 8.4 mm 16.9 mm 33.8 mm 67.6 mm ew-a1 24.55 (±1.19) 18.47 (±2.07) 25.93 (±1.00) 39.12 (±1.57)*** 58.95 (±1.07)*** ew-a2 14.03 (±2.71) 10.79 (±0.39) 11.43 (±0.40) 12.48 (±1.13) 49.93 (±1.80)*** ew-a3 25.03 (±2.09) 18.79 (±0.63) 19.70 (±0.72) 22.85 (±0.20) 44.87 (±0.44)** a ss ay 1 ew-a4 24.85 (±0.14) 19.43 (±1.80) 20.90 (±1.01) 29.75 (±10.7) 45.75 (±2.89)** ew-a5 50.61 (±1.86) 34.53 (±1.39) 39.47 (±2.29) 54.49 (±2.26)* 72.34 (±1.07)*** ew-a6 35.93 (±2.79) 34.84 (±1.62) 35.96 (±2.17) 43.69 (±2.22)* 52.78 (±3.94)** ew-a7 15.48 (±2.05) 11.34 (±1.15) 16.11 (±1.15) 21.15 (±1.47)* 45.75 (±1.71)*** a ss ay 2 ew-a8 20.17 (±3.34) 20.76 (±0.31) 23.69 (±4.15) 29.33 (±1.34)* 57.27 (±1.23)*** to activated caspases in the cell via a covalent cysteine linkage. unbound fam-vad-fmk reagent is washed from the cell while bound reagent is anchored in the cell and fluoresces during flow cytometry, hence enabling the detection of activated caspases in cells undergoing the initial stages of apoptosis. we tested six earthworms in two separate assays. figure 4 shows the results of these assays where coelomocytes were pretreated with 0 (a), 16.9 (b), 33.8 (c) and 67.6 (d) mm h202. the same strategy for excluding necrotic/late apoptotic cells described in fig. 3 was used in these experiments, i.e., relevant cells used for analysis were obtained by gating r1 (amoebocytes) and r6 (pi-negative) positive coelomocytes (fig. 4). a marked increase in necrosis/cell death was observed between 33.8 and 67.6 mm h202 (fig. 4cd). again we observed changes in cellular morphology (as detected by fsc versus ssc profiles) as the concentration of h202 increased, particularly at 67.6 mm (fig. 4, middle panels). statistically significant increases in caspase activation were detected in 100 % (3/3) and 67 % (4/6) of samples exposed to 67.6 and 33.8 mm h202, respectively (fig. 5). morphological changes in coelomocytes ew f4-f6 were subjected to further analysis to determine if morphological changes associated with a decrease in cell volume, another hallmark of cells undergoing apoptosis, was occurring in response to h202 treatment. treated and untreated coelomocytes incubated with fam-vad-fmk flica and pi (as described in caspase assay above) were gated to select for pi-negative (viable and early apoptotic), large coelomocytes. gated cells were analyzed in a single-parameter histogram measuring forward light scatter (fsc). duplicate samples were averaged and p values were determined by student’s t test. figure 6 shows a decrease in cell volume; in all cases except one (16.9 mm h202 for ewf5), decreases in cell volume of h202-treated samples were statistically significant (p < 0.05) compared to untreated samples. the difference in cell volume was much more pronounced in the large coelomocytes (fig. 6) than in the small coelomocytes (data not shown). these results show that in earthworms undergoing biochemical changes (caspase activation), morphological alterations (cell volume) are also occurring. dna fragmentation another hallmark of apoptosis is the fragmentation of dna which occurs when an endonuclease (caspase-activated deoxyribonuclease, cad) is activated as a consequence of programmed cell death induction. genomic dna is cleaved at sites between nucleosomes at ~ 200 base pairs (bp) intervals (nagata, 2000). in this study, total coelomocytes (including amoebocytes and eleocytes) were pretreated with h202 prior to the purification and electrophoresis of dna. in the first assay (fig. 7, right hand gel), h202 was used at 0 and 33.8 mm. dna fragmentation consistent with apoptosis was   98 fig. 4 representative flow cytometry profile of caspase (flica/pi) assay using ew-f6 as an example for data collection. coelomocytes were pretreated with 0 (spontaneous apoptosis) (a), 16.9 (b), 33.8 (c), and 67.6 (d) mm h202. left hand column: fsc (abscissa) versus ssc (ordinate) of total, ungated coelomocyte population. region 1 (r1) depicts the amoebocyte population (hyaline and granular amoebocytes). middle column: fsc (abscissa) versus fl-2 (pi) (ordinate) of r1 gated amoebocytes (excluding eleocytes). region 6 (r6) depicts pi-negative (fl-2 negative), viable amoebocyte population. right hand column: fl-1 (abscissa) versus cell number (ordinate) of amoebocytes gated on r1 and r6 (i.e. only viable amoebocytes that have not taken up pi). region 7 (r7) corresponds to fluorescein negative amoebocytes while region 8 (r8) corresponds to fluorescein positive (early apoptotic) amoebocytes. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of fluorescein, fl-2 = relative fluorescence intensity of pi.   99 fig. 5 caspase activation in untreated and h202-treated coelomocytes. top row: percent caspase positive amoebocytes of ew-f1-f3 treated with 0 mm (spontaneous apoptosis) or 33.8 mm h202. bottom row: percent caspase positive amoebocytes of ew-f4-f6 treated with 0, 16.9, 33.8 and 67.6 mm h202. asterisks denote p ≤ 0.05 compared to control. observed compared to the untreated control. in a second assay (fig. 7, two left hand gels) h202 was used at 0, 33.8, 16.9 and 8.4 mm. laddering was observed only when coelomocytes were exposed to 33.8 and 16.9 mm h202. these results indicate that initiation of the stereotypical internucleosomal degradation of genomic dna characteristic of apoptosis is occurring in earthworm coelomocytes in response to oxidative stress. tunel assay a terminal dutp nick-end labeling (tunel) method was used to enzymatically verify dna fragmentation in h2o2-treated coelomocytes. this method involved labeling the 3’-hydroxyl dna ends generated during dna fragmentation using a terminal deoxynucleotidyl transferase (tdt) and biotin-labeled utp. labeled dna was detected by incubating with fitc-conjugated streptavidin and subjecting the samples to flow cytometric analysis. amoebocytes identified on forward versus side scatter dot plots were gated for fl-1 (fitc) analysis. four earthworms were treated with 33.8 mm h2o2, however, only three exhibited any demonstrable difference in labeling compared to untreated controls(ew t1, ew t2 & ew t4); two of these (ew t1 & ew t2) were statistically significant (p < 0.006). figure 8 shows the results from the three responding earthworms. note the increase in relative fluorescence intensity compared to untreated controls. a nuclease control was included for verification purposes (data not shown). these results complement the agarose gel electrophoresis results and illustrate that flow cytometry combined with the tunel assay is a useful and rapid method to detect dna fragmentation in earthworm coelomocytes. h202 production during phagocytosis our final investigation was aimed at determining whether hyaline amoebocytes of e. hortensis undergo a respiratory burst and produce endogenous h202 upon phagocytosis of the soil bacteria p. stutzeri (gram negative) and b. megaterium (gram positive). we used dhr 123, a fluorogenic molecular probe (excitation 505 nm; emission 534 nm) to detect the generation of h202. dhr 123 is oxidized by h202 and is converted consequently to it fluorescent derivative rhodamine 123, which can be measured intracellularly by the flow cytometer. flow cytometry analysis involved: 1) setting a region around the hyaline amoebocytes (as described in fig. 1, top row, left hand dot plot); 2) generating a fl-1, single-parameter histogram gated on this region; and 3) establishing a boundary (markers) to separate negative events from positive events (as described in fig. 4, right hand histograms). the boundary was established based   100 fig. 6 cell volume changes in untreated and h202-treated coelomocytes. ew f4-f6 samples from the caspase assay were gated on pi-negative events falling in the region corresponding to the large coelomocyte population. h202 concentration (abscissa) versus geometric mean of forward side scatter (ordinate) are plotted for ew f4 (diamonds), ew f5 (squares) and ew f6 (triangles). with the exception of ew f5 at 16.9 mm, p values < 0.05 compared to controls (untreated) were obtained. on the negative control sample which was incubated in the absence of dhr 123 (but with an equivalent amount of dmso compared to experimental samples) to control for autofluorescence. any events exceeding the fl-1 boundary marker were considered positive (percent positive hyaline amoebocytes). eight earthworms were extruded and three (ew 2, 7 and 8) were selected for the phagocytosis assay based on the quality of the coelomocytes (i.e., higher proportion of amoebocytes compared to eleocytes and adequate cell numbers). figure 9 shows the results of these three earthworms and illustrates that dhr 123 is oxidized to rhodamine 123 during phagocytosis of p. stutzeri and b. megaterium. statistical significance was determined based on comparison between control (dhr 123 only) and experimental (bacteria plus dhr 123) samples. p. stutzeri generated statistically significant results in 100 % and 67 % of cases at a m.o.i. of 100:1 and 10:1, respectively. b. megaterium generated statistically significant results in 67 % of cases at a m.o.i. of 100:1 and 10:1. a second assay employing p. stutzeri at a m.o.i. of 1000:1 revealed enhanced h2o2 production in 67% of cases (4 out of 6 earthworms) compared to controls (data not shown). discussion this study demonstrates that exogenous h202 (0.26 8.4 mm) caused an increase in phagocytosis by e. hortensis; 67 % of earthworms tested exhibited statistically significant enhancement of percent specific phagocytosis for one or more of the concentrations of h202 used. bejarano et al. (2007), gamaley et al. (1994) and takeda et al. (1998) examined the effects of oxygen free radicals in cultured human neutrophils, murine macrophages and rat ameboid microglia, respectively, and found that exposure to exogenous h202 also caused an increase in phagocytic function. it is interesting to propose that in our model h202 may be acting as a second messenger involved in evoking a significant elevation of phagocytic function. we are interested in determining if calcium mobilization from agonistsensitive intracellular stores or influx across the plasma membrane accompanies h202-enhanced phagocytosis, an effect reported by others (redondo et al., 2004; bejarano et al., 2007). the use of a calcium quelator (e.g., dimethyl bapta) would help to reveal the role, if any, that calcium mobilization plays in phagocytosis in earthworms, and to facilitate a better understanding of the physiological role of oxygen free radicals in calcium homeostasis. we also plan to examine the effect of pre-treating earthworm amoebocytes with catalase, an enzyme facilitating h202 decomposition into water and oxygen, prior to the addition of exogenous h202 in phagocytosis assays. in parallel with the invertebrate studies of blanco et al. (2005) which examined the effects of h202 on coelomocytes of themiste petricola (the sipunculan marine worm), we also revealed that h202 induced apoptosis-like cell death in coelomocytes of e. hortensis. using pi, we were able to discriminate between viable coelomocytes (nonapoptotic and early apoptotic) and nonviable coelomocytes (necrotic/late apoptotic); pi-positive cells were excluded from flow cytometric analyses due to dna labeling. note that eleocytes were not included in   101 fig. 7 dna fragmentation of h202-treated coelomocytes. these three gels represent two independent dna fragmentation assays where earthworm coelomocytes were treated with h202 for 6 h and dna was isolated and electrophoresed in 1.5 % agarose gels in tbe buffer. lanes m (molecular weight markers in bp are indicated for the nine distinct bands of the 100 bp ladder), lanes c (untreated cells, 0 mm h202), lanes 1 (33.8 mm h202), lane 2 (16.9 mm h202), and lane 3 (8.4 mm h202). the two left-hand gels are from the same assay and same population of coelomocytes. the right-hand gel is from a separate assay with a different population of coelomocytes. these analyses owing to relatively high level of autofluorescence exhibited by this subpopulation. we observed ps translocation and caspase activation when amoebocytes were treated with h202 in the range of 8.4 67.6 mm and 16.9 67.6 mm, respectively, under the conditions specified. it is interesting to note that relatively high concentrations of h202 were required to induce apoptosis, suggestive of high tosc in earthworm coelomocytes. we also observed dose-dependent changes in fsc when amoebocytes were pretreated with h202, a phenomenon consistent with apoptotic volume decrease (avd) (maeno et al., 2000; okada and maeno, 2001) when cells undergoing apoptosis experience cell shrinkage and subsequent cell fragmentation (apoptotic bodies). exposure of ps on the surface of infected coelomocytes may be one mechanism by which phagocytes recognize and remove cellular reservoirs of pathogens during innate immune responses. fadok et al. (1992), for example, showed that murine macrophages rapidly recognize and remove apoptotic lymphocytes following exposure of ps on the outer leaflet of the plasma membrane of apoptotic cells, thereby prevent potential tissue damage from lysis of these cells in vivo. caspases are highly conserved and have been identified in invertebrates; for example the ced-3 protein of the nematode caehorhabditis elegans was the first to be characterized. the gene encoding ced-3 is known to exhibit homology to murine and human caspase-1 (formerly known as interleukin-1 beta converting enzyme) (yuan et al., 1993). several caspases known to participate in apoptosis have also been identified in drosophila melanogaster fig. 8 tunel analysis of h202-treated coelomocytes. coelomocytes treated with 0 mm h2o2 (filled graph line) (control) or 33.8 mm h2o2 (unfilled graph line) were chemically fixed, and the fragmented dna ends were labeled with biotinconjugated utp and stained with streptavidin fitc. the gated amoebocyte population was analyzed for fl-1 (fitc) fluorescence intensity (abscissa) versus cell number (ordinate). ew t1 and ew t2 exhibited statistical significance (p < 0.006) between untreated and treated amoebocytes.   102 fig. 9 endogenous h202 is produced during phagocytosis. the percent hyaline amoebocytes oxidizing dhr 123 to rhodamine 123 for each treatment is shown (percent positive hyaline amebocyes). phagocytosis assays using p. stutzeri and b. megaterium at m.o.i. of 100:1 and 10: 1 compared to control (dhr 123) shows statistically significant (p ≤ 0.05) increases in rhodamine 123 production (*) for ew 2, 7 and 8. (reviewed in boyce et al., 2004). in addition, the crystal structure of sf-caspase-1 from spodoptera frugiperda has been resolved and its overall fold found to be exceedingly similar to active caspases from humans (forsyth et al., 2004). using a fluorescent inhibitor of poly-caspases, our results show that h202 induces caspase activation in earthworm amoebocytes. it would be worthwhile to try to dissect the caspase signaling pathway in e. hortensis using inhibitors known to target caspases with specificity for particular caspase subfamilies (matsura et al., 1999). we also evaluated oligonucleosomal dna fragmentation using agarose gel electrophoresis to resolve dna fragments. a dose-dependent laddering pattern characteristic of apoptotic cells was observed when coelomocytes were treated with 16.9 and 33.8 mm, but not 8.4 mm h202. perhaps a longer incubation period would have resulted in dna laddering at the lower concentration. the presence of discrete ~200 bp dna fragments is consistent with apoptosis and not necrosis as the latter would result in random dna fragmentation causing smears on an agarose gel rather than discrete repetitive oligonucleosomal fragments generated by endonuclease cleavage between nucleosomes (walker et al., 1988). in addition, we conducted tunel assays in conjunction with flow cytometry which provided enzymatic verification of dna fragmentation in h2o2-treated coelomocytes, and a more rapid procedure for detecting dna fragmentation in apoptotic cells. we are also interested in examining mitochondrial transmembrane potential using a mitochondriaspecific probe such as jc-1 which incorporates into mitochondria and undergoes a shift in fluorescence emission spectra during membrane depolarization (cossarizza et al., 1995). finally, our results show that endogenous h202 is produced by hyaline amoebocytes during phagocytosis of gram positive (b. megaterium) and gram negative (p. stutzeri) bacteria at m.o.i. ranging from 10:1 to 1000:1. although further investigation is required in vivo, it is tempting to speculate that during infection in the earthworm, the production of h202 facilitates physiological changes in coelomocytes that induce appropriate innate immune responses needed not only to eradicate pathogen load through ingestion (phagocytosis), but also to eliminate infected and damaged cells, and control cell numbers through a clearance mechanism (apoptosis). engulfment of apoptotic cells would prevent the lytic release of toxic molecules (e.g. enzymes, proteases and oxidizing molecules) and spare or minimize surrounding tissue from inflammatory-type damage, thus enabling immune mechanisms to function effectively. acknowledgments this work was funded by the pennsylvania academy of science, the beta beta beta national biological honor society and the cabrini college science department research fund.   103 references abele d, tesch c, wencke p, pörtner, ho. how does oxidative stress relate to thermal tolerance in the antarctic bivalve yoldia eightsi? antarct. sci. 13: 111-118, 2001. adamowicz a, wojtaszek j. morphology and phagocytotic activity of coelomocytes in dendrobaena veneta (lumbricidae). zoolog. pol. 46: 91-104, 2001. axline sg, reaven ep. inhibition of phagocytosis and plasma membrane mobility of the cultivated macrophage by cytochalasin b; role of subplasmalemmal microfilaments. j. cell biol. 62: 647-659, 1974. bedard k, krause k-h. the nox family of rosgenerating nadph oxidases: physiology and pathophysiology. physiol. rev. 87: 245-313, 2007. bejarano i, terrón mp, paredes, sd, barriga c, rodríguez ab, parienta ja. hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation. mol. cell. biochem. 296: 77-84, 2007. blanco ga, bustamante j, garcia m, hajos se. hydrogen peroxide induces apoptotic-like cell death in coelomocytes of themiste petricola (sipuncula). biol. bull. 209: 168-183, 2005. boyce m, degterev a, yuan j. caspases: an ancient cellular sword of damocles. cell death differ. 11: 29-37, 2004. brenneisen p, steinbrenner h, sies h. selenium, oxidative stress, and health aspects. mol. aspect. med. 26: 256-267, 2005. cairo g, tacchini l, pogliaghi g, anzon e, tomasi a, bernelli-zazzera a. induction of ferritin synthesis by oxidative stress. transcriptional and post-transcriptional regulation by expansion of the “free” iron pool. j. biol. chem. 270: 700703, 1995. coffey md, cole ra, colles sm, chisolm gm. in vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals. j. clin. invest. 96: 1866-1873, 1995. cooper el. earthworm immunity. in: invertebrate immunology. rinkevich b, műller weg (eds), springer verlag, heidelberg, pp 10-45, 1996. cossarizza a, cooper el, quaglino d, salvioli s, kalachnikova g, franceschi c. mitochondrial mass and membrane potential in coelomocytes from the earthworm eisenia foetida: studies with fluorescent probes in single intact cells. biochem. biophys. res. commun. 214: 503510, 1995. cossarizza a, cooper el, suzuki mm, salvioli s, capri m, gri g, et al. earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. exp. cell res. 224: 174-182, 1996. cossarizza a, pinti m, troiano l, cooper el. flow cytometry as a tool for analyzing invertebrate cells. inv. surv. j. 2: 32-40, 2005. csiszar a, labinskyy n, zhao x, hu f, serpillon s, huang z, et al. vascular superoxide and hydrogen peroxide production and oxidative stress resistance in two closely related rodent species with disparate longevity. aging cell 6: 783-797, 2007. di guilo rt, washburn pc, wenning rj, winston gw, jewell cs. biochemical responses in aquatic animals: a review of determinants of oxidative stress. environ. toxicol. chem. 8: 1103-1123, 1989. dovzhenko nv, kurilenko av, bel’cheva nn, chelomin vp. cadmium-induced oxidative stress in the bivalve mollusk modiolus modiolus. russian j. marine biol. 31: 309-313, 2005. dunford hb. free radicals in iron-containing systems. free radic. biol. med. 3: 405-421, 1987. engelmann p, pál j, berki t, cooper el, németh p. earthworm leukocytes react with different mammalian specific monoclonal antibodies. zoology 105: 257-265, 2002. engelmann p, molnár l, pálinkás l, cooper el, németh p. earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge. cell tissue res. 316: 391-401, 2004. engelmann p, palinkas l, cooper el, németh p. monoclonal antibodies identify four distinct annelid leukocyte markers. dev. comp. immunol. 29: 599-614, 2005. fadok va, voelker dr, campbell pa, cohen jj, bratton dl, henson pm. exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. j. immunol. 148: 2207-2216, 1992. fato r, bergamini c, leoni s, lenaz g. mitochondrial production of reactive oxygen species: role of complex i and quinone analogues. biofactors 32: 31-39, 2008. findlay vj, tapiero h, townsend dm. sulfiredoxin: a potential therapeutic agent?: antioxidants in the prevention of human diseases. biomed. pharmacother. 59: 374-379, 2005. flohé l, budde h, hofmann b. peroxiredoxins in antioxidant defense and redox regulation. biofactors 19: 3-10, 2003. forman hj, torres m. reactive oxygen species and cell signaling, respiratory burst in macrophage signaling. am. j. respir. crit. care med. 166: s4-s8, 2002. forsyth cm, lemongello d, lacount dj, friesen pd, fisher aj. crystal structure of an invertebrate caspase. j. biol. chem. 279: 70017008, 2004. fuller-espie sl, goodfield l, hill k, grant k, derogatis n. conservation of cytokinemediated responses in innate immunity: a flow cytometric study investigating the effects of human proinflammatory cytokines on phagocytosis in the earthworm eisenia hortensis. inv. surv. j. 5: 124-134, 2008. gamaley ia, kirpichnikova km, klyubin iv. activation of murine macrophages by hydrogen peroxide. cell. signal. 6: 949-957, 1994. ghezzi p. regulation of protein function by glutathionylation. free rad. res. 39: 573-580, 2005.   104 http://www.ncbi.nlm.nih.gov/pubmed?term=%22floh%c3%a9%20l%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22budde%20h%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22hofmann%20b%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract gorbi s, regoli f. total oxyradical scavenging capacity as an index of susceptibility to oxidative stress in marine organisms. comments toxicol. 9: 303-332, 2003. hartenstein v. blood cells and blood cell development in the animal kingdom. annu. rev. cell dev. biol. 22: 677-712, 2006. helfand sl, rogina b. genetics of aging in the fruit fly, drosophila melanogaster. annu. rev. genet. 37: 329-348, 2003. hermann m, lorenz h-m, voll r, grünke m, woith w, kalden jr. a rapid and simple method for the isolation of apoptotic dna fragments. nucleic acids res. 22: 5506-5507, 1994. imlay ja. pathways of oxidative damage. annu. rev. microbiol. 57: 395-418, 2003. jankov rp, kantores c, pan j, belik j. contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats. am. j. physiol. lung cell. mol. physiol. 294: l223-l245, 2008. kell db. iron behaving badly: inappropriate iron chelation as a major contributor to the aetiology of vascular and other progressive inflammatory and degenerative diseases. bmc med. genomics 2: 2, 2009. kalaç y, ayten k, gűlruh u, çotuk a. the role of opsonin in phagocytosis by coelomocytes of the earthworm dendrobaena veneta. j. cell mol. biol. 1: 7-14, 2002. kang jh, kim ks, choi sy, kwon hy, won mh. oxidative modification of human ceruloplasmin by peroxyl radicals. biochim. biophys. acta 1568: 30-36, 2001. larsen pl. aging and resistance to oxidative damage in caenorhabditis elegans. proc. natl. acad. sci. usa 90: 8905-8909, 1993. laukens d, waeytens a, de bleser p, cuvelier c, devos m. human metallothionein expression under normal and pathological conditions: mechanisms of gene regulation based on in silico promoter analysis. crit. rev. eukaryot. gene expr. 19: 301-317, 2009. lewis dfv. oxidative stress: the role of cytochromes p450 in oxygen activation. j. chem. tech. biotechnol. 77: 1095-1100, 2002. livingstone, dr, garcia martinez p, michel x, narbonne, jf, o’hara s, ribera d et al. oxyradical production as a pollution-mediated mechanisms of toxicity in the common mussel mytilus edulis l, and other molluscs. funct. ecol. 4: 415-424, 1990. livingstone dr, lemaire p, matthews a, peters ld, porte c, fitzpatrick pj, et al. assessment of the impact of organic pollutants on goby (zosterisessor ophiocephalus) and mussel (mytilus galloprovincialis) from the venice lagoon, italy: biochemical studies. mar. environ. res. 39: 235-240, 1995. loo g. redox-sensitive mechanisms of phytochemical-mediated inhibition of cancer cell proliferation. j. nutr. biochem. 14: 64-73, 2003. maeno e, ishizaki y, kanaseki t, hazana a, okada y. normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. proc. natl. acad. sci. usa 17: 9487-9492, 2000. matsura t, kai m, fujii y, ito h, yamada k. hydrogen-peroxide-induced apoptosis in hl-60 cells requires caspase-3 activation. free radic. res. 30: 73-83, 1999. mosiman v, patterson b, canterero l, goolsby c. reducing cellular autofluorescence in flow cytometry: an in situ method. cytometry 30: 151-156, 1997. nagata s. apoptotic dna fragmentation. exp. cell res. 256: 12-18, 2000. nordberg j, arnér e.s.j. reactive oxygen species, antioxidants, and the mammalian thioredoxin system. free rad. biol. med. 31: 1287-1312, 2001. okada y, maeno e. apoptosis, cell volume regulation and volume-regulatory chloride channels. comp. biochem. physiol. a 130: 377383, 2001. panasenko om, osipov an, chekanov av, arnhold j, sergienko vi. peroxyl radical is produced upon the interaction of hypochlorite with tertbutyl hydroperoxide. biochemistry 67: 880-888, 2002. patel m, francis j, cooper el, fuller-espie sl. development of a flow cytometric, nonradioactive cytotoxicity assay in eisenia fetida: an in vitro system designed to analyze immunosuppression of natural killer-like coelomocytes in response to 7, 12 dimethylbenz[a]anthracene (dmba). eur. j. soil biol. 43s1:s97-103, 2007. pichaud n, pellerin j, fournier m, gauthier-clerc s, rioux p, pelletier e. oxidative stress and immunologic responses following a dietary exposure to pahs in mya arenaria. chem. cent. j. 2: 23, 2008. raes mc, toussaint o, remacle j. importance of se-glutathione peroxidase, catalase, and cu/zn-sod for cell survival against oxidative stress. free radic. biol. med. 17: 235-248, 1994. rattan si. theories of biological aging: genes, proteins and free radicals. free radic. res. 40: 1230-1238, 2006. redondo pc, salido gm, rosado ja, parienta ja. effect of hydrogen peroxide on ca2+ mobilisation in human platelets through sulphydryl oxidation dependent and independent mechanisms. biochem. pharmacol. 67: 491-502, 2004. regoli f. total oxyradical scavenging capacity (tosc) is polluted and translocated mussels: a predictive biomarker of oxidative stress. aquatic toxicol. 50: 351-361, 2000. sies h, stahl w, sundquist ar. antioxidant functions of vitamins. vitamins e and c, betacarotene and other carotenoids. ann. n. y. acad. sci. 669: 7-20, 1992. strazzullo p, puig jg. uric acid and oxidative stress: relative impact on cardiovascular risk? nutr. metab. cardiovasc. dis. 17: 409-414, 2007. sundaram c, koster w, schallreuter u. the effect of uv radiation and sun blockers on free radical defence in human and guinea pig epidermis. arch. derm. res. 282: 526-531, 1990.   105 takeda h, tomita m, tanahashi n, kobari m, yokoyama m, takao m, et al. hydrogen peroxide enhances phagocytic activity of ameboid microglia. neurosci. lett. 240: 5-8, 1998. thannickal fj, fanburg bl. reactive oxygen species in cell signaling. am. j. phsiol. lung cell. mol. physiol. 279: l1005-l1028, 2000. tyagi n, sedoris kc, steed m, ovechkin av, moshal ks, tyagi sc. mechanisms of homocysteine-induced oxidative stress. am. j. physiol. heart circ. physiol. 289: h2649h2656, 2005. valko m, rhodes, cj, moncol j, izakovic m, mazur m. free radicals, metals and antioxidants in oxidative stress-induced cancer. chem. biol. interact. 160: 1-40, 2006. walker ni, harmon bv, gobé gc, kerr jf. patterns of cell death. methods achiev. exp. pathol. 13: 18-54, 1988. wang g. nadph oxidase and reactive oxygen species as signaling molecules in carcinogenesis. front. med. china 3: 1-7, 2009. yamaji y, nakazato y, oshima n, hayashi m, saruta t. oxidative stress induced by iron released from transferrin in low ph peritoneal dialysis solution. nephrol. dial. transplant. 19: 2592-2597, 2004. yuan j, shaham, s, ledoux s, ellis hm, horvitz hr. the c. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. cell 75: 641-652, 1993.   106 http://www.ncbi.nlm.nih.gov/pubmed?term=%22gob%c3%a9%20gc%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22kerr%20jf%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract isj 8: 56-58, 2011 isj 8: 56-58, 2011 issn 1824-307x visions and perspectives stress-based modulation of the immune response in molluscan hemocytes: a tworeceptor model r barcia, ji ramos-martínez department of biochemistry and molecular biology, school of veterinary, university of santiago de compostela, campus de lugo, lugo, spain accepted march 23, 2011 abstract in molluscs, hemocytes perform the molecular mechanisms related to immunity. these cells have the ability to respond to the different varieties of stress by modulating their responses. the stressors may be bacterial toxins, cytokines or growth factors, and even physical agents such as changes in temperature or oxygen partial pressure. in the first place, hemocytes synthesise catecholamines, which, in turn, modify the immune response in terms of phagocytosis or nitric oxide synthesis. according to studies on the hemocytes of the mussel mytilus galloprovincialis, we propose a model for a sequential action where the il-2 receptor and its wide agonist specificity play an important role. also, α and β-adrenergic receptors suggest the functioning of a return-to-hemocyte mechanism. the model is proposed taking into account the possible relationship between the pathways mediated by campactivated protein kinase and protein kinase c in hemocytes. key words: molluscs; mytilus galloprovincialis; stress; endocrinology; immunology   introduction in molluscs the immune response is performed by specialized cells termed hemocytes. because molluscs lack an adaptative immune system, the response against toxic agents of diverse origin is performed through an ancestral process that was preserved along the evolution until vertebrates and ___________________________________________________________________________ corresponding author: juan ignacio ramos-martínez department of biochemistry and molecular biology school of veterinary university of santiago de compostela campus de lugo, 2702 lugo, spain e-mail: juanignacio.ramos@usc.es list of abbreviations: acth: adrenocorticotropic hormone; crh: corticotrophin-releasing hormone; erk: extracellular signal-regulated kinase; ifn: interferon; il-2: interleukin-2; (il-2r: il-2 receptor; jnk: c-jun n-terminal kinase; lps: lipopolysaccharide; mapk: mitogen-activated protein kinase; no: nitric oxide; pdgf: plateletderived growth factor; pka: camp-activated protein kinase; pkc: protein kinase c; ros: oxygen radicals; stat: signal transducers and activators of transcription protein; tnf: tumor necrosis factor. is known as innate immunity (medzitov and janeway, 1997). up to date, the system has been admitted to involve a rigid response; however, new data regarding the structures of the recognition elements suggest a certain selective ability, which allows guessing the elaboration of a possible molecular memory (brehélin and roch, 2008; ottaviani, 2011). the immune response of molluscan hemocytes is modulated by stress in a way apparently similar to that described in vertebrates as “hypothalamushypophysis-adrenal axis” (hpa axis), constituting a proto-response to stress centered on these cells, which can be qualified as an “immune-mobile brain” (ottaviani et al. 1993). the receptor-based modulation of molluscan immunity when a foreign element enters the organism, the immune response increases the phagocytic activity, among other actions. phagocytosis develops in several stages, and one of them is the synthesis of ros and no, leading to the generation of peroxynitrite. when molluscan hemocytes are incubated with agonists so different in structure as growth factors (pdgf, tnf), peptidic hormones (acth, crh), interleukins (il-2), or even bacterial toxins (lps), catecholamine and no synthesis is induced (ottaviani and franceschi, 1996). the   56 mailto:juanignacio.ramos@usc.es http://en.wikipedia.org/wiki/corticotropin-releasing_hormone fig. 1 hypothesis on the stress-based molecular mechanisms modulating the immune response in hemocytes of mytilus galloprovincialis lmk. nitric oxide synthase (nos). other abbreviations, in the text. the red lines suggest non demonstrated pathways. effects are detectable, both in freshly extracted hemocytes (therefore, stressed because of extraction procedure) and in mytilus hemocytes stabilized after three days in culture (cao et al., 2004, 2007a). depending on the agonist assayed, differences in the expression of the subunits forming the il-2 receptor were detected, mainly of the α subunit (cao et al., 2004, 2007a). this agrees with the hypothesis about the presence of a unique ancestral receptor with wide specificity (ottaviani and franceschi, 1996). on the other hand, the same agonists also provoke remarkable increases in catecholamine production, which evidencing a stress on these cells (cao et al., 2004, 2007a, b). the catecholamines that the hemocytes or other cells secrete might take a return-to-hemocyte pathway, or else, act directly as suggested by the presence of α and β adrenergic receptors in crassostrea gigas (lacoste et al., 2001). the signals generated by catecholamines are internalized through pka and pkc, as observed in crassostrea hemocytes (lacoste et al., 2001, 2002). also, the signals of lps and other bacterial factors involve the activation of stress kinases, such as p38-mapk, jnk o erk (canesi et al., 2002, betti et al., 2006, ciacci et al., 2010). at the same time, pka and pkc mediate no synthesis (barcia and ramos-martínez, 2008, gonzalez-riopedre et al., 2009). a common intermediation of some protein kinases related to stress (mapk) in phagocytosis and in synthesis of thermal shock proteins has been reported in mytilus hemocytes incubated with toxins and bacteria (canesi et al., 2002; gaitanaki et al., 2004; kefaloyianni et al., 2005), and mapk is known to mediate processes activated through g protein receptors. therefore, there it seems to be a network between the different protein elements involved in the internalization of the stress processes and those evidencing infective or inflammatory actions. canesi and colleagues also reported that the incubation of mytilus hemocytes with the macrophage activator ifn-γ induced stat phosphorylation, which proves the convergence towards mapk-like phosphorylation mediators (canesi et al., 2003). this result leaves open the possibility of the existence in hemocytes of two types of receptor for immune response activating agonists. one of them with wide specificity, similarly to il-2r of vertebrates, and another one similar to the tyrosine kinase receptors, which would internalize stat activating signals. in addition, at least other two adrenergic receptors should operate the modulation of the catecholamine-generated response. the differences in the timing of signal internalization account for the operability of the different types of receptors and suggests a possible consecutive connection of their actions. in this sense, il-2 and lps increase catecholamine synthesis quickly (30 60 min) and then, a return to basal values is detected with longer incubation times (cao et al., 2004, 2007a, b; gonzalezriopedre et al., 2009). on the contrary, no synthesis requires a more prolonged incubation with the agonists (novas et al., 2004). the results obtained suggest that il-2 and lps make pkc activity to increase rapidly (barcia and ramosmartínez, 2008; gonzalez-riopedre et al., 2009). catecholamine secretion and later binding to   57 adrenergic receptors may trigger late no synthesis (24 h). this hypothesis would justify the early pka and pkc increase induced by il-2 or lps (barcia and ramos-martínez. 2008; gonzalez-riopedre et al., 2009). the reduction of no synthesis detected at 30 min of cell incubation with il-2 or lps would confirm an early kinase action (novas et al., 2004). this ensemble of actions can be summarized in the hypothesis shown in figure 1. the timing of successive actions of cytokines must play an important role in the activation of the different mapks. in this sense, and taking into account the implication of mapks in the synthesis of thermal shock proteins (cellura et al., 2006; gourgou et al., 2010), the biphasic expression of the hsp70 when incubating the cells with il-2 or lps confirms the hypothesis unfolded in the present text (gonzalez-riopedre et al., 2007) and opens new perspectives in the study of these cells and their function in cell signalling in molluscan neuroimmunology. references barcia r, ramos-martínez ji. effects of interleukin2 on nitric oxide production in molluscan innate immunity. inv. surv. j. 5: 43-49, 2008. betti m, ciacci c, lorusso lc, canonico b, falcioni t, gallo g, et al. effects of tumour necrosis factor alpha (tnf alpha) on mytilus haemocytes: role of stress-activated mitogenactivated protein kinases (mapks). biol. cell 98: 233-244, 2006. brehélin m, roch p. specificity, learning and memory in the innate immune response. inv. surv. j. 5: 103-109, 2008. canesi l, betti m, ciacci c, scarpato a, citterio b, pruzzo c, et al. signalling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 map kinases. dev. comp. immunol. 26: 325-334, 2002. canesi, l., betti, m, ciacci, c, citterio b, pruzzo c, gallo, g. tyrosine kinase-mediated cell signalling in the activation of mytilus hemocytes: possible role of stat-like proteins. biol. cell 95: 603-613, 2003. cao a, novas a, ramos-martínez ji, barcia r. seasonal variations in haemocytes response in the mussel mytilus galloprovincialis lmk. aquaculture 263: 310-319, 2007a. cao a, ramos-martínez ji, barcia r. in vitro effects of lps, il-2, pdgf and crf on haemocytes of mytilus galloprovincialis lmk. fish shellfish immunol. 16: 215-225, 2004. cao a, ramos-martínez ji, barcia r. in haemocytes from mytilus galloprovincialis lmk., treatment with corticotrophin of growth factors conditions catecholamine release. int. immunopharmacol. 7: 1395-1402, 2007b. cellura c, toubiana m, parrinello n, roch p. hsp70 gene expression in mytilus galoprovincialis hemocytes is triggered by moderate heat shock and vibrio angilarum, but not by v. splendidus or micrococcus lysodeikticus. dev. comp. immunol. 30: 984997, 2006. ciacci c, betti m, canonico b, citterio b, roch p, canesi l. specificity on anti-vibrio immune response through p38 mapk and pkc activation in the hemocytes of the mussel mytilus galloprovincialis. j. invertebr. pathol. 105: 49-55, 2010. gaitanaki c, kefaloyianni e, marmari a, beis i. various stressors rapidly activate the p38mapk signaling pathway in mytilus galloprovincialis (lam.) mol. cell biochem. 260: 119-127, 2004. gonzalez-riopedre m, novás a, dobaño e, ramosmartínez ji, barcia r. effect of termal stress on protein expression in the mussel mytilus galloprovincialis lmk. comp. biochem. physiol. 147b: 531-540, 2007. gonzalez-riopedre m, barcia r, ramos-martínez ji. implication of pkc isozymes in the release of biogenic amines by mussel hemocytes: effect of pdgf, il-2 and lps. j. exp. zool. 311a: 727-734, 2009. gougou e, aggeli ik, beis i, gaitanaki c. hyperthermia-induced hsp70 and mt20 transcritional upregulation are mediated by p38mapk and jnks in mytilus galloprovincialis (lamarck); a prosurvival response. j. exp. biol. 213: 347-357, 2010. kefaloyianni e, gourgou e, ferle v, kotsakis, e, gaitanaki c, beis i. acute thermal stress and various heavy metals induce tissue-specific pro or anti-apoptotic evetnts via the p38-mapk signal transduction pathway in mytilus galloprovincialis (lam.). j. exp. biol. 208: 44274436, 2005. lacoste a, de cian mc, cueff a, poulet sa. noradrenaline and alpha-adrenergic signalling induce the hsp70 gene promoter in mollusk immune cells. j. cell sci. 114: 3557-3564, 2001. lacoste a, malham sk, gelèbart f, cueff a, poulet sa. stress-induced immune changes in the oyster crassotrea gigas. dev. comp. immunol. 26: 1-9, 2002. medzhitov r, janeway cajr. innate immunity: the virtues of a nonclonal system of recognition. cell 91: 295-298, 1997. novas a, cao a, barcia r, ramos-martínez ji. nitric oxide release by hemocytes of the mussel mytilus galloprovincialis lmk kwas provoked by interleukin-2 but not by lipopolysaccharide. int. j. biochem. cell biol. 36: 390-394, 2004. ottaviani e. is the distinction between innate and adaptative immunity in invertebrates still as clear-cut as throught?. ital. j. zool., 2011 [in press]. ottaviani e, caselgrandi e, franchini a, franceschi c. crf provokes the release of norepinephrine by hemocytes of viviparus ater (gastropoda, prosobranchia): further evidence in favour of the evolutionary hypothesis of the mobile immune-brain. biochem. biophys. res. commun. 193: 446-452, 1993. ottaviani e, franceschi c. the neurobiology of stress from invertebrates to man. progr. neurobiol. 48: 421-440, 1996.   58 http://www.ncbi.nlm.nih.gov/pubmed/14720463 http://www.ncbi.nlm.nih.gov/pubmed/14720463 http://www.ncbi.nlm.nih.gov/pubmed/14720463 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 isj 5: xx-yy, 2008 isj 5: 54-74, 2008 issn 1824-307x review immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art l ballarin department of biology, university of padua, padua, italy accepted may 29, 2008 abstract the phylogenetic position of invertebrate chordates closely related to vertebrates explains the increasing interest towards tunicate immunobiology. most of the tunicates are ascidians which, like all other invertebrates, rely only on innate immunity for their defense. compound ascidians differ from solitary species for the presence of colony specificity, i.e. the ability for intraspecific non-self recognition. the immunobiology of compound ascidians has been particularly studied in botryllus schlosseri, which is an emerging model organism for this kind of studies. in b. schlosseri and related species, immunocytes are represented by phagocytes and cytotoxic morula cells, the former able to ingest foreign cell and particles, the latter representing the effectors of the inflammatory reaction which follows the contact between genetically incompatible colonies. activated phagocytes release lectins with opsonic activity and are involved in the clearance of apoptotic cells during the colonial generational change. morula cells recognize the presence of foreign molecules as well as allogeneic soluble factors diffusing from an alien colony and as a consequence they: i) release cytokines in the medium which have chemotactic activity and activate phagocytes; ii) degranulate and release phenoloxidase which induces necrotic cell death by oxidative stress. a better knowledge of botryllus genome will allow a deeper insight into open problems in immunobiology of compound ascidians. key words: colonial ascidians; botryllus; immunobiology; immunocytes; allorecognition; phagocytosis introduction deuterostomes of the phylum chordata feature: i) the presence of a notochord, permanent or temporary, which prevents shortening of the body when longitudinal muscles contract; ii) a dorsal nerve cord, in the form of a hollow tube, enlarged to some extent at the front end to form a brain; iii) a ventral digestive tract, which expands anteriorly to form a pharynx, provided with gill slits or pharyngeal pouches and a ventral gland secreting iodoproteins (endostyle or thyroid). both the notochord and the neural tube extend to the tail, the muscular postanal part of the body. tunicates and cephalochordates, collectively named protochordates, represent the invertebrate relatives of vertebrata (schubert et al., 2006), the major chordate subphylum with nearly 50,000 species. unlike vertebrates, which radiated on lands, ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padua, via u. bassi 58/b, 35100 padova, italy email: loriano.ballarin@unipd.it freshwater and seawater and are active predators and/or grazers, protochordates are marine filterfeeding animals, most with a sedentary life-style. tunicates (or urochordates) owe their name to the tunic or test, the peculiar tissue which embeds the larval and adult body. although of epidermal origin, it resembles connective tissue in having an amorphous matrix in which tunicine fibres, similar to cellulose in composition, and interspersed ameboid cells are found. the results of a recent phylogenetic study, based on analysis of many nuclear genes, suggest that tunicates are the closest living relatives of vertebrates (delsuc et al., 2006), thus increasing interest towards these organisms. the majority of tunicates are represented by ascidians or sea squirts, sessile animals widespread all over the world with approximately 3,000 species, both solitary and colonial. today, the genome of some solitary ascidian species (ciona intestinalis, ciona savignyi, halocynthia roretzi) has been fully or partially sequenced (dehal et al., 2002; yokobori et al., 2003) which renders these organisms important 54 fig. 1 a: dorsal view of a b. schlosseri colony; b: colonial system showing buds and budlets; c: colony at generational change or take-over; d: schematic representation of the main stages of b. schlosseri colonial blastogenetic cycle. days from the beginning of the cycle are in brackets. a: ampullae; b: buds; bl: budlet; cs: cloacal siphon; os: oral siphon; s: system; t: tunic; tv: tunic vessel; z: zooids; bar = 1 mm. models in studying the molecular control of embryogenesis and differentiation (nishida, 2002a, b; oda-ishii et al., 2005; passamaneck and di gregorio, 2005; satoh and levine, 2005; dufour et al., 2006). immunity in ascidians the evolutionary relationships of tunicates with vertebrates explain the increasing interest in the immunobiology of ascidians. unlike vertebrates and like all invertebrates, tunicates rely only on innate immunity, characterized by neither somatic recombination nor long-term immune memory, low discriminative power, and a limited array of effector responses. nevertheless, precursors of vertebrate immune components have been described in ascidians, e.g. proteins of the lectin pathway of the complement activation system (nonaka et al., 1999; nair et al., 2000; nonaka, 2001; sekine et al., 2001; raftos et al., 2002; pinto et al., 2003), orthologues of the c-type lectin-like receptor cd94 (khalturin et al., 2003; zucchetti et al., 2007) and other genes involved in innate immunity (azumi et al., 2003; shida et al., 2003; oren et al., 2007) the immunobiology of solitary ascidians has been studied in the past, mostly in relation with inflammation, cytotoxicity, encapsulation, phagocytosis and allorecognition (parrinello et al., 1977, 1984, 1993; parrinello and patricolo, 1984; raftos et al., 1987a, b; raftos, 1991; peddie and smith, 1993, 1994; ohtake et al., 1994; cammarata et al., 1995; dan-sohkawa et al., 1995; lipari et al., 1995). this kind of research has been improved by the availability of the genomes of ciona and halocynthia (azumi et al., 2003; shida et al., 2003), which allowed to study the molecular control of immune responses. a review on the immunity of solitary ascidians is presented elsewhere in this journal. although less well studied at the molecular level, compound ascidians have some advantages with respect to solitary species. for instance, several developmental pathways (embryogenesis, blastogenesis, and regeneration) leading to adult, filter-feeding zooids may be compared in the same 55 organism and at various levels (morphological, biochemical, molecular). immunity in compound ascidians growing interest in the defense reactions of compound ascidians has been stimulated by the presence of colony specificity, i.e. the ability for intraspecific non-self recognition (allorecognition), and consequent attempts to understand the biological basis of the phenomenon, which frequently results in an inflammatory response (see below). most research has been carried out in botryllid ascidians, mainly in the cosmopolitan species botryllus schlosseri, which is emerging as a model organism for studying immunobiology (manni et al., 2007): i) it is easily found in the field; ii) it can grow and reproduce in laboratory conditions; iii) colonies are embedded in a soft and transparent tunic which allows direct observation of biological processes; iv) large colonies can be cut into clonal fragments which are able to grow and reproduce, so that subclones of the same colony can be used in control and experimental series; v) colonies undergo recurrent generation changes, with diffuse cell death by natural apoptosis in zooid tissues and efferocytosis by circulating phagocytes; vi) colonies have a well-defined tunic circulatory system and hemocytes can easily be collected with glass micropipettes after the tunic vessels have been punctured with a fine tungsten needle. this review, therefore, focuses mainly on this model organism as an important reference species. when available, various data from studies on other compound ascidians are reported. the colony of b. schlosseri three blastogenetic generations are usually present in a colony of b. schlosseri (fig. 1a): i) adult, filter-feeding zooids, 1.5-2 mm in length, are grouped in star-shaped systems of 10-12 individuals and oral siphons are located in the anterior part of each zooid (i.e. towards the periphery of the system), whereas the central cloacal siphon connects the cloacal chamber, into which individual atrial siphons open, with the exterior; ii) primary palleal buds on zooids are able to replace the parental generation, and iii) secondary buds (budlets) on buds grow to buds and, lastly, to zooids (fig. 1b). budding occurs continuously, in an orderly and synchronized way, as well as the cyclical change of adult generations or take-over (fig. 1c). one of the first report of budding and generational changes in botryllus was that of spallanzani in 1784 (in gibin, 1997). after that, botryllus blastogenesis was described by several researchers (metschnikow, 1869; della valle, 1882; oka, 1892; hjort, 1893; pizon, 1893; ärnbäck-christie-linde, 1923; berrill, 1941a, b; watterson, 1945; sabbadin, 1955a; milkman, 1967; izzard, 1973). the time interval between one generation change and the next is called a blastogenetic cycle: its length is temperature-dependent and lasts one week at 19 °c (fig. 1d). at take-over, old zooids close their siphons, cease filtering, contract (fig. 1c), undergo massive, diffuse apoptosis of their tissues (in parallel with some necrosis in the digestive tube), and are gradually resorbed by either wandering professional or fixed occasional phagocytes (burighel and schiavinato, 1984; lauzon et al., 1993, cima et al., 2003). they are replaced by primary buds, which open their siphons 24–36 h after the beginning of take-over; in the meantime, secondary buds become primary buds and give rise to a new budlet generation (sabbadin, 1955a, 1958; manni et al., 2007). therefore, the lifespan of a zooid, from its appearance as a budlet primordium to its resorption at take-over, covers approximately three weeks. as colonies cannot feed until the new adult generation opens its siphons, they rely on recycling of the components of dying zooids, which are used for growth of the developing buds (sabbadin, 1956; lauzon et al., 2002). each colony is a clone, as it derives from a founder oozoid, which is the outcome of the metamorphosis of a tadpole-like, swimming larva (fig. 2a) deriving from sexual reproduction. oozoids (fig. 2b) bear a palleal bud on their right side, which matures into a filter-feeding blastozooids (fig. 2c), replacing the parental zooid and producing two or more palleal buds on each side of the body, able to produce budlets and grow to mature blastozooids (sabbadin, 1969). zooids, buds and budlets are embedded in a common tunic and interconnected by fig. 2 swimming larva (a), oozooid (b) and first blastozooids (c) of b. schlosseri. a: ampullae; b: bud; bl: budlet; o: oozooid; z: blastozooids. bar = 0.5 mm. 56 a network of vessels of epidermal origin crossing the tunic and joined to a colonial marginal vessel which runs along the contour of the colony and gives rise to many sausage-like blind endings, known as ampullae, where blood cells are stored (brunetti and burighel, 1969; fig. 3). since adult zooids are cyclically resorbed and replaced by their growing buds, a healthy colony can be formed of hundreds or thousands of zooids and buds, synchronized in their development by the common vascular system. the development of buds and zooids in botryllus is highly coordinated (berrill, 1941a, b; watanabe, 1953; sabbadin, 1955a; milkman, 1967) so that the developmental stages of a colony during the blastogenetic cycle can be univocally defined by the developmental stages of its blastogenetic generations. stages more than one day from the preceding or the following take-over are called midcycle stages (lauzon et al., 1992). immunocytes of compound ascidians invertebrate immunocytes represent the key component of immunity, responsible for cellmediated immune reactions and, through their secretions, of most humoral responses. they are wandering cells, active in immunosurveillance which, in ascidians, represent a well-defined class of circulating hemocytes (ballarin and cima, 2005). like all other ascidian species, many types of circulating hemocytes are found in colonial ascidians. the morphology of both living and fixed cells has been widely described by many authors (pérès, 1943; sabbadin, 1955b; andrew, 1961; schlumpberger et al., 1984; cima et al., 2001; ballarin and cima, 2005), as well as their ultrastructure (overton, 1966; fujimoto and watanabe, 1976; milanesi and burighel, 1978; burighel et al., 1983; hirose and mukai, 1992; sugino et al., 1993; cima et al., 2001; hirose et al., 2003) and various attempts have been made to find unifying classification criteria (e.g. de leo, 1992; burighel and cloney, 1997). however, doubts still exist about their functions, mutual relationships, and differentiation pathways. in colonial botryllid ascidians, especially in the species botryllus schlosseri, hemocytes have been particularly investigated for their role in immunity (ballarin et al., 1993, 1995; cima et al., 1996, 2001; ballarin and cima, 2005). in this species, circulating hemocytes are grouped into three main categories: i) undifferentiated cells; ii) immunocytes; iii) storage cells (pigment cells and nephrocytes). immunocytes are represented by cytotoxic morula cells (mcs) and phagocytes, the latter including hyaline amebocytes and macrophage-like cells. morula cells mcs represent the most abundant circulating hemocyte type in botryllid ascidians, their frequency ranging from 20 to 60 %. they have a diameter of 10-15 µm, contain many vacuoles of uniform size, approximately 2 μm in diameter (fig. 4a), and, after treatment with aldehydes, assume a yellowish color and shrink (fig. 4b), thus conferring the typical fig. 3 schematic drawing of the tunic circulatory system of a b. schlosseri colony (modified after bunetti and burighel, 1969). a: ampullae; b: bud; z: zooid; t: tunic. berry-like morphology on fixed cells (sabbadin, 1955b; ballarin et al., 1993; ballarin and cima, 2005). the content of mc vacuoles has reducing properties, as demonstrated by its capability to reduce osmium, and shows positivity for peroxidase, phenoloxidase (po) and arylsulfatase (ballarin and cima, 2005). in addition, it can be stained with eosin (shirae et al., 2002), gives positive cytochemical reactions for polyphenols, quinones and dopacontaining proteins (ballarin et al., 1995; ballarin and cima, 2005) and is recognized by antibodies raised against tunichromes of solitary ascidians (ballarin, 2008). the precursors of mcs are probably granular amebocytes, with which they share similar cytochemical properties and enzymatic content (ballarin and cima, 2005). mcs are abundant inside the lumen of the ampullae of the colonial growing edge (cima et al., 2006a), have specific homing sites such as the lacunae inside the tentacles of the oral siphon (rinkevich et al., 1998; rinkevich, 2005), and their frequency inside the contacting ampullae increases abruptly in early stages of the non-fusion reaction (see below). mcs can sense foreign molecules and, on recognizing them, degranulate (fig. 4c) and release their vacuolar content (ballarin et al., 1995, 1998, 2005) consequently inducing cytotoxicity in neighbouring cells. this effect is directly related to the presence in the medium of active po, as demonstrated by the significantly lower cytotoxicity with respect to controls in the presence of po inhibitors such as sodium benzoate, phenylthiourea and dimethyldithiocarbamate (ballarin et al., 1998, 2005). b. schlosseri po has been purified and partially characterized: the monomeric form has a molecular 57 weight of 80 kda and can polymerize to larger complexes (frizzo et al., 1999). using a polyclonal antibody against the purified protein, it was possible to confirm the enzyme location inside mc vacuoles (frizzo et al., 2000). the observation that serine protease inhibitors can decrease po activity suggests that the enzyme is stored, at least partly, as a proenzyme (pro-po) which, as in arthropods, is converted to active po through the removal of a short peptide by serine proteases (söderhäll and cerenius, 1998). polyphenol substrata are probably represented by tunichromes, small peptides containing dopa or topa (oltz et al., 1987; smith et al., 1991), present inside ascidian mcs (dorsett et al., 1987; azumi et al., 1990; ballarin et al., 1995; taylor et al., 1995;. bayer et al., 1997). they are probably present in a masked form inside mc vacuoles, with sulfates, also revealed in mcs, bound to the aromatic ring, and made available to po by the action of the enzyme arylsulfatase which detaches sulfates from phenols (ballarin et al., 1995). phagocytes in all metazoans, phagocytes can recognize and ingest foreign cells or particles entering the organism, thus ensuring their clearance. in b. schlosseri, circulating professional phagocytes are represented by hyaline amebocytes (fig. 4d) and macrophage-like cells (fig. 4e), which represent two diverse morphologies of the same hemocyte type (sabbadin, 1955b; ballarin et al., 1993). the former represent cells active in phagocytosis which, upon ingestion, withdraw their pseudopods and turn to the globular morphology of macrophage-like cells (ballarin et al., 1993, 1994; ballarin and cima, 2005). the two hemocyte types have similar cytochemical properties and common contents of lysosomal and fig. 4 b. schlosseri hemocytes. a, c: living mcs incubated in the absence (a) or in the presence (c) of foreign molecules or cells (from ballarin et al., 2005); b: aldehyde-fixed mcs; d, e: fixed b. schlosseri phagocytes (from menin and ballarin, 2006). d: hyanine amebocyte; e: macrophage-like cell. bar = 10 µm. antioxidant enzymes (such as phosphatases, 5’nucleotidase, β-glucuronidase and esterases), share the same surface carbohydrates, as demonstrated by their labelling with narcissus pseudonarcissus agglutinin, and are immunopositive to anti-cd39 antibody (ballarin and cima, 2005). similar results were obtained in botrylloides leachi (cima et al., 2001). the release of acid phosphatase in the medium has been observed in in vitro phagocytosis by b. schlosseri hemocytes (cima et al., 1996), due to leakage of the enzyme by phagocytes during phagosome formation (davies and bonney, 1980). tunic and external immunocytes the ascidian tunic contains numerous cells deriving from the hemocytes which leave the circulation and colonize the test matrix (burighel and cloney, 1997). a common tunic cell type is represented by spreading cells or amebocytes which closely resemble circulating hyaline amebocytes. in b. schlosseri three distinct types of cells were recognized: fusiform, fibrocytic and vacuolated cells, the latter deriving from morula cells migrated through the epidermis into the tunic (zaniolo, 1981). a role in immune defense has been postulated for some of these cells, confirmed by the observation that, in both solitary and colonial ascidians, they have phagocytic activity (smith, 1970; hirose et al. 1994a; burighel and cloney, 1997). in addition, as demonstrated in solitary ascidians, hemocytes can infiltrate the tunic in response to the presence of non-self cells or particles, where they contribute to form a capsule (parrinello and patricolo, 1984). in botryllid ascidians, massive infiltration of immunocytes into the tunic is associated with the non-fusion reaction between contacting, genetically incompatible colonies (see below). in the colonial ascidian b. schlosseri, we recently reported the presence of amebocytes, completely exposed to seawater, attached to the tunic of siphonal regions. these cells share many cytochemical features with circulating phagocytes, respond to the same lectins and antibodies used as markers of the phagocytic cell line, and can also engulf carmine particles. all these results suggest that these cells guard the entrances to the branchial and atrial chambers, and trigger a systemic defense response toward non-self particles or cells (cima et al., 2006b). non-self recognition phagocytosis invertebrate phagocytes recognize non-self molecular patterns through a series of poorly known receptors. in b. schlosseri, indirect evidence suggests the involvement of a mannose receptor in yeast recognition (ballarin et al., 1994); in addition, yeast-matched hemocytes express molecules cross-reacting with antibodies raised against mammalian toll-like receptor (tlr)-2 and -4, located in phagocytes (menin and ballarin, 2007), which suggests the involvement of tlrs in the recognition of foreign cells. vertebrate phagocytosis implies a zipper-like mechanism involving a close adhesion, mediated by 58 integrins, of phagocyte projections with the surface of foreign particles or cells (griffin et al., 1975a, b) in b. schlosseri, the interaction with yeast cells requires the activation of phagocyte integrins after recognition of the arg-gly-asp (rgd) motif on the surface of target particles, for both pseudopod formation and cell spreading, as suggested by the fact that the soluble antagonist tetrapeptide arg-glyasp-ser (rgds) significantly inhibits both yeast phagocytosis and cell spreading and disrupts cytoskeletal organization (ballarin et al., 2002a). in b. schlosseri, the occurrence of the respiratory burst after the interaction of phagocytes with non-self particles has been clearly documented in the case of yeast phagocytosis, in which an increase in the production of cytotoxic superoxide anions, peroxides and hypochlorite by phagocytes has been reported (ballarin et al., 1994; cima et al., 1996). an increase of nitrite in the culture medium was also observed when hemocytes were incubated with yeast cells (cima et al., 1996), suggesting that, as in vertebrates (hibbs et al., 1987), the activation of inducible nitric oxide synthase (nos) in botryllus phagocytes occurs on recognition of foreign cells and, as a consequence, nitric oxide (no) with microbicidal activity is produced. this hypothesis may be strengthened by the immunocytochemical assay on phagocytes with anti-nos antibodies. non-self recognition by b. schlosseri phagocytes triggers various signal transduction pathways. the observation that h89 and calphostin, inhibitors of protein kinase a (pka) and protein kinase c (pkc), respectively, can decrease the ingestion of yeast cells by phagocytes (menin and ballarin, 2006), suggests the involvement, in phagocytosis, of transduction pathways activated by camp and diacylglycerol, respectively, and controlled by trimeric g proteins (gomperts et al., 2002). pkc activation follows activation, by trimeric g proteins, of a membrane-associated phospholipase c which produces inositol triphosphate (ip3) and diacylglycerol (dag), the former increasing the cytosolic ca2+ concentration, the latter acting on pkc. a transient rise in cytosolic ca2+ concentration is required for ingestion to occur, as demonstrated by the inhibition of phagocytosis in the presence of edta or when hemocytes and target cells are incubated in the presence of either thimerosal, which depletes intracellular calcium stores, or inhibitors of the calmodulin-dependent ca2+-atpase, such as thapsigargin, pimozide or tributiltin (tbt) chloride, which cause a sustained increase in cytosolic ca2+ concentration (cima et al., 1995; ballarin et al., 1997; cima and ballarin, 2000). manumicin, an inhibitor of monomeric g proteins, decreases the fraction of in vitro phagocytosing cells, indicating the involvement of these proteins, also suggested by the expression, revealed by immunohistochemical and immunoblot analysis, of molecules recognized by anti-pan-ras antibodies in yeast-matched phagocytes (menin et al., 2007). we recently demonstrated that various mapk inhibitors, such as pd98059 (erk pathway), sp600125 (jnk pathway) and sb202190 (p38 pathway) can decrease the frequency of yeast-ingesting hemocytes, and an increase in the expression of various activated mapks, such as p38, erk, sapk/jnk, was observed in immunoblot analysis of yeast-matched hemocyte lysates (menin et al., 2007). phosphatidylinositol-3-kinase (pi3k) is also involved in phagocytosis, and its role seems to be related to the cytoskeletal re-organization required for pseudopod formation (ballarin et al., 2002a). immunopositivity to anti-nf-kb antibodies is located in the cytoplasm of unstimulated phagocytes, whereas it migrates into the nucleus of yeastactivated cells (ballarin, 2008) suggesting the involvement of this transcription factor in phagocytosis. efferocytosis, i.e. phagocytosis of apoptotic cells (de cathelineau and henson, 2003; gardai et al., 2005), occurs massively during take-over and is performed by professional, circulating phagocytes massively recruited in the senescent tissues (fig. 5a), and by occasional phagocytes such as cells of the digestive epithelium (tiozzo et al., 2006). during the generational change, there is a significant increase in the frequency of circulating macrophage-like cells with respect to mid-cycle stages (fig. 5b), paralleled by a decrease in the fraction of circulating hyaline amebocytes (fig. 5c) (cima et al., 2003; ballarin et al., 2008), matching the hypothesis that the two hemocyte types represent different stages of the same cell. as a result of intense phagocytosis, there is a significant increase in the fraction of hemocytes showing positivity for acid phosphatase which, as stated above, is a phagocyte marker, and in both the activity of acid phosphatase and the concentration of peroxides in the blood plasma (cima et al., 1996). botryllus phagocytes recognize phosphatidylserine on the surface of effete cells and corpses, as soluble phospho-l-serine can inhibit their ingestion. in addition, clearance of senescent cells requires the expression of molecules recognized by antimammalian-cd34 antibodies on the phagocyte surface, the expression of which increases at takeover (cima et al., 2003). phagocytes of b. schlosseri are also capable of constitutive macropinocytosis, a process generally responsible of the ingestion of bacteria and necrotic material (krysko et al., 2003), at sites of membrane ruffling along their leading edge. this activity is enhanced by the presence, on the substrate, of molecules containing the rgd motif, such as fibronectin or fibrinogen. this suggests that, as in mammals (meier et al., 2002), integrins can also regulate macropinocytosis. the increase in fluidphase endocytosis is associated with a rise in both oxygen consumption, as indicated by the higher production of reactive oxygen species, and increased cytochrome oxidase activity, related to the synthesis of atp (ballarin and burighel, 2006). allorecognition as stressed by buss (1987), clonal organisms are characterized by colony specificity or allorecognition, i.e. the ability for intraspecific nonself recognition. colony specificity of botryllid ascidians has been known since the pioneer observations by bancroft (1903). it was further investigated in japanese species (oka and watanabe, 1957a, 1960; oka, 1970; mukai and watanabe, 1974) and in b. schlosseri (karakashian 59 fig. 5 a: tissues of senescent zooid during take-over. infiltrated macrophage-like cells having ingested apoptotic cells and corpses are marked by asterisks. bar = 15 µm. b, c: frequencies of circulating macrophage-like cells (mlc) and hyaline amebocytes (ha) in 4 colonies, at mid-cycle (red bars) and take-over (yellow bars). significant differences between mid-cycle and take-over in the same colony are marked by asterisks. *p < 0.05; **p < 0.01; ***p < 0.001. and milkman, 1967; sabbadin, 1962). colony specificity manifests itself as either fusion or nonfusion of genetically compatible or incompatible colonies, respectively, contacting each other at their growing edges. the phenomenon has been studied in 14 species of botryllid ascidians (sabbadin, 1962, 1982; tanaka, 1975; taneda et al., 1985; rinkevich, 1992, 2005; saito et al., 1994; hirose, 2003), particularly in the japanese species botryllus primigenus and the cosmopolitan species b. schlosseri, where the outcome of the contact is genetically controlled by a highly polymorphic fu/hc gene, the alleles of which are co-dominantly expressed (oka and watanabe, 1957a, 1960; sabbadin, 1962; oka, 1970). in b. schlosseri, the fu/hc locus also controls the vascularization and the completion of development of secondary buds grafted from a colony in the tunic of an alien colony, which occur only if the two colonies are fusible (sabbadin, 1982; sabbadin et al., 1991). the great majority of botryllid colonies in nature are heterozygous at the fu/hc locus and fusion occurs when at least one allele is shared by the two colonies, otherwise non-fusion is observed. fusion implies the formation of a chimeric colony, with anastomosis of tunic and circulation (katow and watanabe, 1980; zaniolo et al., 2006); non-fusion requires a partial fusion of the facing tunics with the local disappearance of the limiting cuticles (tanaka and watanabe, 1973; taneda et al., 1985; sabbadin et al., 1992; saito et al., 1994; hirose, 2003; rinkevich, 2005; zaniolo et al., 2006) and is usually characterized by the appearance of a series of cytotoxic foci (rejection reaction), called points of rejection, along the contact border (oka and watanabe, 1957a, 1960; sabbadin, 1962; oka, 1970; rinkevich, 1992, 2005). the growth of contacting ampullae to unusually large size (megaloampullae) during allorecognition has been reported in botrylloides leachi (rinkevich et al., 60 1994; zaniolo et al., 2006). as a consequence of the non-fusion reaction, a change in vectorial growth of the contacting colonies (retreat growth) occurs (rinkevich and weissman, 1988). both cells and soluble factors are involved in botryllus allorecognition. in b. primigenus and b. schlosseri, the partners of a chimeric colony separated after a fusion of at least four days show altered fusibility (mukai, 1967; sabbadin and astorri, 1988). in b. schlosseri, sabbadin and astorri (1988) demonstrated that this alteration is related with the persistence, in a colony, of cells from the partner with which it was previously fused. tanaka (1973, 1975), working with colonies of b. primigenus of defined genotype at the fu/hc locus, showed that fusion conferred to the chimera ac-bc the ability to reject a colony bd when contacting the bc partner. the fusibility of b. primigenus colonies was altered by the previous exposure to x-rays which reduces circulating undifferentiated cells, stressing the role of blood cells in allorecognition (taneda and watanabe, 1982c). non-fusion reaction is irreversible after the fusion of the tunic cuticles occurred, even if one colony is removed after the contact, suggesting the involvement of diffusible (circulating) humoral factors from one colony to the other through the fused tunics (tanaka, 1975; watanabe and taneda, 1982). this is supported by the observation that, in early stages of non-fusion reaction, an increase in permeability of the ampullar epithelium occurs (taneda and watanabe, 1982a) and that the reaction can be mimicked by the injection of whole blood and blood plasma from a colony into the vessels of a genetically incompatible host (taneda and watanabe, 1982b; saito and watanabe, 1984). the observation of a simultaneous occurrence of fusion and non-fusion reactions between contacting colonies fits the hypothesis of the recognition of soluble histocompatibility factors diffusing from one colony by effector hemocytes of the facing partner (taneda, 1985). humoral factors involved in the non-fusion reaction have been partially characterized from blood plasma of botrylloides simodensis: they are resistant to dyalisis heat-labile and require divalent cations for their activity (saito and watanabe, 1984). in b. primigenus and b. schlosseri, the non-fusion reaction shares many characteristics with vertebrate inflammation, such as cell recruitment by chemotaxis, extravasation, cell degranulation, and induction of cytotoxicity. as stated before, it is preceded by partial fusion of the contacting tunics, in front of the facing marginal ampullae, which allows the diffusion of soluble factors from one colony to the next. this event induces selective crowding of mcs inside the ampullae of the growing edge, their migration through the epithelium of the ampullar tips into the tunic, and their final degranulation, with the consequent release of their vacuolar content (taneda and watanabe, 1982a; sabbadin et al., 1992; saito et al., 1994; rinkevich et al., 1998; cima et al., 2006c), in particular, the enzyme po and its polyphenol substrata, which contribute to inducing the observed cytotoxicity (fig. 6; ballarin et al., 1995, 1998; cima et al., 2004, 2006c). analogous behavior, although restricted to a very limited region in front of the facing ampullae (subcuticular rejection), has been described in b. simodensis, botrylloides fuscus and botrylloides violaceus (hirose et al., 1988, 1990, 1997; shirae et al., 2002) and b. leachi (zaniolo et al., 2006; ballarin and zaniolo, 2007). in b. schlosseri, mc degranulation and the induction of cytotoxicity may be mimicked in vitro by exposing hemocytes to the blood plasma of incompatible colonies (ballarin et al., 1995; cima et al., 2006c) and prevented by the above-reported po inhibitors (ballarin et al., 1998). cytotoxicity is related to the induction of oxidative stress, as indicated by the reduction of nitroblue tetrazolium when added to incompatible blood plasma in in vitro assays, and the observation that scavengers of reactive oxygen species, such as superoxide dismutase, catalase and sorbitol, can suppress cell death both in vitro, when hemocytes are incubated with incompatible blood plasma, and in vivo, in colony allorecognition, although they have no effects on mc degranulation or po activity (ballarin et al., 1998, 2002b). the role of no in the induction of cell death is suggested by the in vitro production of nitrite ions when hemocytes are exposed to nonself molecules and the decrease of in vitro cytotoxicity in the presence of the nos inhibitor nωnitro-l-arginine methyl ester (cima et al., 2004). the necrotic masses at rejection points share several chemical and cytochemical properties with mc content (ballarin et al., 1995), thus strengthening the assumption that mcs infiltrated into the tunic play a major role in their formation. various properties of the pigment deposited at rejection points are also indicative of its melanic nature (ballarin et al., 1995), and melanins are the end-product of po activity in metazoans (waite, 1992). despite the well-defined role of mcs as effectors of non-fusion reactions in most botryllid ascidians, even phagocytes take part in rejection reactions: i) in b. schlosseri, fusion between allogeneic colonies, sharing only one allele at the fu/hc locus, usually leads to the resorption of one of the partners during take-over of one of the blastogenetic cycles of the chimeric colony following fusion (rinkevich and weissman, 1987, 1992; sabbadin and astorri, 1988; rinkevich, 2002, 2005). colony resorption, therefore, mimicks zooid resorption at take-over, implying the involvement of phagocytes in the elimination of tissues of the “losing” partner; ii) a more direct role of phagocytes in allorejection has been described in botryllus scalaris, which is the only botryllid species reported so far in which phagocytes and not mcs are involved in allorecognition between contacting, incompatible colonies. in this case, the rejection reaction starts after fusion of the ampullae of the facing growing edges and the beginning of blood exchange through the fused vessels. phagocytes crowd inside the fused ampullae and stimulate the aggregation of hemocytes into large clusters which are finally encapsulated by other phagocytes. in this way, hemocyte clusters plug the lumen of the fused ampullae and blood flow is interrupted in a few 61 fig. 6 schematic representation of the rejection reaction in b. schlosseri. for sake of simplicity, the main steps are indicated only on the left colony. 1: fusion of the contacting tunics after the local disappearance of the cuticles; 2: diffusion of histocompatible factor(s) through the fused tunics; 3: recognition of alien factors by mcs inside the tips of ampullae; 4: release of cytokines by activated mcs enhancing recruitment; 5: extravasation of mc and their degranulation in the tunic; 6: release of po; 7: cytotoxicity and melanin formation at points of rejection. modified after hirose (2003). minutes. differently from other botryllid species studied so far, no signs of selective recruitment or degranulation of mcs were observed (shirae et al., 1999). when incompatible colonies of botryllid ascidians are artificially brought into contact at their cut surfaces (cut surface allorecognition assay), an intense rejection reaction is observed in ovoviviparous species (rinkevich, 1992, 2005; saito et al., 1994; hirose, 2003; zaniolo et al., 2006; ballarin and zaniolo, 2007). however, fusion of tunics and blood vessels (surgical fusion) always occurs in the case of the viviparous species studied so far, suggesting that the hemocytes of these species have lost the ability for allorecognition, which persists in tunic cells (saito et al., 1994; hirose et al., 1994b; okuyama et al., 2002; hirose, 2003; rinkevich, 2005). this may be in relation with the necessity to prevent the immune system from attacking the brooded embryos which shares only one allele at the fu/hc locus with the mother colony and might undergo rejection/resorption as they are exposed to the circulation for more than a week (hirose, 2003). in partial confirmation of the above statement, the po activity of the hemolysate of viviparous species is much lower than that of ovoviviparous ones (shirae and saito, 2000; okuyama et al., 2002). humoral factors lectins it is well-known that ascidian hemolymph contains lectins, often revealed by their 62 hemagglutinating activity (hence the name hemagglutinins), with various carbohydrate specificities (vasta et al., 1982; coombe et al., 1984a; parrinello, 1995). a role in immune defense has been postulated for some of them, but clear involvement in cell proliferation after non-self recognition or in the modulation of phagocytosis has been demonstrated in a few cases (coombe et al., 1984b; kelly et al., 1992; pearce et al., 2001). in compound ascidians, the presence of soluble agglutinins has been reported in various species of the genera amaroucium, aplidium, botrylloides, botryllus, didemnum, diplosoma, clavelina and polyandrocarpa (vasta et al., 1982, 1986; coombe et al., 1984a; suzuki et al., 1990; parrinello, 1995). in b. schlosseri, we demonstrated that yeastactivated phagocytes can synthesize and release, through apocrine secretion, lectins with specificity for β-galactosides, which can agglutinate rabbit erythrocytes and yeast cells (ballarin et al., 1999; fig. 7) and which were previously thought to be members of the galectin family on the basis of their ca2+-independence (ballarin et al., 2000). polyclonal antibodies against these molecules recognize the surface of erythrocyte or yeast cells clumped by exposure to affinity-purified lectins (ballarin et al., 1999). these lectins can improve yeast phagocytosis by acting as opsonins and promoting interactions between target cells and phagocytes (ballarin et al., 1999, 2000). recently, in a full-length cdna library from botryllus colonies, we identified five transcripts homologous to known rhamnose-binding lectins (rbls). their predicted amino acid sequences exactly match the sequences of the tryptic fragments previously obtained from the above agglutinins purified by affinity chromatography. they probably represent different isoforms of a novel rbl, called b. schlosseri rbl (bsrbl), with a molecular weight of approximately 11 kda. they contain the eight cysteines which characterize rbls, form four disulfide bonds, and have a single carbohydrate recognition domain; through non-covalent interactions they can form multivalent complexes able to act as bridges between target particles and the phagocyte surface and enhance phagocytosis (gasparini et al., 2008). antiviral, antimicrobial and antitumoral factors various bioactive molecules, with antiviral or cytotoxic activities against microbial or tumoral cells have been described in compound ascidians. most of these compounds were isolated from didemnid ascidians. ulithiacyclamide and ulicyclamide are cyclic peptides from lissoclinum patella showing antineoplastic and antiviral activity (ireland and sheuer, 1980; ireland et al., 1982; wasylyk et al., 1983). in the same species, a lactone, named lissoclinolide, with antibacterial and antitumoral activity has been isolated (davidson and ireland, 1990; richardson and ireland, 2004). lissoclibadins are cytotoxic and antimicrobial alkaloids from the indonesian lissoclinum cf. badium (nakazawa et al., 2007). didemnins and eudistomins are antiviral molecules isolated from whole extracts of colonies of trididemnum sp. and eudistoma olivaceum, respectively. the former are cyclic peptides, able to inhibit the replication of various rna and dna viruses, fig. 7 agglutination of rabbit erythrocytes (a) and yeast cells (c) in the presence of purified bsrbl. immunostaining with polyclonal anti-bsrbl antibodies reveal the presence of the lectin on the surface of clumped erythrocytes (b) or yeast cells (d). bar = 10 µm. which also exert antitumor activity (rinehart et al., 1981a, b), the latter are β-carboline derivatives containing bromine, active against herpex simplex virus (rinehart et al., 1984). lepadins d, e and f are decahydroquinoline derivatives from didemnum sp. with antiplasmodial and antitrypanosomal activity (wright et al., 2002). anticancer hydroquinones derivatives with cytotoxic properties have been described in aplidium californicum (howard and clarkson, 1979; cotelle et al., 1991). in the mediterranean aplidium albicans, the cytotoxic peptide aplidin induces perturbations of the cell cycle and apoptosis of human leukaemia cells (erba et al., 2002). another antitumoral peptide, vitilevuamide, isolated from didemnum cuculiferum and polysyncraton lithostrotum, was able to inhibit tubulin polymerization required for the organization of the mitotic spindle (edler et al., 2002). metabolites inducing apoptosis of human cancer cells were recently isolated from the japanese ascidian diplosoma virens (ogi et al., 2008). the enzyme po, as stated above, is also involved in preventing microbial infections through its cytotoxic activity and b. schlosseri mcs degranulate and release cytotoxic active po in response to the recognition of yeast cells or bacterial spores (ballarin et al., 2005). cytokines the presence of soluble immunomodulatory molecules, cross-reacting with antibodies raised against mammalian il-1, has been reported in ascidians since the pioneering work by beck et al. (1989). b. schlosseri and a member of the genus didemnum were among the tunicate species investigated, showing the presence of lymphocyte activation factors, the activity of which was neutralized by anti-il-1 antibodies. 63 in b. schlosseri, mcs are the main source of molecules recognized by antibodies raised against the mammalian pro-inflammatory cytokines il-1-α and tnf-α: when hemocytes are exposed to nonself molecules, such as mannan or incompatible blood plasma, or particles, such as yeast cells or microbial spores, mcs acquire immunopositivity to the above antibodies (ballarin et al., 2001, 2005). immunopositive mcs are also observed inside the facing ampullae of contacting incompatible colonies (cima et al., 2004) in early stages of the non-fusion reaction. in b. leachi, mcs are recognized by anti-il-1-α antibodies, whereas positivity to anti-tnf-α antibodies has been unexpectedly observed in another type of hemocytes which, according to cima et al. (2001), were classified as granular cells (ballarin and zaniolo, 2007). in b. schlosseri, the above antibodies inhibit both the rise in cell death observed when hemocytes are incubated with incompatible blood plasma (cima et al., 2004) and mc chemotaxis (cima et al., 2006c), suggesting that the recognized molecules stimulate both the recruitment of mcs inside the tips of facing ampullae and their degranulation with the consequent release of po (fig. 7). the supernatants from cultures of b. schlosseri hemocytes, matched with non-self particles such as yeast cells or zymosan (conditioned media) enhance the phagocytosis of yeast cells (menin et al., 2005). these effects were abolished by the addition of anti-il-1-α or antitnf-α antibodies, suggesting that molecules recognized by the above antibodies are involved in immunomodulation and may be considered as cytokines in the broad sense of the term. using cell fractionation by density gradient centrifugation in ficoll 400, we obtained four hemocyte bands, with different immunocyte distribution, from which we prepared four different conditioned media, one for each band. in this way, we could demonstrate that the enhancing effect on phagocytosis was present in conditioned media derived from hemocyte bands enriched in mcs, but that it was not present in conditioned media from bands rich in phagocytes (fig. 8), in agreement with previous results indicating mcs as the source of molecules crossreacting with anti-mammalian cytokine antibodies (menin et al., 2005). immunoblot analysis of the supernatant from zymosan-matched hemocytes showed a 60 kda band cross-reacting with both anti-il-1-α and antitnf-α. in addition, a 37 kda band, recognized by anti-bsrbl antibodies, was detected in the above conditioned media, and the presence of the lectins was confirmed by hemagglutinating assay (menin and ballarin, 2008). all the above results fit a scenario in which mcs are the main organism sentinel cells which sense non-self molecules, being recruited as a consequence of the recognition and, on the basis of the nature of the foreign molecules, they are able to trigger a cytotoxic response or stimulate phagocytes to ingest foreign cells and release agglutinins which potentiate their activity. immunity and xenobiotics b. schlosseri is one of the most diffuse encrusting organisms in the lagoon of venice, where it characterizes the relative climax of the ecological succession of hard-substratum macrobenthos (cima et al., 2006c). as colonial ascidians have been reported to be very susceptible to antifoulants (henderson, 1986), we studied the effects of short-term exposure of b. schlosseri hemocytes to biocides such as organotin compounds (tributyltin, triphenyltin) and new organic compounds used in antifouling paint formulations after the ban on tin-based antifoulants, on cell functions in order to reveal the cellular targets of these molecules (cima et al., 1995, 1997, 1998, 2008; cima and ballarin, 1999, 2000, 2004). organotin compounds alter the morphology of phagocytes, their capability to ingest target yeast cells, and to induce the respiratory burst in a dosedependent manner, and these changes are related to disruption of cytoskeletal components (cima and ballarin, 2000, 2008; cima et al., 1995, 1997, 1998). tributyltin can interact with calmodulin and alter the activity of calmodulin-dependent ca2+-atpase (cima et al., 2002a) or react directly with cytosolic thiols (cima and ballarin, 2004). as a consequence, they alter cytosolic calcium and thiol homeostasis which cause the observed alterations in cell morphology (cima et al., 1995, 1997), inhibition of hydrolytic, detoxifying and mitochondrial enzymes (cima et al., 2002b), and eventually lead to apoptosis (cima and ballarin, 1999), probably due to the severe oxidative stress following the reduction of cytosolic thiols (cima et al., 2004). among the new antifoulants, we assayed seaninetm (4,5 dichloro-2-n-octyl-4-isothiazoline-3-one), chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and tcms pyridine (2,3,5,6-tetrachloro-4(methylsulfonyl)pyridine). both sea-ninetm and chlorothalonil have a negative effect on the phagocyte cytoskeleton, altering cell morphology and severely hindering phagocytosis. both compounds decrease intracellular reduced glutathione, inducing oxidative stress which is probably the cause of the observed cell death by apoptosis (cima et al., 2008), and perturb the mitochondrial respiratory chain, whereas only sea-ninetm disrupts cytosolic calcium homeostasis (cima et al., 2008). diuron and tcms pyridine inhibit phagocytosis and cell spreading in a dose-dependent manner, suggesting cytoskeletal alteration, and can induce apoptosis at the higher concentrations assayed (100 and 20 μm, for diuron and tcms pyridine, respectively). the two biocides did not have any negative effect on esterase or cytochrome-c oxidase activities, or on the homeostasis of cytosolic calcium (menin et al., 2008). conclusions and perspectives today, the study of ascidian immunobiology is a topical subject and it is a widespread opinion among scientists that protochordates can greatly 64 fig. 8 density gradient centrifugation of b. schlosseri hemocytes. a: enriched fractions of cells obtained after centrifugation in discontinuous gradient of ficoll 400. b: concentration of mcs (yellow bars) and phagocytes (brown bars) in each fraction. c: percentage of phagocytizing cells in hemocyte cultures exposed to a suspension of yeast cells in seawater (control) or in the supernatants from cultures of hemocytes of each band obtained after density gradient centrifugation; significant differences with respect to the control are marked by asterisks. *p < 0.05; **p < 0.01. modified after menin et al. (2005). contribute to answering the still unresolved problems of the origin of the highly sophisticated and complex vertebrate immune system. among compound ascidians, b. schlosseri is diffuse and studied worldwide, and represents an optimal animal model for immunobiological studies, despite the size of its zooids, thanks to good knowledge of the biology of its immunocytes (manni et al., 2007). in addition, this kind of study has been frequently motivated by interest in understanding the cellular basis of allorecognition. as in other invertebrates, immune responses in ascidians rely mainly on circulating immunocytes which act as the effector arms of immunity, and can ingest or kill non-self cells or release opsonins and immunomodulatory molecules which enhance or reinforce cellular responses. as reported, two distinct immunocyte differentiation lines are present in botryllid ascidians: phagocytes and cytotoxic, phenoloxidase-containing morula cells. nevertheless, despite the abundance of data gathered in the last two decades, unlike solitary ascidians of the genus ciona and halocyntyhia (dehal et al., 2002; yokobori et al., 2003), compound ascidians and b. schlosseri in particular still lack an adequate molecular approach to the study of their genomes. few genes other than the bsrbl genes reported above (gasparini et al., 2008), which might be involved in non-self recognition, have been isolated. examples are a cd94 orthologue of the vertebrate cd94 receptor on nk cells, expressed on a subset of b. schlosseri blood cells, probably phagocytes, mainly in the contacting ampullae (khalturin et al., 2003), and genes for putative homologue of c-type lectin (pancer et al., 1997), human eb1 (pancer et al., 1996a), fk506-binding protein (pancer et al., 1993), vertebrate receptor for antigens (pancer et al., 1996b) and hsp70 (fagan and weissman, 1996). recently, a subtraction cdna library of contacting, genetically incompatible colonies of b. schlosseri allowed the identification of more than 100 genes differentially expressed and related to immunity (oren et al., 2007). for deeper insight into the immunobiology of compound ascidians, better knowledge of the genome of these organisms, at least of botryllus, on which many of the studies have been concentrated, is required. in addition, there are still some open questions regarding basic biological processes related to immunity, which represent good challenges for future research on compound ascidians. some of them are listed below. origin and differentiation hemocytes (and immunocytes) ascidian hemocytes derive from the embryonic mesenchyme (cowden, 1968; sala, 1973; sabbadin et al., 1999). undifferentiated cells are known as hemoblasts and are characterized by high nucleocytoplasmic ratio, a well-defined nucleolus and a basophilic cytoplasm whereas the term lymphocyte has been used either as a synonymous of hemoblast or to indicate a cell type thought to represent an immature differentiating hemocyte (pérès, 1943; sabbadin, 1955b; ermak, 1976; kawamura et al., 1988). however, the real nature of the lymphocyte and the differentiation pathways of hemocytes are not clear and require further investigations. it is known that, in b. schlosseri, almost 30 % of hemocytes die by apoptosis at take-over and are replaced by new hemoblasts entering the circulation 65 at the same stage of the blastogenetic cycle (ballarin et al., 2008). however, their origin is still unknown as no structures comparable to the branchial hemopoietic nodules, reported by ermak (1977) in solitary species, have been described in compound ascidians. in certain experimental conditions, mitosis figures have been observed in circulating hemocytes of (cima and ballarin, 2007). undifferentiated blood cells are directly involved in asexual reproduction by stolonal (freeman, 1964) or vascular budding (oka and watanabe, 1957b). the latter occurs spontaneously in b. primigenus (oka and watanabe, 1957b) and in dormant colonies of b. leachi (burighel et al., 1976) and can lead to the re-building of a whole colony from small colony fragments, in botrylloides violaceus (oka and watanabe, 1959) and b. leachi (rinkevich et al., 2007), or from the colonial matrix deprived of zooids and buds, in b. schlosseri (milkman, 1967; sabbadin et al., 1975). both blood and epithelial cells can be cultured in vitro in appropriate conditions, and this represents an interesting tool in studying cell differentiation (sala, 1973; rinkevich and rabinowitz, 1993, 1994; rabinowitz and rinkevich, 2003). allorecognition: old problems, new questions i) some japanese authors, in comparing the non-fusion reactions of various botryllid ascidians, hypothesized the key role of tunic cells and the epithelium of the facing ampullae in allorecognition (taneda et al., 1985; saito et al., 1994; hirose, 2003). the fu/hc gene of b. schlosseri has recently been characterized (de tomaso et al., 2005), as well as that of the putative receptor involved in allorecognition (nyholm et al., 2006). the receptor is expressed in facing ampullae: intense labelling is observed in the epithelium of the ampullar tips but not in mcs (nyholm et al., 2006). in addition, the epithelium of the ampullar tips express high levels of bs cadherin in early stages of the non-fusion reaction (rosner et al., 2007). indeed, the ampullar epithelium plays a fundamental role in botryllus allorecognition in allowing the diffusion of non-self factors from the alien colony into the ampullar lumen, where they can alert mcs. during the non-fusion reaction, it is highly fenestrated and increases its permeability in both b. primigenus and b. schlosseri (taneda et al., 1982a; sabbadin et al., 1992), which suggests that it perceives signals which are absent in the fusion reaction. as all these events precede mc activation, represent prerequisites for the following inflammatory reaction and are worthy of study. ii) in botryllid ascidians, in addition to allorecognition, the fu/hc gene also controls the recognition between sperm and egg. it has been reported that the sperm of the japanese species botryllus primigenus cannot fertilize eggs when it shares a fu/hc allele with the diploid, maternallyderived, egg envelope (oka, 1970; saito et al., 1994). this limitation also seems to exist in some populations of b. schlosseri (scofield et al., 1982) but not in others (sabbadin, 1971, 1982; grosberg 1987). however, at least in the population from the lagoon of venice, high levels of inbreeding depression have been reported (sabbadin, 1971). this offers the possibility of investigating relationships among allorecognition, fertilization and inbreeding depression, and their implications in the ecology of natural populations. iii) as stated before, in laboratory conditions, resorption of one partner in the chimera usually follows colony fusion (rinkevich and weissman, 1987; rinkevich, 2002, 2005). in natural populations, multichimeras form as the result of kin recognition, controlled by the fu/hc gene, allowing larvae to settle near parental of genetically compatible adult colonies with which, once metamorphosed, they can fuse (grosberg and quinn, 1986; grosberg, 1987; ben-shlomo et al., 2008). this natural multichimerism gives colonies greater fitness in terms of faster growth rate, maximum size reached, better competition for the substrate, earlier achievement of sexual maturity and increased genetic diversity (sabbadin, 1994; rinkevich and shapira, 1999; paz and rinkevich, 2002; de tomaso, 2006). in addition, stem cells can be maintained and proliferate for a long time within a chimeric colony, although the corresponding soma has been resorbed (sabbadin and zaniolo, 1979; sabbadin and astorri, 1988; stoner et al., 1999; laird et al., 2005; de tomaso, 2006). according to sabbadin (1994) chimerism has the important role of maintaining the genetic structure of a population allowing the preservation of the genomes of fusible colonies, even in the case of their resorption by a partner, and seems to be diffuse in colonies collected from the field (ben-shlomo et al., 2008). for this reason, botryllus is an interesting model for the study of the evolution of microchimerism (rinkevich, 2001), which seems to be responsible for various human pathologies (adams and nelson, 2004), and its relationships with the immune system. apoptosis and efferocytosis at take-over despite the abundance of in vitro model systems, mainly represented by selected cell lines, there is an increasing need for reliable models for in vivo investigations of the biological role of apoptosis in organisms. with its spontaneous and recurrent apoptosis of zooid tissues at the end of each blastogenetic cycle, b. schlosseri is an interesting model organism for the study of apoptosis and its genetic control. two genes changing their expression during take-over have already been described (lauzon et al., 1996), but we do not know the nature of the cyclical signal inducing weekly cell death. in addition, phagocytes have been reported to play an important role in regulating the clearance of senescent cells, the extent of growth of buds and their budding activity (voskoboynik et al., 2004), but the molecular mechanisms underlying phagocyte recruitment in senescent tissues and recognition of effete cells are still largely unclear. tunicate cytokines: what is their relationship with their vertebrate counterparts? vertebrate cytokines are immunomodulatory proteins secreted by activated immunocytes after the recognition of foreign molecules, and they take part in several immunological processes such as inflammation, apoptosis, clearance of effete cells 66 and corpses, cytotoxicity and phagocytosis. in addition, they guarantee fine cooperation between sentinel cells, which recognize non-self molecules, and effector cells, which can mount active responses towards foreign cells or particles, in order to ensure the health and survival of individuals (abbas et al., 2000). in tunicates, molecules recognized by antibodies raised against the mammalian proinflammatory cytokines il-1 have been reported in various ascidian species (beck et al., 1989; raftos et al., 1991, 1992; ballarin et al., 2001, cima et al., 2004; parrinello et al., 2007). they have been partially characterized and their molecular weights range between 12 and 59 kda, as resolved by sdspage and gel chromatography (beck et al., 1989; raftos et al., 1992; parrinello et al., 2007). it is common opinion that invertebrate cytokines share no homologies with their vertebrate counterparts (beck, 1998; beschin et al., 2001, 2004) and this may explain the absence of orthologues of vertebrate pro-inflammatory cytokine genes in the genome of ciona instestinalis (azumi et al., 2003). however, it has been reported that various vertebrate cytokines have a lectin domain and can bind carbohydrates (cebo et al., 2002; beschin et al., 2004) and this probably represents the evolutionary link between tunicate and vertebrate cytokines (beschin et al., 2001, 2004). the availability of the sequenced genome of botryllus may allow us to clarify the evolution of vertebrate cytokines. acknowledgements i would like to thank prof a sabbadin who, more than 50 years ago, decided to use b. schlosseri, this wonderful organism, as a model in the laboratories of the institute of zoology and comparative anatomy (now department of biology) of the university of padova. i am indebted to him for having introduced me to the field of ascidian immunobiology, for his continuous support to my research and his stimulating and helpful discussions. thanks are due also to prof p burighel for the critical reading of the manuscript and his useful suggestions. references abbas k, lichtman h, pober js. cellular and molecular immunology. wb saunders company, philadelphia, 2000. adams cm, nelson jl. microchimerism: an investigative frontier in autoimmunity and transplantation. j. am. med. ass. 291: 11271131, 2004. andrew w. phase microscope studies of living blood-cells of the tunicates under normal and experimental conditions, with a description of a new type of motile cell appendage. quart. j. microsc. sci. 102: 89-105, 1961. azumi k, yokosawa h, ishii s. presence of 3,4dihydroxyphenylalanine-containing peptides in hemocytes of the ascidian halocynthia roretzi. experientia 46: 1020-1023, 1990. azumi k, de santis r, de tomaso a, rigoutsos i, yoshizaki f, pinto mr, et al. genomic analysis of immunity in a urochordate and the emergence of the vertebrate immune system: “waiting for godot”. immunogenetics 55: 570581, 2003. ärnbäck-christie-linde a. northern and arctic invertebrates in the collection of the swedish state museum. ix: tunicata. 2. botryllidae: reproductive organs of metrocarpa (n. gen.) leachi savigny and botryllus schlosseri pallas. kungl. svenska vet. akad. handl. 43: 1-25, 1923. ballarin l. the immune system of compound ascidians. inv. surv. j. 5: 30, 2008. ballarin l, cima f. cytochemical properties of botryllus schlosseri haemocytes: indications for morpho-functional characterisation. eur. j. histochem. 49: 255-264, 2005. ballarin l., burighel p. rgd-containing molecules induce macropinocytosis in ascidian hyaline amoebocytes. j. invertebr. pathol. 91: 124-130, 2006. ballarin l, zaniolo g. colony specificity in botrylloides leachi. ii. cellular aspects of the non-fusion reaction. inv. surv. j. 4: 38-44, 2007. ballarin l, cima f, sabbadin a. histoenzymatic staining and characterization of the colonial ascidian botryllus schlosseri hemocytes. boll. zool. 60: 19-24, 1993. ballarin l, cima f, sabbadin a. phagocytosis in the colonial ascidian botryllus schlosseri .dev. comp. immunol. 18: 467-481, 1994. ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995. ballarin l, cima f, sabbadin a. calcium homeostasis and yeast phagocytosis in hemocytes of the colonial ascidian botryllus schlosseri. comp. biochem. physiol. 118a: 153-158, 1997. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri .dev. comp. immunol. 22: 479-492, 1998. ballarin l, tonello c, guidolin l, sabbadin a. purification and characterization of a humoral opsonin, with specificity for d-galactose, in the colonial ascidian botryllus schlosseri. comp. biochem. physiol. 123b: 115-123, 1999. ballarin l, tonello c, sabbadin a. humoral opsonin from the colonial ascidian botryllus schlosseri as a member of the galectin family. mar. biol. 136: 823-827, 2000. ballarin l, franchini a, ottaviani e, sabbadin a. morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species botryllus schlosseri. biol. bull. 201: 59-64, 2001. ballarin l, scanferla m, cima f, sabbadin a. phagocyte spreading and phagocytosis in the compound ascidian botryllus schlosseri: evidence for an integrin-like, rgd-dependent recognition mechanism. dev. comp. immunol. 26: 39–48, 2002a. ballarin l, cima f, floreani m, sabbadin a. oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian botryllus schlosseri. comp. biochem. physiol. 133c: 411-418, 2002b. 67 ballarin l, menin a, franchi n, bertoloni g, cima f. morula cells and non-self recognition in the compound ascidian botryllus schlosseri. inv. surv. j. 2: 1-5, 2005. ballarin l, menin a, tallandini l, matozzo v, burighel p, basso g, et al. haemocytes and blastogenetic cycle in the colonial ascidian botryllus schlosseri: a matter of life and death. cell tissue res. 331: 555-564, 2008. bancroft fw. variation and fusion of colonies in compound ascidians. proc. calif. acad. sci. 3: 137-186, 1903. bayer sw, kammerer b, nicholson gj, pusecker k, walk t, bayer e, et al. morulin pm: a modified polypeptide containing topa and 6.bromotryptophan from the morula cells of the ascidian, phallusia mammillata. arch. biochem. biophys. 348: 278-288, 1997. beck g. macrokines: invertebrate cytokine-like molecules? front. biosci. 3: d559-d569, 1998. beck g, vasta r, marchalonis j, habicht gs. characterization of interleukin-1 activity in tunicates. comp. biochem. physiol. 92b: 93-98, 1989. ben-shlomo r, motro u, paz g, rinkevich b. pattern of settlement and natural chimerism in the colonial urochordate botryllus schlosseri. genetica 132: 51-58, 2008. berrill nj. the development of the bud in botryllus. biol. bull. 80: 169-184, 1941a. berrill nj. size and morphogenesis in the bud of botryllus. biol bull 80: 185-193, 1941b. beschin a, bilej m, torreele e, de baetselier p. on the existence of cytokines in invertebrates. cell. mol. life sci. 58: 801-814, 2001. beschin a, bilej m, magez s, lucas r, de baetselier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on a similar lectin-like activity. prog. mol. subcell. biol. 34: 145-163, 2004. brunetti r, burighel p. sviluppo dell’apparato vascolare coloniale in botryllus schlosseri. pubbl. staz. zool. napoli 37: 137-148, 1969. burighel p., schiavinato a. degenerative regression of the digestive tract in the colonial ascidian botryllus schlosseri (pallas). cell tissue res. 235: 309-318, 1984. burighel p, cloney ra. urochordata: ascidiacea. in: fw harrison, ee ruppert (eds), microscopic anatomy of invertebrates, vol. 15, wiley-liss inc., new york, pp 221-347, 1997. burighel p, brunetti r, zaniolo g. hibernation of the colonial ascidian, botrylloides leachi (savigny): histological observations. boll. zool. 43: 293301, 1976. burighel p, milanesi c, sabbadin a. blood cell ultrastructure of the ascidian botryllus schlosseri. ii. pigment cells. acta zool. (stockh.) 64: 15-23, 1983. buss lw. the evolution of individuality. pr.eton university press, princeton, nj, usa, 1987. cammarata m, candore g, arizza v, caruso c, parrnello n. cytotoxic activity of styela plicata hemocytes against mammalian cell targets: ii. properties of the in vitro reaction against human tumour cell lines. anim. biol. 4: 139144, 1995. cebo c, vergoten g, zanetta jp. lectin activities of cytokines: function and putative carbohydraterecognition domains. biochim. biophys. acta 1572: 422-434, 2002. cima f, ballarin l. tbt-induced apoptosis in tunicate haemocytes. appl. organometal. chem. 13: 97-703, 1999. cima f, ballarin l. tributyltin induces cytoskeletal alterations in the colonial ascidian botryllus schlosseri phagocytes via interaction with calmodulin. aquat. toxicol. 48: 419-429, 2000. cima f, ballarin l. tbt-sulphydryl interaction as a cause of immunotoxicity in tunicates. ecotoxicol. environ. saf. 58: 386-395, 2004. cima f, ballarin l. undifferentiated cells in the blood of the colonial ascidian botryllus schlosseri: a morpho-functional characterisation. inv. surv. j. 4: 29, 2007. cima f, ballarin l, bressa g, sabbadin a. immunotoxicity of butyltins in tunicates. appl. organometal. chem. 9: 567-572, 1995. cima f, ballarin l, sabbadin a. new data on phagocytes and phagocytosis in the compound ascidian botryllus schlosseri. ital. j. zool. 63: 357-364, 1996. cima f, ballarin l, bressa g, sabbadin a, burighel p. triphenyltin pesticides as immunotoxins for tunicates. mar. chem. 58: 27-273, 1997. cima f, ballarin l, bressa g, burighel p. cytoskeleton alteration by tributyltin (tbt) in tunicate phagocytes. ecotoxicol. environ. saf. 40: 10-165, 1998. cima f, perin a, burighel p, ballarin l. morphofunctional characterisation of haemocytes of the compound ascidian botrylloides leachi (tunicata, ascidiacea). acta zool. 82: 261-274, 2001. cima f, dominici d, mammi s, ballarin l. butyltin and calmodulin: which interaction? appl. organometal. chem. 16: 182-186, 2002a. cima f, dominici d, ballarin l, burighel p. influence of tbt on activity of detoxifying enzymes from haemocytes of a colonial ascidian. fresen. environ. bull. 11: 573-577, 2002b. cima f, basso g, ballarin l. apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian botryllus schlosseri. cell tissue res. 312: 369-376, 2003. cima f, sabbadin a, ballarin l. cellular aspects of allorecognition in the compound ascidian botryllus schlosseri. dev. comp. immunol. 28: 881-889, 2004. cima f, sabbadin a, zaniolo g, ballarin l. colony specificity and chemotaxis in the compound ascidian botryllus schlosseri. comp. biochem. physiol. 145a: 376-382, 2006a. cima f, ballarin l, gasparini f, burighel p. external amoebocytes guard the pharynx entry in a tunicate (ascidiacea). dev. comp. immunol. 30: 463-472, 2006b. cima f, burighel p, ballarin l. temporal and biotic evolution of “botryllus biocoenosis” in the presence of antifouling paints. in: p campostrini (ed), scientific research and safeguarding of venice 2005, corila research program 20042006, volume iv, 2005 results, corila, venice, pp 239-246, 2006c. 68 cima f, bragadin m, ballarin l. toxic effects of new antifouling compounds on tunicate haemocytes. i. sea-nine 211tm and chlorothalonil. aquat. toxicol. 8: 299-312, 2008. coombe dr, ey pl, jenkin cr. ascidian haemagglutinins: incidence in various species, binding specificities and preliminary characterisation of selected agglutinins. comp. biochem. physiol. 77b: 811-819, 1984a. coombe dr, ey pl, jenkin cr. particle recognition by haemocytes from the colonial ascidian botrylloides leachi: evidence that the b. leachi ha-2 is opsonic. j. comp. physiol. 154: 509521, 1984b. cotelle n, moreau s, cotelle p, catteau jp, bernier jl, hénichart jp. generation of free radicals by simple pregnylated hydroquinone derivatives, natural antitumor agents from the marine urochordate aplidium californicum. chem. res. toxicol. 4: 300-305, 1991. cowden rr. the embryonic origin of blood cells in the tunicate clavelina. trans. am. microsc. soc. 87: 521-524, 1968. dan-sohkawa m, morimoto m, kaneko h. in vitro reactions of coelomocytes against sheep red blood cells in the solitary ascidian halocynthia roretzi. zool. sci. 12: 411-417, 1995. davidson bs, ireland cm. lissoclinolide, the first non-nitrogenous metabolite from a lissoclinum tunicate. j. nat. prod. 53: 1036-1038, 1990. davies p, bonney rj. the secretion of hydrolytic enzymes by mononuclear phagocytes. in: g. weissman (ed), the cell biology of inflammation. elsevier, amsterdam, new york, oxford, pp 497-542, 1980. de cathelineau am, henson pm. the final step in programmed cell death: phagocytes carry apoptotic cells to the grave. essays biochem. 39: 105-117, 2003. de leo g. ascidian haemocytes and their involvement in defense reactions. boll. zool. 59: 195-213, 1992. dehal p, satou y, campbell rk, chapman j, degnan b, de tomaso a, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157-2167, 2002. della valle a. sur le bourgeonnement des didemnides et des botryllides et sur le type enterocoelien des ascidies. arch. it. biol. 2: 5072, 1882. delsuc f, brinkmann h, chourrout d, hervé p. tunicates and not cephalochordates are the closest living relatives of vertebrates. nature 439: 965-968, 2006. de tomaso aw. allorecognition polymorphism versus parasitic stem cells. trends genet. 22: 485-490, 2006. de tomaso aw, nyholm sv, palmeri kj, ishizuka kj, ludington wb, mitchel k, et al. isolation and characterization of a protochordate histocompatibility locus. nature 438: 454-459, 2005. dorsett lc, hawkins cj, grice ja, lavin mf, merefield pm, parry dl, et al. ferreascidin: a highly aromatic protein containing 3,4dihydroxyphenylalanine from the blood cells of a stolidobranch ascidian. biochemistry 26: 8078-8082, 1987. dufour hd, chettouh zc, deyts c, de rosa r, goridis c, joly js. precraniate origin of cranial motoneurons. proc. natl. acad. sci. usa 103: 8727-8732, 2006. edler mc, fernandez am, lassota p, ireland cm, barrows lr. inhibition of tubulin polymerization by vitilevuamide, a bicyclic marine peptide, at a site distinct from colchicine, the vinca alkaloids, and dolastatin 10. biochem. pharmacol. 63: 707-715, 2002. erba e, bassano l, di liberti g, muradore i, chiorino g, ubezio p, et al. cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine. brit. j. cancer 86: 15101517, 2002. ermak th. the hematogenic tissues of tunicates. in: rk wright, el cooper (eds), philogeny of thymus and bone marrow bursa cells. elsevier, amsterdam, pp 45-46, 1976. fagan mb, weissman il. sequence and characterization of two hsp70 genes in the colonial protochordate botryllus schlosseri. immunogenetics 44: 134-142, 1996. freeman g. the role of blood cells in the process of asexual reproduction in the tunicate perophora viridis. j. exp. zool. 156: 157-184, 1964. frizzo a, guidolin l, ballarin l, sabbadin a. purification and partial characterisation of phenoloxidase from the colonial ascidian botryllus schlosseri. mar. biol. 135: 483-488, 1999. frizzo a, guidolin l, ballarin l., baldan b, sabbadin a. immunolocation of phenoloxidase in vacuoles of the compound ascidian botryllus schlosseri morula cells. ital. j. zool. 67: 273276, 2000. fujimoto h, watanabe h. the characterization of granular amoebocytes and their possible roles in the asexual reproduction of the polystyelid ascidian, polyzoa vesiculiphora. j. morphol. 150: 623-638, 1976. gardai sj, mcphillips ka, frasch sc, janssen wj, starefeldt a, murphy-ullrich je, et al. cellsurface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of lrp on the phagocyte. cell 123: 321-334, 2005. gasparini f, franchi n, spolaore b, ballarin l. novel rhamnose-binding lectins from the colonial ascidian botryllus schlosseri. dev. comp. immunol. [in press], 2008 (doi: 10.1016/j.dci.2008.03.006). gibin c. l’esplorazione del golfo di venezia. i disegni del naturalista chioggiotto stefano chiereghin per lazzaro spallanzani. t & g edizioni, conselve (pd), 1997. gomperts bd, tatham per, kramer im. signal transduction. academic press, san diego, ca, 2002. griffin fm, griffin ja, leider je, silverstein sc. studies on the mechanism of phagocytosis. i. requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane. j. exp. med. 142: 1263-1282, 1975a. 69 griffin fm, griffin ja, silverstein sc. studies on the mechanism of phagocytosis. ii. the interaction of macrophages with anti.immunoglobulin iggcoated bone marrow-derived lymphocytes. j. exp. med. 144: 788-809, 1975b. grosberg rk. limited dispersal and proximitydependent mating success in the colonial ascidian botryllus schlosseri. evolution 41: 372384, 1987. grosberg rk, quinn jf. kin recognition and colony fusion in the colonial ascidian botryllus schlosseri. nature 322: 457-459, 1986. henderson rs. effects of organotin antifouling paint leachates on pearl harbour organisms: a site specific flowthrough bioassay. in: proceedings of the organotin symposium. ocens ’86 conference, washington dc usa, september, 23-25, 1986. new york, the institute of electrical and electronic engineers, inc., vol. 4, pp 1226-1233, 1986. hibbs jb, taintor rr, vavrin z. macrophage cytotoxicity: role of l-arginine deaminase and imino nitrogen oxidation to nitrite. science 235: 473-476, 1987. hirose e. colonial allorecognition, hemolytic rejection, and viviparity in botryllid ascidians. zool. sci. 20: 387-394, 2003. hirose e, mukai h. an ultrastructural study on the origin of glomerulocytes, a type of blood cell in a styelid ascidian, polyandrocaropa misakiensis. j. morphol. 211: 269-273, 1992. hirose e, saito y, watanabe h. a new type of the manifestation of colony specificity in the compound ascidian, botrylloides violaceus oka. biol. bull. 175: 240-245, 1988. hirose e, saito y, watanabe h. allogeneic rejection induced by cut surface contact in the compound ascidian, botrylloides simodensis. invertebr. reprod. dev. 17: 159-164, 1990. hirose e, ishii t, saito y, taneda y. phagocytic activity of tunic cells in the colonial ascidian aplidium yamazii (polyclinidae, aplousobranchia). zool. sci. 11: 203-208, 1994a. hirose e, saito y, watranabe h. surgical fusion between incompatible colonies of the compound ascidian, botrylloides fuscus. dev. comp. immunol. 18: 287-294, 1994b. hirose e, saito y, watanabe h. subcuticular rejection: an advanced mode of the allogeneic rejection in the compound ascidians botrylloides simodensis and b. fuscus. biol. bull. 192: 53-61, 1997. hirose e, shirae m, saito y. ultrastructure and classification of circulating hemocytes in 9 botryllid ascidians (chordata: ascidiacea). zool. sci. 20: 647-656, 2003. hjort j. über den entwicklungscyclus der zusammengesetzen ascidien. mitt. zool. stn. neapel 10: 584-617, 1893. howard bm, clarkson k. simple pregnylated hydroquinone derivatives from the marine urochordate aplidium californicum. natural anticancer and antimutagenic agents. tetrahedron lett. 46: 4449-4452, 1979. ireland c, scheuer pj. ulicyclamide and ulithiacyclamide, two new small peptides from a marine tunicate. j. am. chem. soc. 102: 56915692, 1980. ireland cm, durso ar, newman ra, hacker mp. antineoplastic cyclic peptides from the marine tunicate lissoclinum patella. j. org. chem. 47: 1807-1811, 1982. izzard cs. development of polarity and bilateral asymmetry in the palleal bud of botryllus schlosseri (pallas). j. morphol. 139: 1-26, 1973. karakashian s, milkman r. colony fusion compatibility types in botryllus schlosseri. biol. bull. 133: 473, 1967. katow h, watanabe h. fine structure of fusion reaction in the compound ascidian botryllus primigenus oka. dev. biol. 76: 1-14, 1980. kawamura k, ohtake s.i, michibata h, nakauki m. autoradiographic studies on cell population kinetics in epithelial and haemopoietic stem cell lines in the process of palleal budding of ascidians. mem. fac. sci. kochi univ. a9: 1-12, 1988. kelly kl, cooper el, raftos da. purification and characterization of a humoral opsonin from the solitary urochordate styela clava. comp. biochem. physiol. 103b: 749-753, 1992. khalturin k, becker m, rinkevich b, bosch tcg. urochordates and the origin of natural killer cells: identification of a cd94/nkr-p1-related receptor in blood cells of botryllus. proc. natl. acad. sci. usa 100: 622-627, 2003. krysko dv, brouckaert g, kalai m, vandenabeele p, d'herde k. mechanisms of internalization of apoptotic and necrotic l929 cells by a macrophage cell line studied by electron microscopy. j. morphol. 258: 336-345, 2003. laird dj, de tomaso aw, weissman il. stem cells are units of natural selection in a colonial ascidian. cell 123: 1351-1360, 2005. lauzon rj, ishizuka kj, weissman il. a cyclical, developmentally-regulated death phenomenon in a colonial urochordate. dev. dyn. 194: 71-83, 1992. lauzon rj, patton cw, weissman il. a morphological and immunohistochemical study of programmed cell death in botryllus schlosseri (tunicata, ascidiacea). cell tissue res. 272: 115-127, 1993. lauzon rj, chang w-t, dewing ls. evidence for transcriptional modulation but not acid phosphatase expression during programmed cell death in the colonial tunicate botryllus schlosseri . microsc. res. tech. 34: 218-227, 1996. lauzon rj, shizuka kj, weissman il. cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration. dev. biol. 249: 333-348, 2002. lipari l, cammarata m, arizza v, parrinello d. cytotoxic activity of styela plicata hemocytes against mammalian cell targets: i. properties of the in vitro reaction against erythrocytes. anim. biol. 4: 131-137. manni l, zaniolo g, cima f, burighel p, ballarin l. botryllus schlosseri : a model ascidian for the study of asexual reproduction. dev. dyn. 236: 335-352, 2007. 70 meier o, boucke k, hammer sv, keller s, stidwill rp, hemmi s, et al. adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. j. cell biol. 158: 1119-1131, 2002. menin a., ballarin l. exogenous il-8 induces phagocyte activation in the compound ascidian botryllus schlosseri. inv. surv. j. 3: 18-24, 2006. menin a., ballarin l. toll-like receptors in haemocytes of the colonial ascidian botryllus schlosseri: preliminary results. inv. surv. j. 4: 31, 2007. menin a, chemello e, ballarin l. signal transduction in phagocytosis of the colonial ascidian botryllus schlosseri: a preliminary approach. inv. surv. j. 4: 31-32, 2007. menin a, ballarin l, marin mg, cima f. immunotoxicity in ascidians of antifouling compounds alternative to organotins the case of diuron and tcms pyridine. j. environ. sci. health a [in press], 2008. menin a, ballarin l. immunomodulatory molecules in the compound ascidian botryllus schlosseri: evidence from conditioned media. j. invertebr. pathol. [submitted], 2008. menin a, del favero m, cima f, ballarin l. release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian. mar. biol. 148: 225230, 2005. metschnikow e. entwicklungsgeschichtliche beiträge. vii. über die larven und knospen von botryllus. bull. acad. imp. sc. st. petersbourg 13: 284-300, 1869. milanesi c, burighel p. blood cell ultrastructure of the ascidian botryllus schlosseri. i. hemoblast, granulocytes, macrophage, morula cell and nephrocyte. acta zool. (stockh.) 59: 135-147, 1978. milkman r. genetic and developmental studies on botryllus schlosseri. biol. bull. 132: 229-243, 1967. mukai h. experimental alteration of fusibility in compound ascidians. sci. rep. tokyo daigaku b13: 51-73, 1967. mukai h, watanabe h. on the occurrence of colony specificity in some compound ascidians. biol. bull. 147: 411-421, 1974. nair sv, pearce s, green pl, mahajan d, newton ra, raftos ds. a collectin-like protein from tunicates. comp. biochem. physiol. 125b: 279289, 2000. nakazawa t, xu j, nishikawa t, oda t, fujita a, ukai k, et al. lissoclibadins 4-7, polysulfur aromatic alkaloids from the indonesian ascidian lissoclinum cf. badium. j. nat. prod. 70: 439442, 2007. nishida h. patterning the marginal zone of early ascidian embryos: localized maternal mrna and inductive interactions. bioessay 24: 613624, 2002a. nishida h. specification of developmental fates in ascidian embryos: molecular approach to maternal determinants and signaling molecules. int. rev. cytol. 217: 227-276, 2002b. nonaka m. evolution of the complement system. curr. opin. immunol. 13: 69-73, 2001. nonaka m, azumi k, ji x, namikawa-yamada c, sasaki m, saiga h, et al. opsonic complement component c3 in the solitary ascidian, halocynthia roretzi. j. immunol. 162: 387-391, 1999. nyholm sv, passegue e, ludington wb, voskoboynik a, mitchel k, weissman il, et al. fester, a candidate allorecognition receptor from a primitive chordate. immunity 25:163-173, 2006. oda-ishii i, bertrand v, matsuo i, lemaire p, saiga h. making very similar embryos with divergent genomes: conservation of regulatory mechanisms of otx between the ascidians halocynthia roretzi and ciona intestinalis. development 132: 1663-1674, 2005. ogi t, taira j, margiastuti p, ueda k. cytotoxic metabolites from the okinawan ascidian diplosoma virens. molecules 13: 595-602, 2008. oka a. über die knospung der botrylliden. z. wiss. zool. 54: 521-547, 1892. oka h. colony specificity in compound ascidians. the genetic control of fusibility. in: h yukawa (ed), profiles of japanese science and scientists, kodanska, tokyo, pp 196-206, 1970. oka h, watanabe h. colony specificity in compound ascidians as tested by fusion experiments (a preliminary report). proc. japan. acad. 33: 657-659, 1957a. oka h, watanabe h. vascular budding, a new type of budding in botryllus. biol. bull. 112: 225-240, 1957b. oka h, watanabe h. vascular budding in botrylloides. biol. bull. 117: 340-346, 1959. oka h, watanabe h. problems of colony specificity in compound ascidians. bull. mar. biol. stat. ashamiushi 10: 153-155, 1960. okuyama m. saito y, hirose e. fusion between incompatible colonies of a viviparous ascidian, botrylloides lentus. invertebr. biol. 121: 163169, 2002. oltz em, bruening rc, smith mj, kustin k, nakanishi k. the tunichromes. a class of reducing blood pigments from sea squirts: isolation, structures, and vanadium chemistry. j. am. chem. soc. 110: 6162-6172, 1987. oren m, douek j, fishelson z, rinkevich b. identification of immune-relevant genes in histoincompatible rejecting colonies of the tunicate botryllus schlosseri. dev. comp. immunol. 31: 889-902, 2007. ohtake s-i, abe t, shishikura f, tanaka k. the phagocytes in hemolymph of halocynthia roretzi and their phagocytic activity. zool. sci. 11: 681-691, 1994. overton j. the fine structure of blood cells in the ascidian perophora viridis. j. morphol. 119: 305-326, 1966. pancer z, gershon h, rinkevich b. cdna cloning of a putative protochordate fk506-binding protein. biochem. biophys. res. commun. 197: 199220, 1993. pancer z, cooper l, müller weg. a urochordate putative homolog of human eb1, the protein which binds apc. cancer lett. 109: 155-160, 1996a. 71 pancer z, cooper l, müller weg. a tunicate (botryllus schlosseri) cdna reveals similarity to vertebrate antigen receptor. immunogenetics 45: 69-72, 1996b. pancer z, diehl-siefert b, rinkevich b, müller weg. a novel tunicate (botryllus schlosseri) putative c-type lectin features an immunoglobulin domain. dna cell biol. 16: 801-806, 1997. parrinello n. humoral and cellular lectins of ascidians. j. mar. biotechnol. 3: 29-34, 1995. parrinello n, patricolo e. inflammatory-like reaction in the tunic of ciona intestinalis (tunicata). ii. capsule components. biol. bull. 167: 238-250, 1984. parrinello n, patricolo e, canicattì c. tunicate immunobiology. tunic reaction of ciona intestinalis l. to erythrocyte injection. boll. zool. 44: 373-381, 1977. parrinello n, patricolo e, canicattì c. inflammatorylike reaction inthe tunic of ciona intestinalis (tunicata). i. encapsulation and tissue injury. biol. bull. 167: 229-237, 1984. parrinello n, arizza v, cammarata m, parrinello d. cytotoxic activity of ciona intestinalis (tunicata) hemocytes: properties of the in vitro reaction against erythrocyte targets. dev. comp. immunol. 17: 19-27, 1993. parrinello n, arizza v, cammarata m, giaramita ft, pergolizzi m, bazzana m, et al. inducible lectins with galectin properties and human il1α epitopes opsonize yeast during the inflammatory response of the ascidian ciona intestinalis. cell tissue res. 329: 379-390, 2007. passamaneck yj, di gregorio a. ciona intestinalis: chordate development made simple. dev. dyn. 233: 1-19, 2005. paz g, rinkevich b. morphological consequences for multi-partner chimerism in botrylloides, a colonial urochordate. dev. comp. immunol. 26: 615-622, 2002. pearce s, newton ra, nair sv, raftos da. humoral opsonins of tunicate, pyura stolonifera. dev. comp. immunol. 25: 377-385, 2001. peddie cm, smith vj. in vitro spontaneous cytotoxic activity against mammalian target cells by the hemocytes of the solitary ascidian, ciona intestinalis. j. exp. zool. 267: 616-623, 1993. peddie cm, smith vj. mechanism of cytotoxic activity by hemocytes of solitary ascidian, ciona intestinalis. j. exp. zool. 270: 335-342, 1994. pérès jm. recherches sur le sang et les organes neuraux des tuniciers. ann. inst. oceanogr. (monaco) 21: 229-359, 1943. pinto mr, chinnici cm, kimura y, melillo d, marino r, spruce la, et al. cic3-1a-mediated chemotaxis in the deuterostome invertebrate ciona intestinalis (urochordata). j. immunol. 171: 5521-5528, 2003. pizon a. histoire de la blastogénèse chez les botryllides. ann. sc. nat. s. 7, zool. 14: 1-386, 1893. rabinowitz c, rinkevich b. epithelial cell cultures from botryllus schlosseri palleal buds: accomplishments and challenges. methods cell sci. 25: 137-148, 2003. raftos da. cellular restriction of histocompatibility resposnses in the solitary urochordate, styela plicata. dev. comp. immunol. 15: 93-98, 1991. raftos da, tait nn, briscoe da. allograft rejection and alloimmune memory in the solitary urochordate, styela plicata. dev. comp. immunol. 11: 343-351, 1987a. raftos da, tait nn, briscoe da. cellular basis of allograft rejection in the solitary urochordate, styela plicata. dev. comp. immunol. 11: 713725, 1987b. raftos da, cooper el, habicht gs, beck g. invertebrate cytokines: tunicate cell proliferation stimulated by an interleukin 1-like molecule. proc. natl. acad. sci. usa 88: 9518-9522, 1991. raftos da, cooper el, stillman dl, habicht gs, beck g. invertebrate cytokines ii: release of interleukin-1-like molecules from tunicate hemocytes stimulated with zymosan. lymph. cyt. res. 11: 235-240, 1992. raftos da, nair sv, robbins j, newton ra, peters r. a complement c3-like protein from the tunicate, styela plicata. dev. comp. immunol. 26: 307-312, 2002. richardson ad, ireland cm. a profile of the in vitro antitumor activity of lissoclinolide. toxicol. appl. pharm. 195: 55-61, 2004. rinehart kl jr, gloer jb, cook jc jr. structures of the didemnins, antiviral and cytotoxic depsipeptides from a caribbean tunicate. j. am. chem. soc. 103: 1857-1859, 1981a. rinehart kl jr, gloer jb, hughes rg jr, renis he, mcgovern jp, swynenberg eb, et al. didemnins: antiviral and antitunor depsipeptides from a caribbean tunicate. science 212: 933-935, 1981b. rinehart kl jr, kobayashi j, harbour gc, hughes rg jr, mizsak sa, scahill ta. eudistomins c, e, k, and l, potent antiviral compounds containing a novel oxathiazepine ring from the caribbean tunicate eudistoma olivaceum. j. am. chem. soc. 106: 1524-1526, 1984. rinkevich b. aspects of the incompatibility nature in botryllid ascidian. anim. biol. 1: 17-28, 1992. rinkevich b. human natural chimerism: an acquired character or a vestige of evolution? hum. immunol. 62: 651-657, 2001. rinkevich b. the colonial urochordate botryllus schlosseri: from stem cells and natural tissue transplantation to issues in evolutionary ecology. bioessay 24: 730-740, 2002. rinkevich b. rejection pattern in botryllid ascidian immunity: the first tier of allorecognition. can. j. zool. 83: 101-121, 2005. rinkevich b, rabinowitz c. in vitro culture of blood cells from the colonial protochordate botryllus schlosseri . in vitro dev. biol. 29: 79-85, 1993. rinkevich b, rabinowitz c. acquiring embryoderived cell cultures and aseptic metamorphosis of larvae from the colonial protochordate botryllus schlosseri. invertebr. reprod. dev. 25: 59-72, 1994. rinkevich b, shapira m. multi-partner urochordate chimeras outperform two-partner chimerical entities. oikos 87: 315-320, 1999. 72 rinkevich b, weissman il. a long-term study on fused subclones in the ascidian botryllus schlosseri: the resorption phenomenon (protochordata: tunicata). j. zool. lond. 213: 717-733, 1987. rinkevich b, weissman il. retreat growth in the ascidian botryllus schlosseri: a consequence of nonself recognition. in: rk grosberg, d hedgecock, k nelson (eds), invertebrate historecognition. plenum press, new york, pp 93-109, 1988. rinkevich b, weissman il. allogeneic resorption in colonial protochordates – consequences of nonself recognition. dev. comp. immunol. 16: 275-286, 1992. rinkevich b, lilker-levav t, goren m. allorecognition/xenorecognition responses in botrylloides (ascidiacea) subpopulations from the mediterranean coast of israel. j. exp. zool. 270: 302-313, 1994. rinkevich b, tartakover s, gershon h. contribution of morula cells to allogeneic responses in the colonial ascidian botryllus schlosseri. mar. biol. 131: 227-236, 1998. rinkevich y, paz g, rinkevich b, reshef r. systemic bud induction and retinoic acid signaling underlie whole body regeneration in the urochordate botrylloides leachi. plos biol. 5: e71, 2007. rosner a, rabinowitz c, moiseeva e, voskoboynik a, rinkevich b. bs-cadherin in the colonial urochordate botryllus schlosseri: one protein, many functions. dev. biol. 304: 687-700, 2007. sabbadin a. osservazioni sullo sviluppo, l’accrescimento e la riproduzione di botryllus schlosseri (pallas), in condizioni di laboratorio. boll. zool. 22: 243-265, 1955a. sabbadin a. studio sulle cellule del sangue di botryllus schlosseri (pallas) (ascidiacea). arch. ital. anat. embriol. 60: 33-67, 1955b. sabbadin a. effetti dell’estirpazione delle gemme sulla durata del ciclo vitale in botryllus schlosseri (pallas). boll. zool. 23: 331-342, 1956. sabbadin a. analisi sperimentale dello sviluppo delle colonie di botryllus schlosseri (pallas) [ascidiacea]. arch. it. anat. embriol. 63:178221, 1958. sabbadin a. le basi genetiche della capacità di fusione fra colonie in botryllus schlosseri (ascidiacea). rend. accad. naz. lincei 32: 1021-1035, 1962. sabbadin a. the compound ascidian botryllus schlosseri in the field and in the laboratory. pubbl. staz. zool. napoli 37 (suppl.): 62-72, 1969. sabbadin a. selfand cross-fertilization in the compound ascidian botryllus schlosseri. dev. biol. 24: 379-391, 1971. sabbadin a. formal genetics of ascidians. amer. zool. 22: 765-773, 1982. sabbadin a. coloniality in ascidians and its adaptive value, with special reference to botryllus schlosseri. anim. biol. 3: 157-163, 1994. sabbadin a, zaniolo g. sexual differentiation and germ cell transfer in the colonial ascidian botryllus schlosseri . j. exp. zool. 207: 289304, 1979. sabbadin a, astorri c. chimeras and histocompatibility in the colonial ascidian botryllus schlosseri. dev. comp. immunol. 12: 737-747, 1988. sabbadin a, zaniolo g, majone f. determination of polarity and bilateral asymmetry in palleal and vascular buds of the ascidian botryllus schlosseri. dev. biol. 46:79-87, 1975. sabbadin a, burighel p, zaniolo g. studies on the colonial ascidian botryllus schlosseri. a review of some lines of research. acta embryol. morphol. exper. n. s. 12: 259-279, 1991. sabbadin a, zaniolo g, ballarin l. genetic and cytological aspects of histocompatibility in ascidians. boll. zool. 59: 167-173, 1992. sabbadin a, zaniolo g, ballarin l, manni l, martinucci gb, burighel p. differentiation of blood and germ cell lines in ascidians. in: embriologia sperimentale, biologia dello sviluppo ed evoluzione, in ricordo di silvio ranzi. incontro di studio n. 13. istituto lombardo di scienze e lettere, milano, pp 3960, 1999. saito y, watanabe h. partial biochemical characterization of humoral factors involved in the nonfusion reaction of a botryllid ascidian, botrylloides simodensis. zool. sci. 1: 229-235, 1984. saito y, hirose e, watanabe h. allorecognition in compound ascidians. int. j. dev. biol. 38: 237247, 1994. sala m. differenziamento in vitro di cellule della linea sanguigna da elementi mesenchimali di larva di botryllus schlosseri . atti mem. acc. pat. sci. lett. arti 85: 275-293, 1973. satoh n, levine m. surfing with the tunicates into the post-genome era. genes dev. 19: 24072411, 2005. schlumpberger jm, weissman il, scofield vl. separation and labeling of specific subpopulations of botryllus blood cells. j. exp. zool. 229: 401-411, 1984. schubert m, escriva h, xavier-neto j, laudet v. amphioxus and tunicates as evolutionary model systems. trends ecol. evol. 21: 269277, 2006. scofield vl, schlumpberger jm, west la, weissman il. protochordate allorecognition is controlled by a mhc-like gene system. nature 295: 499-502, 1982. sekine h, kenjo a, azumi k, ohi g, takahashi m, kasukawa r, et al. an ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, halocynthia roretzi. j. immunol. 167: 4504-4510, 2001. shida k, terajima d, uchino r, ikawa s, ikeda m, asano k, et al. hemocytes of ciona intestinalis express multiple genes involved in innate immune host defense. biochem. biophys. res. comm. 302: 207-218, 2003. shirae m, hirose e, saito y. behavior of hemocytes in the allorejection reaction in two compound ascidians, botryllus scalaris and symplegma reptans. biol. bull. 197: 188-197, 1999. 73 shirae m, saito y. a comparison of hemocytes and their phenoloxidase activity among botryllid ascidians. zool. sci. 17: 881-891, 2000. taneda y, saito y, watanabe h. self or non-self discrimination in ascidians. zool. sci. 2: 433442, 1985. shirae m, ballarin l, frizzo a, saito y, hirose e. involvement of quinones and phenoloxidase in the allorejection reaction in a colonial ascidian, botrylloides simodensis: histochemical and immunohistochemical study. mar. biol. 141: 659-665, 2002. taylor sw, ross mm, waite jh. novel 3,4-diand 3,4,5-trihydroxyphenylalanine-containing polypeptides from the blood cells of the ascidian ascidia ceratodes and molgula manhattensis. arch. biochem. biophys. 324: 228-240, 1995. tiozzo s, ballarin l, burighel p, zaniolo g. programmed cell death in vegetative development: apoptosis during the colonial life cycle of the ascidian botryllus schlosseri. tissue cell 38: 193–201, 2006. smith mj. the blood cells and tunic of the ascidian halocynthia aurantium (pallas). i. hematology, tunic morphology, and partitioning of cells between blood and tunic. biol. bull. 138: 354378, 1970. vasta gr, warr gw, marchalonis jj. tunicate lectins: distribution and specificity. comp. biochem. physiol. 73b: 887-900, 1982. smith mj, kim d, horenstein b, nakanishi k, kustin k. unraveling the chemistry of tunichrome. acc. chem. res. 24: 117-124, 1991. vasta gr, marchalonis jj, decker jm. binding and mitogenic properties of a galactosylspecific lectin from the tunicate didemnum candidum for murine thymocytes and splenocytes. j. immunol. 137: 3216-3233, 1986. söderhäll k, cerenius l. role of the prophenoloxidase-activating system in invertebrate immunity. curr. opin. immunol. 10: 23-28, 1998. stoner ds, rinkevich b, weissman il. heritable germ and somatic cell lineage competitions in chimeric colonial protochordates. proc. natl. acad. sci. usa 96: 9148-9153, 1999. voskoboynik a, rinkevich b, weiss a, moiseeva e, reznick az. macrophage involvement for successful degeneration of apoptotic organs in the colonial urochordate botryllus schlosseri . j. exp. biol. 207: 2409-2416, 2004. sugino ym, tsuji y, kawamura k. an ultrastructural study of blood cells in the ascidian, polyandrocarpa mysakiensis: their classification and behavioral characteristics. mem. fac. sci. kochi univ. series d (biology) 14: 33-41, 1993. waite jh. the dopa ephemera: a recurrent motif in invertebrates. biol. bull. 183: 178-184, 1992. suzuki t, takagi t, furokuohri t, kawamura k, nakauchi ma. calcium-dependent, galactosebinding lectin from the tunicate, polyandrocarpa misakiensis. j. biol. chem. 265: 1274-1281, 1990. wasylyk jm, biskupiak je, costello ce, ireland cm. cyclic peptide structures from the tunicate lissoclinum patella by fab mass spectrometry. j. org. chem. 48: 4445-4449, 1983. watanabe h, taneda y. self or non-self recognition in compound ascidians. amer. zool. 22: 775782, 1982. tanaka k. allogeneic inhibition in a compound ascidian, botryllus primigenus oka. ii. cellular and humoral responses in “nonfusion” reaction. cell. immunol. 7: 427-443, 1973. watterson rl. asexual reproduction in the colonial tunicate, botryllus schlosseri (pallas) savigny, with special reference to the developmental history of intersiphonal bands of pigment cells. biol. bull. 88: 71-103, 1945. tanaka k. allogeneic distinction in botryllus primigenus and other colonial ascidians. in: wa hildemann, aa benedict (eds), immunologic phylogeny. plenum press, new york, london, pp 115-124, 1975. wright ad, goclik e, könig gm, kaminsky r. lepadins d-f: antiplasmodial and antitrypanosomal decahydroquinoline derivatives from the tropical marine tunicate didemnum sp. j. med. chem. 45: 3067-3072, 2002. tanaka k, watanabe h. allogeneic inhibition in a compound ascidian, botryllus primigenus oka. i. processes and features of “nonfusion” reaction. cell. immunol. 7: 410-426, 1973. taneda y. simultaneous occurrence of fusion and nonfusion reaction in two colonies in contact of the compound ascidian, botryllus primigenus. dev. comp. immunol. 9: 370-375, 1985. yokobori si, watanabe y, oshima t. mitochondrial genome of ciona savignyi (urochordata, ascidiacea, enterogona): comparison of gene arrangement and trna genes with halocynthia roretzi mitochondrial genome. j. mol. evol. 57: 574–587, 2003. taneda y, watanabe h. studies on colony specificity in the compound ascidian, botryllus primigenus oka. i. initiation of “nonfusion” reaction with special reference to blood cell infiltration. dev. comp. immunol. 6: 43-52, 1982a. zaniolo g. histology of the ascidian botryllus schlosseri tunic: in particular, the test cells. boll. zool. 48: 169-178, 1981. zaniolo g, manni l, ballarin l. colony specificity in botrylloides leachi. i. morphological aspects. inv. surv. j. 3: 125-136, 2006. taneda y, watanabe h. studies on colony specificity in the compound ascidian, botryllus primigenus oka. ii. in vivo bioassay for analyzing the mechanism of “nonfusion” reaction. dev. comp. immunol. 6: 243-252, 1982b. zucchetti i, marino r, pinto mr, lambris jd, du pasquier l, de santis r. cicd94-1, an ascidian multipurpose c-type lectin-like receptor expressed in ciona intestinalis hemocytes and larval neural structures. j. immunol. 162: 387-391, 2007. taneda y, watanabe h. effects of x-rradiation on colony specificità in the compound ascidian, botryllus primigenus oka. dev. comp. immunol. 6: 665-673, 1982c. 74 review immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art immunity in ascidians immunity in compound ascidians immunocytes of compound ascidians non-self recognition phagocytosis humoral factors cytokines immunity and xenobiotics acknowledgements references isj 6: xx-yy, 2009 isj 6: 59-77, 2009 issn 1824-307x review the lipopolysaccharide-activated innate immune response network of the horseshoe crab s kawabata, t koshiba, t shibata department of biology, faculty of sciences, kyushu university, fukuoka 812-8581, japan accepted may 20, 2009 abstract primary stimulation of the horseshoe crab innate immune system by bacterial lipopolysaccharide (lps) activates a network of responses to ensure host defense against invading pathogens. granular hemocytes selectively respond to lps via a g protein-dependent exocytic pathway that critically depends on the proteolytic activity of the lps-responsive coagulation factor c. in response to stimulation by lps, the hemocyte secretes transglutaminase (tgase) and several kinds of defense molecules, such as coagulation factors, lectins, antimicrobial peptides, and protein substrates for tgase. lps-induced hemocyte exocytosis is enhanced by a feedback mechanism in which the antimicrobial peptide tachyplesin serves as an endogenous mediator. the coagulation cascade triggered by lps or β-1,3-d-glucans results in the formation of coagulin fibrils that are subsequently stabilized by tgase-dependent cross-linking. a cuticle-derived chitin-binding protein additionally forms a tgase-stabilized mesh at sites of injury. invading pathogens are agglutinated by both hemocyteand plasma-derived lectins. in addition, the proclotting enzyme and tachyplesin functionally convert hemocyanin to phenoloxidase. in the plasma, coagulation factor c acts an lps-sensitive complement c3 convertase on the surface of gram-negative bacteria. in this manner, lps-induced hemocyte exocytosis leads not only to coagulation but also activates a sophisticated innate immune response network that coordinately effects pathogen recognition, prophenoloxidase activation, pathogen clearance, and tgase-dependent wound healing. key words: horseshoe crab; lipopolysaccharide; innate immunity; hemolymph coagulation; transglutaminase; complement c3 introduction innate immunity, which defends the host against infectious pathogens, is an ancient and ubiquitous immune system in both vertebrates and invertebrates. each species employs a variety of environment-specific adaptations to ensure host defense, whereas a generalized recognition strategy against invading pathogens underlies the innate immune reaction. the innate immune system recognizes broadly conserved microbial cell wall components known as pathogen-associated molecular patterns (pamps), such as lipopolysaccharides (lps) of gram-negative bacteria, peptidoglycans of gram-positive bacteria, and β-1,3-d-glucans of fungi via pattern-recognition ___________________________________________________________________________ corresponding author: shun-ichiro kawabata 6-10-1 hakozaki higashi-ku, department of biology faculty of sciences, kyushu university fukuoka 812-8581, japan e-mail: skawascb@kyudai.jp proteins (janeway, 1989; hoffmann, 2003; akira et al., 2006). innate immune systems in invertebrates consist of pathways that promote recognition of pathogen-associated macromolecules, hemolymph coagulation, phenoloxidase-mediated melanization, cell agglutination, antimicrobial activity, and phagocytosis (nappi et al., 2004; theopold et al., 2004; iwanaga and lee, 2005; kurata et al., 2006; nakanishi and shiratsuchi, 2006). the horseshoe crab belongs to the class merostomata and is phylogenetically more closely related to arachnoidea than it is to crustacea. fossils of horseshoe crabs, such as mesolimulus walchi and limulus coffini, have been found in deposits from the paleozoic era to the cenozoic era in europe and north america (størmer, 1952). extant horseshoe crabs comprise four species, limulus polyphemus, tachypleus tridentatus, t. gigas, and carcinoscorpius rotundicauda, each having a distinct geographic distribution; l. polyphemus is distributed along the east coast of north america, 59 fig. 1 lps-activated innate immune response network of the horseshoe crab. granular hemocytes selectively respond to lps via a g protein-dependent exocytic pathway that critically depends on the proteolytic activity of the lps-responsive coagulation factor c. in response to stimulation by lps, the hemocyte secretes several kinds of defense molecules, such as coagulation factors, lectins, antimicrobial peptides, and protein substrates for tgase involved in protein cross-linking. lps-induced hemocyte exocytosis is enhanced by a feedback mechanism in which the antimicrobial peptide tachyplesin serves as an endogenous mediator. the coagulation cascade triggered by lps or β-1,3-d-glucans results in the formation of coagulin fibrils that are subsequently stabilized by tgase-dependent cross-linking with stablin and proxin. a cuticle-derived chitin-binding protein caraxin additionally forms a tgase-stabilized mesh at sites of injury. invading pathogens are agglutinated by both hemocyteand plasma-derived tachylectins and crp. in addition, the proclotting enzyme and tachyplesin functionally convert hemocyanin to phenoloxidase. in the plasma, coagulation factor c also acts an lps-sensitive complement c3 convertase on the surface of gram-negative bacteria. an immunocompetent cell with phagocytotic activity against gram-negative bacteria has not been identified in the horseshoe crab and the complement-dependent clearance system of invading pathogens remains to be examined. po, phenoloxidase; plc, phospholipase c; pip2, phosphatidylinositol-4,5-biphosphate; ip3, inositol-1,4,5-triphosphate; er, endoplasmic reticulum. and the other three species are mainly distributed throughout southeast asia. in japan, t. tridentatus inhabits coastal areas of the northern part of kyushu island as well as the inland sea. t. tridentatus has proven to be a suitable model system for the investigation of arthropod immunity, since, in addition to having a sophisticated innate immune system, it is relatively long-lived; the embryo molts four times within the fertilized egg, and after hatching it molts every year over 15 years to become a mature adult (sekiguchi et al., 1988). here we review our current knowledge of horseshoe crab innate immunity at the molecular level with an emphasis on the importance of hemocytes and hemolymph plasma. lps-induced hemocyte exocytosis and its endogenous amplification system in t. tridentatus, granular hemocytes, as determined by morphological classification, constitute 99 % of all hemocytes, and play a key role in the innate immune system (iwanaga et al., 1998; iwanaga, 2002; kawabata and tsuda, 2002). horseshoe crab hemocytes respond selectively to lps but not to other pamps, such as β-1,3-d-glucans and peptidoglycans (ariki et al., 2004). a variety of defense molecules are stored in the secretory granules of the hemocyte; large granules contain serine protease zymogens for hemolymph coagulation (factor c, factor g, factor b, 60 fig. 2 factor c is a membrane associated lps sensor on hemocytes. hemocytes were treated without (left panel) and with (right panels) fitc-labeled lps (green), and stained with a monoclonal antibody (2c12) against factor c. for the detection, cy3-conjugated anti-mouse secondary antibody was used (red). arrowheads indicate lps co-localized with membrane bound factor c. bar = 10 μm. and the proclotting enzyme), the clottable protein coagulogen, serine protease inhibitors (serpins), lectins, and substrates for transglutaminase (tgase), whereas small granules contains antimicrobial peptides. in response to stimulation by lps, these defense molecules are rapidly secreted by the hemocyte (fig. 1). factor c is a unique lps-responsive serine protease zymogen that is stored in the large granules of hemocytes and acts as an lps sensor to potentiate hemocyte exocytosis. upon activation by lps, factor c initiates hemocyte exocytosis via a g-protein-dependent exocytic pathway that is dependent upon the proteolytic activity of factor c. in this respect, the activation of hemocytes by factor c is analogous to the thrombin-thrombin receptor (the protease-activated g protein-coupled receptor, par) signaling axis in mammalian platelets (ariki et al., 2004). hemocyte exocytosis can be quantitatively assayed by elisa using an antibody against a granular component such as coagulogen in the presence of 50 mm mg2+ and 10 mm ca2+, equivalent to the concentrations of these cations in hemolymph plasma. exclusion of divalent cations from the assay buffer inhibits exocytosis even at high concentrations of lps. moreover, in the absence of lps, hemocyte exocytosis can be induced by synthetic hexapeptides corresponding to the tethered ligands of mammalian pars, supporting the notion of a par-like receptor on the hemocyte surface. immunofluorescence microscopy of the hemocyte using an anti-factor c antibody detects factor c, which is localized in a punctate distribution on the hemocyte surface (kurata et al., 2006; koshiba et al., 2007) (fig. 2). when hemocytes are incubated with fitc-labeled lps, the factor c antigen co-localizes with fitc-lps accumulated on the cell surface (koshiba et al., 2007). the accumulation of fitc-lps is not observed on hemocytes fixed with formaldehyde, as would be expected with chemical modification and inactivation of cell surface proteins. these results also suggest that factor c is a membrane-bound lps sensor on the hemocyte surface. factor c has no obvious transmembrane domain within in its sequence, whereas it strongly interacts with acidic phospholipids (nakamura et al., 1988a). surface plasmon resonance analyses indicate that factor c interacts with phosphatidylserine (kd = 2.5×10 -9 m) and phosphatidylinositol (kd = 4.7×10 -9 m) as well as with cholesterol (kd = 1.4×10 -9 m) (ariki et al., 2004). the interaction of factor c with lps (kd = 7.6×10 -10 m) is 61 62 competitively inhibited by the addition of the acidic phospholipids. in contrast, cholesterol does not inhibit the interaction of factor c with lps, suggesting that factor c interacts with cholesterol through a binding site that is distinct from that for lps, and raising the possibility that factor c may be localized on cholesterol-rich microdomains or lipid rafts on the hemocyte membrane. the horseshoe crab hemocyte has an endogenous positive feedback mechanism for lps-induced hemocyte exocytosis (ozaki et al., 2005). the hemolymph contains hemocytes at ~106 cells/ml. lps-induced hemocyte exocytosis is highly dependent on the cell density, namely, an increase in cell density from 0.05×106 to 0.8×106 cells/ml results in a 106-fold change in the apparent lps sensitivity (from 10-7 to 10-13 g/ml of lps), suggesting the presence of feedback mechanism for secretion via an unknown secretagogue secreted from hemocytes in response to the stimulation by lps. interestingly, tachyplesin in the exocytosed fluid acts as a secondary secretagogue, thereby dramatically enhancing the sensitivity of the hemocyte to lps. tachyplesin (17 residues) is a potent antimicrobial peptide and one of the most abundant components stored in the hemocyte (nakamura et al., 1988b; shigenaga et al., 1990). the effective concentration of tachyplesin required for hemocyte exocytosis ranges from 5 to 10 μm, indicating that a high concentration of tachyplesin is required to act as an effective endogenous secretagogue. tachyplesin has structural properties in common with mastoparan, a basic tetradecapeptide from wasp venom. mastoparan interacts directly with g proteins without direct stimulation of the upstream receptor, and induces exocytosis in the mast cell (higashijima et al., 1988). consistent with these findings, mastoparan is able to induce hemocyte exocytosis in t. tridentatus (ariki et al., 2004). moreover, tachyplesin binds to bovine g protein an equilibrium dissociation constant (kd) of 8.8×10 -7 m (ozaki et al., 2005). these data suggest that tachyplesin interacts with g-proteins in the hemocyte in a manner similar to that of mastoparan. in addition, tachyplesin has an ability to bind to hemocyanin (nagai et al., 2001), the major protein in the hemolymph plasma, suggesting that hemocyanin may serve as a sink for tachyplesin released from hemocytes, thereby spatially restricting its hemocyte-stimulating effect to the site of infection. on the other hand, insect and mammals conserve a signaling pathway of the innate immune system through cell-surface receptors called tolls and toll-like receptors (hoffmann, 2003; akira et al., 2006). a toll-like receptor (ttoll) has been identified in t. tridentatus, which is most closely related to drosophila toll in both domain architecture and overall length (inamori et al., 2000, 2004). the two receptors show a significant sequence identity between their tir domains (39 %). interestingly, spätzle, a protein ligand for drosophila toll, shows significant structurally similarity to horseshoe crab coagulogen (see "hemolymph coagulation"). moreover, ttoll is nonspecifically expressed in all tissues examined (inamori et al., 2004), suggesting that ttoll does not act as an lps receptor on granular hemocytes. in addition, nf-κb and iκb homologues (crnf-κb and criκb) have been identified in the horseshoe crab c. rotundicauda (wang et al., 2006). gram-negative bacteria infection causes degradation of criκb and nuclear translocation of crnf-κb, leading to up-regulation of immune-related gene expression, including nitric oxide synthase and factor c, indicating that the nf-κb/iκb signaling cascade remains well conserved from horseshoe crabs to mammals, playing a fundamental role in regulating the expression of critical immune defense molecules. hemolymph coagulation the hemocyte releases coagulation factors by lps-induced exocytosis, leading to the activation of the proteolytic coagulation cascade (fig. 1). factor c secreted from hemocytes is autocatalytically activated in the presence of gram-negative bacteria or lps. the resulting activated factor c activates coagulation factor b, which in turn converts the proclotting enzyme into the clotting enzyme. the clotting enzyme then promotes the proteolytic conversion of coagulogen to coagulin, which spontaneously forms an insoluble polymer. alternatively, activated factor g in the presence of β-1,3-d-glucans triggers the activation of the proclotting enzyme to the clotting enzyme. in this manner, factor c and factor g independently serve to couple the recognition of lps and β-1,3-d-glucans, respectively, to the formation of a physical barrier at the site of microbial invasion. the vertebrate coagulation system acts locally on the phospholipid surface in cooperation with ca2+ at the site of vascular injury. in an analogous fashion, hemolymph coagulation in the horseshoe is restricted to the surfaces of invading pathogens, such as gram-negative bacteria and fungi. this mechanistic similarity between the coagulation cascades of vertebrates and horseshoe crabs may lead to the erroneous assumption of a common evolutionary origin (fig. 3). in fact, fibrinogen homologues of the horseshoe crab, named tachylectins-5a and -5b, act as non-self recognizing proteins rather than as target proteins of the coagulation cascade (gokudan et al., 1999). also coagulogen has no structural similarity or evolutionary relatedness to fibrinogen (bergner et al., 1996). a protease cascade in drosophila has been well characterized as the morphogenetic cascade for determining embryonic dorsal-ventral polarity, leading to the production of the toll ligand spätzle (belvin and anderson, 1996). the drosophila toll pathway additionally controls resistance to fungal and gram-positive bacterial infections (ferrandon et al., 2007). spätzle, belongs to nerve growth factor family and possesses a structural similarity to horseshoe crab coagulogen (smith and delotto, 1992; bergner et al., 1996; bergner et al., 1997). in addition, a clip-like domain located in the n-terminal region of horseshoe crab coagulation factor b and the proclotting enzyme, originally identified in the proclotting enzyme as a disulfide-knotted domain, fig. 3 comparison of proteolytic cascades between horseshoe crab hemolymph coagulation, mammalian blood coagulation, and drosophila toll pathway. the serine protease zymogens are indicated by asterisks. homologous proteins are connected by ladders. has been identified in the proteins snake and easter of the drosophila toll pathway (muta et al., 1990, 1993). the structural similarity between coagulogen to spätzle, as well as that between the serine protease zymogens participating in the two cascades, suggests that the two functionally distinct cascades may have a common evolutionary origin (fig. 3) (krem and di cera, 2002; kawabata et al., 2003). the coagulation cascade in the horseshoe crab is regulated by three types of serpins that form stable 1:1 covalent complexes with target coagulation proteases (fig. 1); serpins-1, -2, and -3 inhibit activated factor c, the clotting enzyme, and activated factor g, respectively (miura et al., 1994, 1995; lal agarwala et al., 1996). all three serpins are stored in the large granules of hemocytes and are secreted upon hemocyte exocytosis in response to stimulation by lps. these serpins appear to prevent diffusion of the activated forms of coagulation factors by scavenging activated proteases that escape into the hemolymph from the surface of microbes at the site of injury, and thereby prevent unnecessary clot formation. horseshoe crab serpins are more closely related to mammalian serpins than they are to insect serpins. for example, serpin-1 shows higher sequence identities to human plasminogen activator inhibitor (40 %) and human neutrophil elastase inhibitor (39 %) than to an elastase inhibitor from manduca sexta (29 %) and silkworm antichymotrypsin (27 %) (miura et al., 1994). mammalian serpin-protease complexes are hypothesized to be rapidly cleared through a cell-surface receptor, which recognizes a hydrophobic consensus sequence that is selectively exposed on the complexed forms of serpins; for instance, phe-val-phe-leu-met in the c-terminal region of α1-antitrypsin (joslin et al., 1991). this consensus sequence is conserved in horseshoe crab serpins in the corresponding region (ex. phe-val-phe-phe-ile for serpin-1), suggesting that a similar clearance mechanism exists in horseshoe crabs. lps recognition by factor c factor c is a multidomain glycoprotein with an apparent molecular mass of 120 kda. in addition to a typical serine protease domain at the c-terminus, factor c contains a cys-rich region, an epidermal factor (egf)-like domain, five complement control protein (ccp) modules, a c-type lectin domain, and an lccl module (derived from a conserved domain of limulus factor c, coch-5b2, and lg11) (muta et al., 1991; trexler et al., 2000) (fig. 4a). structure-function analyses reveal that the lps-binding site in factor c is present in the n-terminal cys-rich region of the molecule and contains a tripeptide sequence (-arg36-trp37-arg38-) consisting of an aromatic residue flanked by two basic residues (koshiba et al., 2007). a recombinant version of the cys-rich/egf fragment (positions 1-116) with paired substitutions of arg36/arg38 to 63 fig. 4 schematic domain structure of horseshoe crab innate immune proteins. (a) domain structure of factor c. egf, epidermal growth factor; ccp, complement control protein; lccl, limulus factor c, coch-5b2, and lg11. (b) domain structure of factor g. z1 and z2, xylanase z-like modules. (c) domain structures of tachylectins. tl, tachylectin. (d) domain structure of ttc3. cub, complement-urchin-bone. glu36/glu38 (r36e/r38e mutant) is incapable of binding to lps. moreover, a mutant protein with substitution of trp37 to ala37 also lacks the ability to bind lps. these data indicate the essential nature of the tripeptide motif for lps recognition by factor c. the cys-rich/egf fragment interacts with lps with kd = 2.0×10 -10 m, which is similar to that of the wild-type protein (shibata et al., unpublished data). this tripeptide motif is conserved in horseshoe crab anti-lps factor (aketagawa et al., 1986) and several mammalian lps-recognizing proteins such as lps-binding protein (schumann et al., 1990) and bactericidal/permeability-increasing protein (marra et al., 1990) (fig. 5a). anti-lps factor (102 residues) has been identified as an inhibitor for lps-mediated hemolymph coagulation. anti-lps factor is stored in the large granule of hemocytes, and shows sequence similarity to the α-lactoalbumin/lysozyme family. the crystal structure shows that anti-lps factor has a single domain consisting of three α-helices packed against a four-stranded β-sheet to form a wedge-shaped molecule with a striking charge distribution and amphipathicity (hoess et al., 1993). the binding site for lps likely encompasses the extended amphiphilic loop with the tripeptide motif (-lys43-trp44-lys45-) (pristovsek et al., 2005) (fig. 5b). in addition to its physical association with factor c, lps promotes its autocatalytic activation upon binding to its cys-rich region. in contrast to wild-type factor c, the r36e/r38e variant is incapable of autoactivation in the presence of lps (fig. 6) (koshiba et al., unpublished data). β-1,3-d-glucan recognition by factor g factor g is another pattern-recognition protein in the coagulation cascade that acts as a sensitive and specific sensor for β-1,3-d-glucans. in other arthropods, such as crustaceans and insects, the recognition of β-1,3-d-glucans also triggers a serine protease cascade, leading to the activation of prophenoloxidase, a key enzyme in the melanization of pathogens and damaged tissues (cerenius and söderhäll, 2004; kanost et al., 2004; vetvicka and sima, 2004). in vertebrates, the recognition of β-1,3-d-glucans by dectin-1, a c-type lectin family member, potentiates the production of cytokines and antifungal reactive oxygen species by dendritic cells and macrophages (brown, 2006). factor g is a heterodimeric serine protease zymogen composed of two non-covalently associated subunits, α and β (seki et al., 1994) (fig. 4b). the β subunit contains a serine protease domain, 64 fig. 5 a model of bacterial recognition by membrane associated factor c via n-terminal cys-rich region. (a) the n-terminal sequence of factor c containing cys-rich and egf-like regions (r1-g116) is shown. the tripeptide motifs in the cys-rich region are highlighted. hlbp, human lps-binding protein; hbpi, human bacterial permeability-increasing protein; hmd-2, human md-2; lalf, limulus anti-lps factor; and rcap18, rabbit cationic antimicrobial protein. (b) the recognition of lps on gram-negative bacteria via membrane-associated factor c initiates the horseshoe crab innate immune response. the modeled 3d structure of the lalf-lps complex is shown in the right panel (pristovsek et al., 2005) and this model may resemble that of the complex between n-terminal elements of factor c and lps. and the α subunit acts as a pattern-recognition subunit. the α subunit comprises three types of non-catalytic glycosidase-like modules: a single β-1,3-d-glucanase a1-like module, three tandem xylanase a-like modules, and two tandem xylanase z-like modules. of the three types of glycosidase-like modules, the two xylanase z-like modules (z1 and z2) have been identified as independent binding sites for β-1,3-d-glucans (takaki et al., 2002). this observation, taken together with the high degree of sequence identity between z1 and z2 (91 %), suggests that duplicated binding sites for β-1,3-d-glucans may increase avidity to allow stable and specific recognition of pathogens. 65 both z1 and z2 show significant sequence similarity to a carbohydrate-binding module of endoglucanase 5a from the aerobic soil bacterium cellvibrio mixtus (45 % sequence identity). endoglucanase 5a from c. mixtus contains an n-terminal catalytic domain and two tandem repeats of non-catalytic family 6 carbohydrate-binding modules, cmcbm6-1 and cmcbm6-2 (fontes et al., 1998). our recent structural studies of recombinant z2 domain by nuclear magnetic resonance spectroscopy clearly indicate that the ligand-binding site in z2 is located in a cleft on a β-sheet in a predicted β-sandwich structure, which is superimposed onto cleft b in cmcbm6-2 (ueda et al., unpublished data). pattern recognition for β-1,3-d-glucans by factor g may be accomplished by a carbohydrate-binding cleft that is evolutionally conserved among invertebrates and bacteria. a crystal structure of the extracellular domain of mouse fig. 6 the r36/38e mutant of factor c abolishes the autocatalytic activation of factor c by gram-negative bacteria. (a) the wild-type factor c was specifically activated by gram-negative bacteria (e. coli b) but not by gram-positive bacteria (s. aureus) and fungi (pichia. pastoris), whereas the r36/38e mutant was not converted into the active form by e. coli b. the wild-type and the mutant factor c contained a myc-epitope tag at their c-terminal ends and were identified by western blotting with the anti-myc monoclonal antibody (9e10). the zymogen form and the active form (the light chain containing the protease domain) of factor c are indicated by the arrows a and b, respectively. (b) the time course for the autocatalytic activation of the wild-type and mutant factor c in the presence of e. coli b. dectin-1 has been determined (brown et al., 2007), and the putative ligand-binding site of dectin-1 exhibits no structural similarity to that of the predicted structure of z2. the “sliding” of factor g molecules on β-1,3-d-glucans seems be essential to increase the frequency of collision between factor g molecules and resultant autocatalytic activation (takaki et al., 2002). an insoluble β-1,3-d-glucan (curdlan) activates factor g at a minimum concentration of 0.01 ng/ml, whereas a 1000-fold higher concentration of a soluble β-1,3-d-glucan (laminarin) is required for activation (tanaka et al., 1991). β-1,3-d-glucans have specific molecular structures, and high molecular weight β-1,3-d-glucans have higher ordered structures such as triple helices (bohn and bemiller, 1995). therefore, to induce the autoactivation efficiently, the dual binding of the repeating modules may occur in the case of the interaction of factor g with curdlan but not with laminarin. the importance of dual binding of repeating modules to β-1,3-d-glucans for the autoactivation of factor g remains to be examined. pamp recognition by tachylectins given the complexity and diversity of pathogen-associated carbohydrate moieties and the essential nature of specific carbohydrate recognition in innate immunity, it has been proposed that a third biochemical alphabet, the sugar code, is indispensable (gabius et al., 2002). in t. tridentatus, four types of lectins have been identified in the large granule of hemocytes, including tachylectin-1 (saito et al., 1995a), tachylectin-2 (okino et al., 1995), tachylectin-3 (saito et al., 1997), tachylectin-4 (inamori et al., 1999), all of which are secreted upon lps-induced hemocyte exocytosis (fig. 1). although each tachylectin recognizes pamps, their carbohydrate ligand specificities differ. tachylectin-1 interacts with 2-keto-3-deoxyoctonate on the surface of gram-negative bacteria, whereas tachylectin-2 binds to glcnac or galnac and recognizes lipoteichoic acids of gram-positive bacteria. in contrast, tachylectin-3 specifically recognizes a certain sugar moiety on o-antigens of s-type lps from several specific gram-negative bacteria, such as escherichia coli o111:b4. tachylectin-4 also recognizes the o-antigen of e. coli o111:b4 and shows the ligand specificity for colitose (3-deoxy-l-fucose), a unique sugar present in the o-antigen. in addition, an isoprotein of tachylectin-1, named tachylectin-p, has also been found in the perivitelline space of the egg (nagai et al., 1999). the crystal structure of tachylectin-2 clearly points out the importance of multivalency for achieving high specificity and high affinity (beisel et al., 1999). tachylectin-2 (236 residues) belongs to the so-called wd-protein family, characterized by the repetitive sequence of 40-60 residues containing trp and asp residues (neer et al., 1994). tachylectin-2 contains five tandem wd-repeats of 47 residues and adopts a five-bladed β-propeller structure with five equivalent glcnac/galnac-binding site (fig. 7a). each propeller blade has an independent binding site for the ligand. the specific recognition by tachylectin-2 is reinforced by the short distance between the individual binding sites (25 å), according 66 fig. 7 crystal structures of tachylectins-2 and -5a. (a) the 5-fold β-propeller structure of tachylectin-2 in complex with glcnac. (b) oligomeric arrangement of tachylectin-5a subunits around the 4-fold crystallographic axis. to the pentagonal geometry, required to interact with pamps. tachylectin-1 (221 residues) and tachylectin-3 (123 residues), despite lacking significant overall sequence similarity to tachylectin-2, also consist of wd repeats, with six-wd-repeats for tachyectin-1 and two-wd-repeats for tachylectin-3, suggesting β-propeller structures analogous to that of tachylectin-2. ultracentrifugation analysis shows that tachylectin-3 is present in dimer in solution. in contrast, tachylectin-4 (232 residues) is an oligomeric glycoprotein of 470 kda and is homologous to the n-terminal domain of xenopus pentraxin-1 (fig. 4c). the function of the n-terminal domain of pentraxin-1 is unknown. hemolymph plasma also contains several lectins, such as isoforms of tachylectin-1 (chiou et al., 2000; chen et al., 2001) and three types of c-reactive proteins (crps), all of which exhibit functional and structural diversity (iwaki et al., 1999). human crp is an acute-phase reactant, its concentration increasing rapidly in response to stress, injury or infection, and it belongs to a protein family of pentraxin (osmand et al., 1977; tennent and pepys, 1994). horseshoe crab crp is a predominant lps-binding protein and is up-regulated at transcript levels by pseudomonas infection, suggesting the importance of horseshoe crab crp as a conserved molecule for pathogen recognition (ng et al., 2004). crp from the horseshoe crab l. polyphemus forms extended fibrilar structures that encapsulate liposomes in the presence of ca2+ (harrington et al., 2009). furthermore, the membranes of limulus crp-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria, suggesting the protein's role as a primary defense molecule, acting in the entrapment and killing of potential pathogens. in addition, tachylectins-5a and -5b, with binding specificity for acetyl-group have been identified in hemolymph plasma (gokudan et al., 1999). the overall sequence identity between tachylectins-5a (269 residues) and -5b (289 residues) is 45 %. about two thirds of the total hemagglutinating activity in the hemolymph plasma can be attributed to tachylectins-5 a and -5b, which strongly agglutinate all types of human erythrocytes at a minimal concentration of 0.004 μg/ml, and also agglutinate both gram-negative and gram-positive bacteria. the concentration of tachylectins-5a or -5b in the plasma is at least 10 μg/ml, suggesting that they play an important role in the recognition of invading pathogens at the forefront of the innate immune system. tachylectins-5a and -5b show significant sequence similarity to the c-terminal globular domain of the γ-chain of vertebrate fibrinogens and fibrinogen-related proteins, with pairwise identities ranging from 35 to 51 % and highest for the fibrinogen-like domain of human ficolin. ficolin is composed of an n-terminal cys-containing oligomerization segment followed by a collagen-like domain and the c-terminal fibrinogen-like domain, forming an overall structure that resembles a bundle-of-tulips (matsushita and fujita, 2001; fujita, 2002). in plasma, ficolin exists as a complex with specific serine proteases, the mannose-binding lectin-associated serine proteases, through the collagen-like domain, leading to complement activation after the recognition of invading pathogens. an analogous collagenous domain is absent in the corresponding regions of tachylecins-5a and 5b. 67 68 the crystal structure of tachylecitn-5a is readily superimposed onto that of the c-terminal polymerization domain of the γ-chain of fibrinogen (kairies et al., 2001) (fig. 7b). there is a dramatic structural similarity between these proteins, not only with respect to the overall topology, but also within their ca2+-binding sites. during the final stage of mammalian blood coagulation, thrombin cleaves the n-terminal portion of the fibrinogen α-chain, and the newly created n-terminal sequence containing gly-pro-arg-prois recognized by the polymerization pocket on the γ-chain to form fibrin protofibrils (pratt et al, 1997; spraggon et al., 1997). the polymerization pocket within the γ-chain structurally corresponds to the acetyl group-binding site of tachylectin-5a, a finding that highlights the evolutionary connection between hemostasis and non-self recognition. tachylectin-5a is present in oligomer in hemolymph plasma, and its propeller-like arrangement is evident by electron microscopy (gokudan et al., 1999). given that tachylectins -2 and -5 exhibit virtually no sideor main-chain conformational changes upon ligand binding, it is likely that the polyvalent nature of these molecules underlies their avidity by allowing them to recognize specific densities or clustering patterns ligands on the surface of pathogens. roles of antimicrobial peptides in innate immunity in arthropods, antimicrobial peptides are widely recognized to be very important for host defense against, and killing of, invasive microbes. however, under isotonic conditions (0.5 m nacl concentration for horseshoe crabs), the apparent antimicrobial activities of horseshoe crab antimicrobial peptides are dramatically reduced to those under hypotonic conditions. therefore, the antimicrobial peptides of horseshoe crabs may act as endogenous mediators and regulators in the innate immune system by enhancing such processes as hemocyte exocytosis and conversion of hemocyanin to phenoloxidase (described below). in t. tridentatus, the small granules of hemocytes contain several kinds of cysteine-rich peptides with antimicrobial activities, including tachyplesin (nakamura et al., 1988b), tachystatins (osaki et al., 1999), big defensin (saito et al., 1995b; kawabata et al., 1997), and tachycitin (kawabata et al., 1996). the ic50 values for these antimicrobial peptides under hypotonic conditions are listed in table 1 (osaki et al., 1999). all of the antimicrobial peptides identified in the horseshoe crab have an affinity for chitin, primary target of the innate immune system. tachyplesin significantly inhibits the growth of gram-negative and gram-positive bacteria and fungi (table 1). tachyplesin forms a rigid hairpin loop constrained by two disulfide bridges and adopts the conformation comprising an antiparallel β-sheet connected to a β-turn (kawano et al., 1990; laederach et al., 2002; mizuguchi et al., 2005) (fig. 8a). when viewed as a planar surface, six basic residues are localized on one face, and six hydrophobic residues are distributed on the opposing face. this amphiphilic structure is presumed table 1 antimicrobial activities of horseshoe crab antimicrobial peptides -------------------------------------------------------------------- e. coli s. aureus c. albicans -------------------------------------------------------------------- ic50 (μg/ml) tachyplesin 2.5 0.3 0.2 tachystatin a 25 4.2 3.0 tachystatin b no inhibition 7.4 3.0 tachystatin c 1.2 0.8 0.9 big defensin 2.5 2.5 20 tachycitin 33 56 52 -------------------------------------------------------------------- to be closely associated with its antimicrobial activity. tachystatins a (44 residues), b (42 residues), and c (41 residues) also exhibit a broad spectrum of antimicrobial activity (table 1). of the tachystatins, tachystatin c shows the most potent antimicrobial activity, and tachystatin c, but not tachystatins a and b, additionally exhibits hemolytic activity against sheep erythrocytes. furthermore, tachystatin c has strong cell lysis activity against budding yeast. tachystatins a and b have 42 % sequence identity. tachystatin c contains a disulfide motif analogous to that found in tachystatin a, but otherwise shows very low sequence similarity to tachystatin a. tachystatin a is homologous to ω-agatoxin-iva of funnel web spider venom, a potent blocker of voltage-dependent ca2+ channels (mintz et al., 1992). despite this similarity, tachystatin a exhibits no blocking activity toward p-type ca2+ channel in rat purkinje cells. tachystatin a consists of a cysteine-stabilized triple-stranded β-sheet and shows an amphiphilicity commonly observed in membrane-interactive peptides (fujitani et al., 2002) (fig. 8b). tachystatin a shares structural similarity with ω-agatoxin-iva. interestingly, ω-agatoxin-iva also has antimicrobial and chitin-binding activities. the three dimensional structure of tachystatin a shows no structural homology with well known chitin-binding motifs, suggesting that tachystatin a belongs to a new family of chitin-binding peptides. as expected, tachystatin b shows a significant three dimensional structural similarity to tachystatin a (42 % sequence identity between tachystatins a and b ) (fujitani et al., 2007) (fig. 8c). big defensin has potent antimicrobial activities against both gram-negative and gram-positive bacteria, but not against fungi (table 1). big defensin (79 residues) is distinct from the mammalian defensins with respect to molecular size (29-34 residues for the latter). it is proteolytically cleaved by trypsin into two domains, the hydrophobic n-terminal domain and a c-terminal domain that is homologous to β-defensins. interestingly, the n-terminal domain possesses a more potent antimicrobial activity against gram-positive bacteria than the c-terminal domain. in contrast, the c-terminal domain displays more fig. 8 nmr structures of antimicrobial peptides of the horseshoe crab. (a) ball-and-stick model of tachyplesin. (b) ribbon representation of the energy-minimized average structure of tachystatin a. (c) ribbon representation of the energy-minimized average structure of tachystatin b. (d) ribbon representation of the energy-minimized average structure of big defensin. (e) ribbon representation of the energy-minimized average structure of tachycitin. potent antimicrobial activity than the n-terminal domain against gram-negative bacteria. these data suggest a new sub-class within the defensin family that possesses two discrete functional domains with different antimicrobial activities. it is noteworthy that tachylectins-5a and -5b enhance the antimicrobial activity of big defensin against gram-positive bacteria (gokudan et al., 1999). structurally, big defensin represents a new class within the defensin family; the c-terminal domain adopts a β-defensin structure, whereas the n-terminal domain forms a unique globular conformation (kouno et al., 2008) (fig. 8d). interestingly, the hydrophobic n-terminal domain, but not the c-terminal domain, undergoes a conformational change in micelle solution, which may be associated with the antimicrobial activity against gram-positive bacteria. 69 tachycitin (73 residues) contains five disulfide bridges and has no significant sequence similarity to known antimicrobial peptides. the antimicrobial activity of tachycitin is not strong by itself (table 1). however, tachycitin synergistically enhances the antimicrobial activity of big defensin as demonstrated by a 50-fold reduction in the ic50 value of big defensin against gram-negative bacteria when a small amount of tachycitin is present. the structure of tachycitin is largely divided into the n-terminal domain and the c-terminal domain (suetake et al., 2000) (fig. 8e). in the c-terminal domain, tachycitin forms a hairpin loop connecting a two-stranded β-sheet, a structural motif shared by the chitin-binding site of hevein, an antifungal peptide from the rubber tree hevea brasiliensis (broekaert et al., 1990). in hevein, the aromatic side chains of the two trp residues in this loop directly interact with chitin-derived oligosaccharides. the side chain of tyr and val residues in the corresponding loop of tachycitin are structurally analogous to these two trp residues in hevein. tachyplesin also contains the similar hairpin loop that connects a two-stranded β-sheet. the hydrophobic residues clustered on the one face of their β-hairpin loops possibly participate in chitin-binding sites. roles of tgase-dependent protein cross-linking in the innate immune system in mammals, the blood coagulation cascade culminates in the proteolytic conversion of soluble fibrinogen into insoluble fibrin. the resulting fibrin fibrils are further stabilized by intermolecular ε-(γ-glutamyl) lysine cross-linking by the plasma fig. 9 stablin as a component of the clotting mesh. (a and b) co-localization of stablin with coagulin fibrils. hemocytes were treated with 1 mg/ml lps (salmonella minnesota r595) for 1 h and stained with a monoclonal antibody against coagulogen (a) and a polyclonal antibody against stablin (b). as secondary antibodies, alexa fluor® 488 goat anti-mouse immunoglobulin and rhodamine-conjugated swine anti-rabbit immunoglobulins were used. bar = 10 μm. (c and d) bacterial immobilization by the clotting mesh. hemocytes were treated with e. coli expressing enhanced green fluorescence protein for 1 h, stained with the monoclonal antibody against coagulogen (c), and detected by a cy3-conjugated anti-mouse secondary antibody. e. coli was identified by the fluorescence of enhanced green fluorescence protein (d). bar = 10 μm. (e and f) involvement of stablin in the formation of the clotting mesh. hemocytes were preincubated with 0.1 mg/ml of an antibody against stablin (f) or without the antibody (e). hemocytes were treated with e. coli for 1 h, stained with the monoclonal antibody against coagulogen, and detected by the cy3-conjugated secondary antibody. bar = 100 μm. tgase, factor xiiia (davie et al., 1991). in crustaceans, hemolymph coagulation directly depends on the tgase-mediated cross-linking of a specific clotting protein (a vitellogenin-related protein) without prior proteolytic cleavage (doolittle and riley, 1990; hall et al., 1999; theopold et al., 2004). in horseshoe crabs, hemolymph coagulation cascade triggered by lps or β-1,3-d-glucans promotes in the conversion of coagulogen to coagulin. in the resulting coagulin homopolymers, coagulin monomers associate non-covalently in a head-to-tail manner (bergner et al., 1996; kawasaki et al., 2000). horseshoe crab tgase is not present in hemolymph plasma. it is ordinarily restricted to cytoplasm of the hemocyte and secreted via an unknown mechanism in response to stimulation by lps (tokunaga et al., 1993a, 1993b; osaki et al., 2002). although horseshoe crab tgase neither catalyzes monodansylcadaverine incorporation into coagulin nor cross-links coagulin intermolecularly, it cross-links coagulin to other hemocyte-derived proteins, such as the proline-rich protein, proxin (osaki et al., 2002) and the cysteine-rich protein stablin (matsuda et al., 2007a), resulting coagulin fibrils with enhanced stability. an anti-stablin antibody strongly inhibits the proper formation of the clotting fibrils. proxin and stablin have been detected in the large granules of hemocytes and are secreted by lps-induced exocytosis. in the absence of tgase, proxin non-covalently interacts with coagulin but not coagulogen. moreover, proxin exhibits the specific interaction with stablin (kd = 4.0×10 -9 m). in contrast, stablin interacts with lps and lipoteichoic acids and exhibits bacterial agglutinating activity against both gram-negative and gram-positive bacteria. consequently, stablin co-localizes with coagulin fibrils that are cross-linked with proxin, effectively trapping bacteria (fig. 9). in addition, stablin binds to chitin, a major component of the arthropod cuticle (kd = 1.5×10 -8 m). these data suggest that proxin and stablin promote not only the formation of the stable clotting fibrils but also the immobilization of invading microbes at injured sites. horseshoe crab tgase additionally cross-links carapace-derived chitin-binding proteins, known as caraxins, that are specifically localized to the sub-cuticular epidermis (matsuda et al., 2007b). one of these homologues, caraxin-1, comprises 135 amino acid residues and consists of nand c-terminal heptad repeats that flank a central domain consisting of a pentapeptide tandem repeat structure. recombinant caraxin-1 exists as an oligomer (~20-mer) in solution, and these oligomers are cross-linked by tgase to form an elaborate mesh 70 fig. 10 scanning electron microscopy of caraxin mesh. (a and c) caraxin-1 was incubated with tgase at 37 °c for 16 h. also, hemocytes were stimulated with 1μg/ml lps for 1 h (b and d). both samples were fixed in 4 % paraformaldehyde for 1 h. bars were 20 μm (a and c) and 2 μm (b and d). of honeycomb structures that is distinguishable, by electron microscopy, from the clotting mesh triggered by lps (fig. 10). the α-helical structures of the nand c-terminal domains of caraxin-1 are essential for proper mesh formation. horseshoe crab hemocytes are actively motile, and one of the principal functions of the hemocyte is to seal scars in the cuticle. this function is fulfilled in part by the adherence of hemocytes to injured sites (armstrong, 1985). the wound repair process of l. polyphemus was observed for 180 days by making a cut (1×40×3 mm) on the carapace (bursey, 1977). according to these observations, a coagulation plug is formed within 10 min, and the coagulum is then infiltrated by hemocytes to form a cellular plug within 24 h. the sub-cuticular epithelial cells begin to migrate into the wound after 15 days, and the epithelial cells span the wound between the cut ends of the exoskeleton by day 30, and then probably secrete cuticular components to complete the wound repair process. at the initial stage of this wound repair process sufficient quantities of tgase may be secreted from hemocytes recruited to the site of injury, and immediately activated by ca2+ in hemolymph plasma. at the same time caraxin may be secreted from the sub-cuticular epithelial cells. the resulting cross-linked clotting fibrils and caraxin mesh may function to seal the wound to stop bleeding, serve as a barrier to the entry of pathogens into the interior of the animal via the wound, and operate as a transient extracellular matrix for the migration of epithelial cells that facilitate wound healing (fig. 1). the complement system the vertebrate complement system consists of three activation pathways: the classical, lectin, and alternative pathways (klein and horejsi, 1997; fujita, 2002). the classical and the lectin pathways are serine protease cascades initiated by the recognition of microbes by antibodies and lectins, respectively. although the alternative pathway can be activated spontaneously, it is potently activated upon microbial infection. activation of each of the three pathways results in generation of a proteolytic complex known as the c3 convertase (c4bc2a in the classical and lectin pathways, or c3bbb in the alternative pathway) that plays a central role in promoting downstream responses. the c3 convertase cleaves c3 to generate c3a, a peptide also known as anaphylatoxin that attracts inflammatory cells to sites of infection. the c3 convertase concomitantly generates c3b, a large polypeptide that covalently 71 72 attaches to microbial surfaces and promotes c3 receptor-dependent phagocytosis by leukocytes as well as the formation of the membrane attack complex. vertebrate complement factors d (df) and b (bf) are involved in the activation of the alternative pathway. df is a single-chain serine protease circulating in the blood as an active protease that cleaves bf, resulting in the formation of c3bbb. although several homologs of vertebrate complement factors are known to be present in ascidians, cyclostomes, and echinoderms, the functional analog of df that triggers the alternative pathway has not been identified in protostomes (endo et al., 2006; dodds and matsushita, 2007). the complement-related protein, α2-macroglobulin, has previously been identified in horseshoe crabs (iwaki et al., 1996). recently, a homolog of complement component c3 has been identified in the horseshoe crab c. rotundicauda (crc3), indicating the presence of a complement system capable of promoting the phagocytosis of invading microbes in protostomes (zhu et al., 2005). in a phylogenetic tree of reactive thioester-containing proteins from vertebrates and invertebrates, crc3 shows the highest similarity to c3 sequences from lower deuterostomes and forms a clade with amphioxus c3, cnidaria c3-like protein, and sea urchin c3. however, despite the identification of the key complement component in protostomes, the molecular mechanism underlying c3 activation has remained unknown. in an effort to elucidate the mechanism of c3 activation in horseshoe crabs, we have isolated and characterized an ortholog of c3 (ttc3) from t. tridentatus (ariki et al., 2008). ttc3 consists of 1,716 residues with an overall domain structure that is identical to vertebrate c3, including α2-macroglobulin domains, complement-urchin-bone (cub) domains, a thioester-containing domain, an anaphylatoxin domain, and a c345c domain (fig. 4d). a thioester motif (-cys-gly-glu-gln-) at residues 1004 to 1007 and a catalytic his at the position 1116 are conserved in the deduced sequence. the sequence identity between ttc3 and crc3 (98 %) is considerably higher than that between coagulogens from the two species (90 %) (srimal et al., 1985). ttc3 forms a disulfide-linked three-chain structure (α-, β-, and γ-chains) in hemolymph plasma, unlike vertebrate c3 which is present in a two-chain form. the horseshoe crab complement system promotes the deposition of ttc3b on the surface of gram-negative or gram-positive bacteria in hemolymph plasma. however, evaluation of the ability of pamps to promote the proteolytic conversion of ttc3 to ttc3b revealed that lps, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to lps stimulation. factor c stored in hemocytes has been identified as an lps sensor and an initiator of hemolymph coagulation in the horseshoe crab innate immune system. as would be expected from these findings, an anti-factor c antibody inhibits both the proteolytic conversion of ttc3 and the deposition of ttc3b on the surface of gram-negative bacteria in hemolymph plasma. moreover, activated factor c present on the surface of gram-negative bacteria directly catalyzes the proteolytic conversion of the purified ttc3, thereby promoting ttc3 deposition. these data indicate that factor c acts as an lps-sensitive c3 convertase on the surface of invading gram-negative bacteria in the initial phase of horseshoe crab complement activation (fig. 1). in the alternative pathway of the vertebrate complement system, the interaction between c3b and bb is essential to form c3 convertase (c3bbb). a homolog of complement bf has been identified in c. rotundicauda (zhu et al., 2005). although physiological function of this bf homolog in the horseshoe crab complement system remains unknown, it is likely that it may be responsible for the formation of the second c3 convertase. ttc3 binds to factor c at kd = 4.9×10 -8m (ariki et al., 2008). ttc3 is present at a concentration of at least 300 μg/ml in hemolymph plasma, whereas the amount of factor c in hemolymph plasma is very low (~10 μg/ml). the relatively high concentration of ttc3 and its high affinity to factor c suggest that factor c exists in a complex with ttc3 in hemolymph plasma, and that formation of this complex is a prerequisite for the immediate activation of ttc3 by factor c on the surface of gram-negative bacteria. factor c antigen has been shown to be present all the tissues examined by western blotting. in contrast, factor g and the proclotting enzyme are not detectable in hemolymph plasma. in contrast to these findings, the anti-factor c antibody exhibits no effect on the deposition of ttc3b on staphylococcus aureus, suggesting the presence of a factor c-independent pathway to initiate the opsonization of gram-positive bacteria (ariki et al., 2008). pamps or complement factors in hemolymph plasma required for the deposition of ttc3b on gram-positive bacteria remain to be examined. factor c and complement bf from c. rotundicauda interact with plasma-derived lectins, including galactose-binding protein, carcinolectin-5, and c-reactive protein (saux et al., 2008). also in t. tridentatus, hemocytes and hemolymph plasma contain several lectins responsible for microbial agglutination (kawabata and tsuda, 2002). therefore, the protease-lectin complexes on the surface of gram-positive bacteria may enhance the deposition of ttc3b. although the complement-dependent clearance system of invading pathogens in horseshoe crabs remains to be examined, phagocytosis of gram-positive bacteria by hemocytes both in vivo and in vitro is inhibited by protease inhibitors, raising the possibility that the proteolytic dependence of opsonization by c3b may underlie phagocytosis by hemocytes (zhu et al., 2005). roles of hemocyanin in the innate immune system in crustaceans and insects, the prophenoloxidase activation system is an important part of the innate immunity, where it acts to detect and kill invading pathogens as well as to synthesize melanin for wound healing and encapsulation of 73 pathogens (cerenius and söderhäll, 2004; nappi et al., 2004). arthropod prophenoloxidases are known to be non-enzymatically activated by treatment with detergents, lipids, or organic solvents (ashida and yamasaki, 1990). in the horseshoe crab as well, the induction of phenoloxidase activity in hemolymph plasma is evident upon similar treatment (nellaiappan and sugumaran, 1996), yet prophenoloxidase has not been identified in horseshoe crabs. arthropod prophenoloxidases have been sequenced, and show significant homology to hemocyanins (aspan et al., 1995; fujimoto et al., 1995; kawabata et al., 1995). both prophenoloxidase and hemocyanin contain two functional copper-binding sites capable of reversibly binding an oxygen molecule (solomon et al., 1996). moreover, the origin of arthropod hemocyanins appears to be an ancient prophenoloxidase-like protein (burmester and scheller, 1996). under physiological conditions, arthropod prophenoloxidases require a proteolytic cleavage for activation by a specific protease (aspan et al., 1995). interestingly, tarantula hemocyanin is found to express phenoloxidase activity after limited proteolysis with trypsin or chymotrypsin (decker and rimke, 1998). in horseshoe crabs, the clotting enzyme or activated coagulation factor b efficiently produces phenoloxidase activity in hemolymph plasma, whereas activated factor c, activated factor g, or trypsin do not (nagai et al., 2000). the phenoloxidase activity in hemolymph plasma disappears upon removal of hemocyanin by ultracentrifugation, suggesting that hemocyanin is involved in the prophenoloxidase activation system in horseshoe crabs. horseshoe crab hemocyanin is composed of six subunits (α, β, γ, δ, ε, and ζ), each of which have a molecular mass of 70 kda and, in their purified forms, independently express phenoloxidase activity. the clotting enzyme converts the α-subunit of hemocyanin to phenoloxidase in a dose-dependent manner, and the resulting phenoloxidase activity reaches a plateau at a 1:1 molar ratio. the proteolytic cleavage of the hemocyanin subunit is not required for the functional conversion, and the zymogen forms of the coagulation proteases, the proclotting enzyme and coagulation factor b, are effective activators. a common structural feature of these two coagulation factors is the presence of an n-terminal clip domain (muta et al., 1990, 1993). homologous clip domains are present in the n-terminal regions of insect prophenoloxidase-activating enzymes (jiang et al., 1998; lee et al., 1998; satoh et al., 1999). therefore, the clip domain of the proclotting enzyme or coagulation factor b may promote the interaction of these factors with hemocyanin effect its functional conversion to an active phenoloxidase. horseshoe crab hemocyanin is also converted to phenoloxidase by treatment with amphiphilic substances such as sds and phosphatidylethanolamine (nagai and kawabata, 2000). consistent with the amphiphilic nature of the antimicrobial peptides secreted from the horseshoe crab hemocyte, tachyplesin interacts with the α-subunit of hemocyanin (kd = 3.4×10 -6 m) and induces its intrinsic phenoloxidase activity. the phenoloxidase activity induced by tachyplesin is inhibited by phenylthiourea, a typical inhibitor of phenoloxidase. although tachyplesin is the most effective activator of hemocyanin, other hemocyte-derived antimicrobial peptides such as tachystatins and tachycitin significantly induce its phenoloxidase activity. mutation of tachyplesin at trp2 or tyr8 and try13 on its hydrophobic face, but not mutation of basic residues on its cationic face, significantly impair its interaction with the α-subunit, implicating the hydrophobic face of tachyplesin in the functional conversion of hemocyanin to a phenoloxidase. horseshoe crab antimicrobial peptides are all capable of binding to chitin, a cell wall component of fungi and also the major structural component of the arthropod cuticle. the antimicrobial peptides likely recognize chitin exposed at sites of injury as well as on the surface of invading microbes. in addition, hemocyanin binds to tachyplesin-coated chitin. although horseshoe crab hemocyanin present at high concentration in hemolymph plasma (~70 mg/ml) and acts as an oxygen carrier under normal physiological conditions, it may be selectively converted to phenoloxidase by the antimicrobial peptides. we hypothesize that the chitin coated with antimicrobial peptides may serve as a scaffold for the binding of hemocyanin and that the resulting phenoloxidase activity acts as trigger for wound healing in the cuticle (fig. 1). in the crayfish pacifastacus leniusculus, an antibacterial peptide astacidin 1 has been identified (lee et al., 2003). interestingly, astacidin 1 is released from the c-terminal part of hemocyanin by a cysteine-like protease and is up-regulated by injection of lps or β-1,3-d-glucans, indicating that hemocyanin acts as a multifunctional protein in the innate immune system. recently, horseshoe crab hemocyanin and human hemoglobin have been found to be activated by microbial proteases and to be enhanced by lps, resulting in the production of reactive oxygen species as an antimicrobial strategy (jiang et al., 2007). acknowledgements we thank dr j kulman (puget sound blood center, seattle, usa) for helpful discussion and suggestions on this manuscript. references aketagawa j, miyata t, ohtsubo s, nakamura t, morita t, hayashida h, et al. primary structure of limulus anticoagulant anti-lipopolysaccharide factor. j. biol. chem. 261: 7357-7365, 1986. akira s, uematsu s, takeuchi o. pathogen recognition and innate immunity. cell 124: 783-801, 2006. ariki s, koori k, osaki t, motoyama k, inamori k, kawabata s. a serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides. proc. natl. acad. sci. usa 101: 953-958, 2004. ariki s, takahara s, shibata t, fukuoka t, ozaki a, endo y, fujita t, koshiba t, kawabata s. factor c acts as a lipopolysaccharide-responsive c3 convertase in horseshoe crab complement activation. j. immunol. 181: 7794-8001, 2008. 74 armstrong, pb. adhesion and motility of the blood cells of limulus. in: cohen wd (ed), blood cells of marine invertebrates: experimental systems in cell biology and comparative physiology, alan r. liss, inc., new york, usa, pp 77-124, 1985. ashida m, yamazaki, h. biochemistry of the phenoloxidase system in insects: with special reference to its activation. in: ohnishi e, ishizaki h (eds), molting and metamorphosis, springer-verlag, berlin, germany, pp 239-265,1990. aspan a, huang ts, cerenius l, söderhäll k. cdna cloning of prophenoloxidase from the freshwater crayfish pacifastacus leniusculus and its activation. proc. natl. acad. sci. usa 92: 939-943, 1995. beisel h-g, kawabata s, iwanaga s, huber r, bode w. tachylectin-2: crystal structure of a specific glcnac/galnac-binding lectin involved in the innate immunity host defense of the japanese horseshoe crab tachypleus tridentatus. embo j. 18: 2312-2322, 1999. belvin mp, anderson kv. a conserved signaling pathway-the drosophila toll-dorsal pathway. annu. rev. cell dev. biol. 12: 393-416, 1996. bergner a, muta t, iwanaga s, beisel h-g, delotto r, bode w. horseshoe crab coagulation is an invertebrate protein with a nerve growth factor-like domain. biol. chem. 378: 283-287, 1997. bergner a, oganessyan v, muta t, iwanaga s, typke d, huber r, et al. crystal structure of coagulogen, the clotting protein form horseshoe crab: a structural homologue of nerve growth factor. embo j. 15: 6789-6797, 1996. bohn ja, bemiller jn. (l→3)-β-d-glucans as biological response modifiers: a review of structure-functional activity relationships. carbohydr. poym. 28: 3-14, 1995. broekaert w, lee hi, kush a, chua nh, raikhel n. wound-induced accumulation of mrna containing a hevein sequence in laticifers of rubber tree (hevea brasiliensis). proc. natl. acad. sci. usa 87: 7633-7637, 1990. brown gd. dectin-1: a signaling non-tlr pattern-recognition receptor. nat. rev. immunol. 6: 33-43, 2006. brown j, o’callaghan ca, marshall asj, gilbert rjc, siebold c, gordon s, et al. structure of the fungal β-glucan-binding immune receptor dectin-1: implications for function. protein sci. 16: 1042-1052, 2007. burmester t, scheller k. common origin of arthropod tyrosinase, arthropod hemocyanin, insect hexamerin, and dipteran arylphorin receptor. j. mol. evol. 42: 713-728, 1996. bursey cr. histological response to injury in the horseshoe crab, limulus polyphemus. can. j. zool. 55: 1158-1165, 1977. cerenius l, söderhäll k. the prophenoloxidase-activating system in invertebrates. immunol. rev. 198: 116-126, 2004. chen sc, yen ch, yeh ms, huang cj, liu ty. biochemical properties and cdna cloning of two new lectins from the plasma of tachypleus tridentatus-tachypleus plasma lectin 1 and 2. j. biol. chem. 276: 9631-9639, 2001. chiou st, chen yw, chen sc, chao cf, liu ty. isolation and characterization of proteins that bind to galactose, lipopolysaccharide of escherichia coli, and protein a of staphylococcus aureus from the hemolymph of tachypleus tridentatus. j. biol. chem. 275: 1630-1634, 2000. davie ew, fujikawa k, kisiel w. the coagulation cascade: initiation, maintenance, and regulation. biochemistry 30: 10363-10370, 1991. decker h, rimke t. tarantula hemocyanin shows phenoloxidase activity. j. biol. chem. 273: 25889-25892, 1998. dodds aw, matsushita m. the phylogeny of the complement system and the origins of the classical pathway. immunobiol. 212: 233-243, 2007. doolittle rf, riley m. the amino-terminal sequence of lobster fibrinogen reveals common ancestry with vitellogenins. biochem. biophys. res. commun. 167: 16-19, 1990. endo y, takahashi m, fujita t. lectin complement system and pattern recognition. immunobiol. 211: 283-293, 2006. ferrandon d, imler j-l, hetru c, hoffmann, ja. the drosophila systemic immune response: sensing and signaling during bacterial and fungal infections. nat. rev. immunol. 7: 862-874, 2007. fontes cmga, clarke jh, hazlewood gp, fernandes th, gilbert hj, ferreira lma. identification of tandemly repeated type vi cellulose-binding modules in an endoglucanase from the aerobic soil bacterium cellvibrio mixtus. appl. microbiol. biotechnol. 49: 552-559, 1998. fujita t. evolution of the lectin-complement pathway and its role in innate immunity. nat. rev. immunol. 2: 346-353, 2002. fujimoto k, okino n, kawabata s, iwanaga s, ohnishi e. nucleotide sequence of the cdna encoding the proenzyme of phenol oxidase a1 of drosophila melanogaster. proc. natl. acad. sci. usa 92: 7769-7773, 1995. fujitani n, kawabata s, osaki t, kumaki y, demura m, nitta k, et al. structure of the antimicrobial peptide tachystatin a. j. biol. chem. 277: 23651-23657, 2002. fujitani n, kouno t, nakahara t, takaya k., osaki t, kawabata s, et al. the solution structure of horseshoe crab antimicrobial peptide tachystatin b with an inhibitory cystine-knot motif. j. pep. sci. 13: 269-279, 2007. gabius hj, andre s, kaltner h, siebert h-c. the sugar code: functional lectinomics. biochim. biophys. acta 1572: 165-177, 2002. gokudan s, muta t, tsuda r, koori k, kawahara t, seki n, et al. horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen. proc. natl. acad. sci. usa 96: 10086-10091, 1999. hall m, wang r, van antwerpen r, sottrup-jensen l, söderhäll k. the crayfish plasma clotting protein: a vitellogenin-related protein responsible for clot formation in crustacean blood. proc. natl. acad. sci. usa 96: 1965-1970, 1999. 75 harrington jm, chou h-t, gutsmann t, gelhaus c, stahlberg h, leippe m, et al. membrane activity of a c-reactive protein. febs lett. 583: 1001-1005, 2009. higashijima t, uzu s, nakajima t, ross em. mastoparan, a peptide toxin from wasp venom, mimics receptors by activating gtp-binding regulatory proteins (g proteins). j. biol. chem. 263: 6491-6494, 1988. hoess a, watson s, siber gr, liddington r. crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, limulus anti-lps factor, at 1.5 å resolution. embo j. 12: 3351-3356, 1993. hoffmann, ja. the immune response of drosophila. nature 426: 33-38, 2003. inamori k, ariki s, kawabata, s. a toll-like receptor in horseshoe crabs. immunol. rev. 198: 106-115, 2004. inamori k, koori, k, mishima c, muta t, kawabata, s. a horseshoe crab receptor structurally related to drosophila toll. j. endotoxin res. 6: 397-399, 2000. inamori k, saito t, iwaki d, nagira t, iwanaga s, arisaka s, et al. a newly identified horseshoe crab lectin with specificity for blood group a antigen recognizes specific o-antigens of bacterial lipopolysaccharides. j. biol. chem. 274: 3272-3278, 1999. iwaki d, kawabata s, miura y, kato a, armstrong pb, quigley jp, et al. molecular cloning of limulus α2-macroglobulin. eur. j. biochem. 242: 822-831, 1996. iwaki d, osaki t, yoshimitsu m, wai, sn, iwanaga s, kawabata, s. functional and structural diversities of c-reactive proteins present in horseshoe crab hemolymph plasma. eur. j. biochem. 264: 314-326, 1999. iwanaga s. the molecular basis of innate immunity in the horseshoe crab. curr. opin. immunol. 14: 87-95, 2002. iwanaga s, kawabata s, muta t. new type of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions. j. biochem. 123: 1-15, 1998. iwanaga s, lee bl. recent advances in the innate immunity of invertebrate animals. j. biochem. mol. biol. 38: 128-150, 2005. janeway ca jr. approaching the asymptote? evolution and revolution in immunology. cold spring harbor symp. quant. biol. 54: 1-13, 1989. jiang h, wang y, kanost mr. pro-phenol oxidase activating proteinase from an insect, manduca sexta: a bacteria-inducible protein similar to drosophila easer. proc. natl. acad. sci. usa 95: 12220-12225, 1998. jiang n, tan ns, ho b, ding jl. respiratory protein-generated reactive oxygen species as an antimicrobial strategy. nat. immunol. 8: 1114-1122, 2007. joslin g, fallon rj, bullock j, adams sp, perlmutter dh. the sec receptor recognizes a pentapeptide neodomain of alpha 1-antitrypsin-proteiase complex. j. biol. chem. 266: 11282-11288, 1991. kairies n, beisel h-g, fuentes-prior p, tsuda r, muta t, iwanaga s, et al. the 2.0-å crystal structure of tachylectin 5 provides evidence for the common origin of the innate immunity and the blood coagulation systems. proc. natl. acad. sci. usa 98: 13519-13524, 2001. kanost mr, jiang h, yu w-q. innate immune response of a lepidopteran insect, manduca sexta. immunol. rev. 198: 97-105, 2004. kawabata s, nagayama r, hirata m, shigenaga t, lal agarwala k, saito t, et al. tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity. j. biochem. 120: 1253-1260, 1996. kawabata s, osaki t, iwanaga s. innate immunity in the horseshoe crab. in: innate immunity. ezekowitz rab, hoffmann ja (eds), humana press, totowa, usa, pp 109-125, 2003. kawabata s, tsuda r. molecular basis of non-self recognition by the horseshoe crab tachylectins. biochim. biophys. acta 1572: 414-421, 2002. kawabata s, saito t, saeki k, okino n, mizutani a, toh y, et al. cdna cloning, tissue distribution, and subcellular localization of horseshoe crab big defensin. biol. chem. 378: 289-292, 1997. kawabata t, yasuhara y, ochiai m, matsuura s, ashida m. molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin. proc. natl. acad. sci. usa 92: 7774-7778, 1995. kawano k, yoneya t, miyata t, yoshikawa k, tokunaga f, terada y, et al. antimicrobial peptide, tachyplesin i, isolated from hemocytes of the horseshoe crab (tachypleus tridentatus). j. biol. chem. 265: 15365-15367, 1990. kawasaki h, nose t, muta, t, iwanaga, s, shimohigashi, y, kawabata s. head-to-tail polymerization of coagulogen, a clottable protein of the horseshoe crab. j. biol. chem. 275: 35297-35301, 2000. klein j, horejsi v. complement and complement receptors. in: immunology. klein j, horejsi v (eds), blackwell science, oxford, uk, pp 348-392, 1997. koshiba t, hashii t, kawabata s. a structural perspective on the interaction between lipopolysaccharide and factor c, a receptor involved in recognition of gram-negative bacteria. j. biol. chem. 282: 3962-3967, 2007. kouno t, fujitani n, mizuguchi m, osaki t, nishimura s, kawabata s, et al. a novel β-defensin structure: a potential strategy of big defensin for overcoming resistance by gram-positive bacteria. biochemistry 47: 10611-10619, 2008. krem m. m., di cera e. evolution of enzyme cascades from embryonic development to blood coagulation. trends biochem. sci. 27: 67-74, 2002. kurata s, ariki s, kawabata, s. recognition of pathogens and activation of immune response in drosophila and horseshoe crab innate immunity. immunobiol. 211: 237-249, 2006. laederach a, andreotti ah, fulton db. solution and micelle-bound structures of tachyplesin i and its active aromatic linear derivatives. biochemistry 41: 12359-12368, 2002. 76 lal agarwala k, kawabata s, miura y, kuroki y, iwanaga s. limulus intracellular coagulation inhibitor type 3. purification, characterization, cdna cloning, and tissue localization. j. biol. chem. 271: 23768-23774, 1996. lee s-y, cho my, hyun jh, lee km, homma k, natori s, et al. molecular cloning of cdna for pro-phenol-oxidase-activating factor i, a serine protease is induced by lipopolysaccharide or 1,3-β-glucan in coleopteran insect, holotrichia diomphalia larvae. eur. j. biochem. 257: 615-621, 1988. lee sy, lee bl, söderhäll k. processing of an antibacterial peptide from hemocyanin of the freshwater crayfish pacifastacus leniusculus. j. biol. chem. 278: 7927-7933, 2003. marra mn, wilde cg, griffith je, snable jl, scott rw. bactericidal/permeability-increasing protein has endotoxin-neutralizing activity. j. immunol. 144: 662-666, 1990. matsuda y, osaki t, hashii t, koshiba t, kawabata s. a cysteine-rich protein from an arthropod stabilizes clotting mesh and immobilizes bacteria at injured sites. j. biol. chem. 282: 33545-33552, 2007a. matusda y, koshiba t, osaki t, suyama h, arisaka f, toh y, et al. an arthropod cuticular chitin-binding protein endows injured sites with transglutaminase-dependent mesh. j. biol. chem. 282: 37316-37324, 2007b. matsushita m, fujita t. ficolins and the lectin complement pathway. immunol. rev. 180: 78-85, 2001. mintz im, venema vj, swiderek km, lee td, bean bp, adam me. p-type calcium channels blocked by the spider toxin ω-aga iva. nature 355: 827-829, 1992. miura y, kawabata s, iwanaga s. a limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily, purification, characterization, and cdna cloning. j. biol. chem. 269: 542-547, 1994. miura y, kawabata s, wakamiya y, nakamura t, iwanaga s. a limulus intracellular coagulation inhibitor type 2. purification, characterization, cdna cloning, and tissue localization. j. biol. chem. 270: 558-565, 1995. mizuguchi m, kamata s, kawabata s, fujitani n, kawano k. in protein data bank (pdb id 1wo0), rcsb protein data bank (2005). muta t, hashimoto r, miyata t, nishimura h, toh y, iwanaga s. proclotting enzyme from horseshoe crab hemocytes. j. biol. chem. 265: 22426-22433, 1990. muta t, miyata t, misumi y, tokunaga f, nakamura, t, toh y, et al. limulus factor c: an endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains. j. biol. chem. 266: 6554-6561, 1991. muta t, oda t, iwanaga s. horseshoe crab coagulation factor b. j. biol. chem. 268: 21384-21388, 1993. nagai t, kawabata s. a link between blood coagulation and prophenoloxidase activation in arthropod host defense. j. biol. chem. 275: 29264-29267, 2000. nagai t, kawabata s, shishikura f, sugita h. purification, characterization, and amino acid sequence of an embryonic lectin in perivitelline fluid of the horseshoe crab. j. biol. chem. 274: 37673-37678, 1999. nagai t, osaki t, kawabata s. functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. j. biol. chem. 276: 27166-27170, 2001. nakamura t, tokunaga f, morita t, iwanaga s, kusumoto, s, shiba t, et al. intracellular serine-protease zymogen, factor c, from horseshoe crab hemocytes: its activation by synthetic lipid a analogues and acidic phospholipids. eur. j. biochem. 176: 89-94, 1988a. nakamura t, furunaka h, miyata t, tokunaga, f, muta t, iwanaga s, et al. tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (tachypleus tridentatus). j. biol. chem. 263: 16709-16713, 1988b. nakanishi y, shiratsuchi a. mechanisms and roles of phagocytosis in drosophila and caenorhabditis elegans. inv. surv. j. 3: 89-96, 2006. nappi a, kohler l, mastore m. signaling pathways implicated in the cellular innate immune responses of drosophila. inv. surv. j. 1: 5-33, 2004. neer ej, schmidt cj, nambudripad r, smith tf. the ancient regulatory-protein family of wd-repeat proteins. nature 371: 297-300, 1994. nellaiappan k, sugumaran m. on the presence of prophenoloxidase in the hemolymph of the horseshoe crab, limulus. comp. biochem. physiol. 113b: 163-168, 1996. ng pml, jin zx, tan ssh, ho b, ding jl. c-reactive protein: a predominant lps-binding acute phase protein responsive to pseudomonas infection. j. endotoxin res. 10: 163-174, 2004. okino n, kawabata s, saito t, hirata m, iwanaga s. purification, characterization, and cdna cloning of a 27-kda lectin (l10) from horseshoe crab hemocytes. j. biol. chem. 270: 31008-31015, 1995. osaki t, okino n, tokunaga f, iwanaga s, kawabata s. proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin. j. biol. chem. 277: 40084-40090, 2002. osaki t, omotezako m, nagayama r, hirata m, iwanaga s, kasahara j, et al. horseshoe crab hemocyte-derived antimicrobial peptides, tachystatins, with sequence similarity to spider neurotoxins. j. biol. chem. 274: 26172-26178, 1999. osmand ap, friedenson b, gewurz h, painter rh, hofmann t, shelton e. characterization of c-reactive protein and the complement subcomponent c1t as homologous proteins displaying cyclic pentameric symmetry (pentraxins). proc. natl. acad. sci. usa 74: 739-743, 1977. ozaki a, ariki s, kawabata s. an antimicrobial peptide tachyplesin acts as a secondary 77 secretagogue and amplifies lipopolysaccharide-induced hemocyte exocytosis. febs j. 272: 3863-3871, 2005. pratt kp, cote hcf, chung dw, stenkamp re, davie ew. the primary fibrin polymerization pocket: the three-dimensional structure of a 30-kda c-terminal γ-chain fragment complexed with the peptide gly-pro-arg-pro. proc. natl. acad. sci. usa 94: 7176-7181, 1997. pristovsek p, feher k, szilagyi l, kidric j. j. structure of a synthetic of the lalf protein when bound to lipopolysaccharide. med. chem. 48: 1666-1670, 2005. saito t, kawabata s, hirata m, iwanaga s. a novel type of limulus lectin-l6. j. biol. chem. 270: 14493-14499, 1995a. saito t, kawabata s, shigenaga t, takayenoki y, cho j, nakajima h, et al. a novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity. j. biochem. 117: 1131-1137, 1995b. saito t, hatada s, iwanaga s, kawabata s. a newly identified horseshoe crab lectin with specificity for blood group a antigen recognizes specific o-antigens of bacterial lipopolysaccharides. j. biol. chem. 272: 30703-30708, 1997. satoh d, horri a, ochiai m, ashida m. prophenoloxidase-activating enzyme of the silkworm, bombyx mori. j. biol. chem. 274: 7441-7453, 1999. saux al, ng pml, koh jjy, low dhp, leong gell, ho b, et al. the macromolecular assembly of pathogen-recognition receptors is impelled by serine proteases, via their complement control protein modules. j. mol. biol. 377: 902-913, 2008. schumann rr, leong sr, flaggs gw, gray pw, wright sd, mathison jc, et al. structure and function of lipopolysaccharide binding protein. science 249: 1429-1431, 1990. seki n, muta t, oda t, iwaki d, kuma k, miyata t, et al. horseshoe crab (1,3)-β-d-glucan-sensitive coagulation factor g. j. biol. chem. 269: 1370-1374, 1994. sekiguchi k, yamamichi y, seshimo h, sugita h. normal development. in: sekiguchi k (ed), biology of horseshoe crabs, science house, tokyo, japan, pp133-224,1988. shigenaga t, muta t, toh y, tokunaga f, iwanaga s. antimicrobial tachyplesin peptide precursor. j. biol. chem. 265: 21350-21354, 1990. smith cl, delotto r. a common domain within the proenzyme regions of the drosophila snake and easter proteins and tachypleus proclotting enzyme defines a new subfamily of serine proteases. protein sci. 1: 1225-1226, 1992. solomon ei, sundaram um, machonkin te. multicopper oxidases and oxygenases. chem. rev. 96: 2563-2605, 1996. spraggon g, everse sj, doolittle rf. crystal structure of fragment d from human fibrinogen and its crosslinked counterpart from fibrin. nature 389: 455-462, 1997. srimal s, miyata t, kawabata s, miyata t, iwanaga s. the complete amino acid sequence of coagulogen isolated from southeast asian horseshoe crab, carcinoscorpius rotundicauda. j. biochem. 98: 305-318, 1985. størmer l. phylogeny and taxonomy of fossil horseshoe crabs. j. paleontol. 26: 630-640, 1952. suetake t, tsuda s, kawabata s, miura k, iwanaga s, hikichi k, et al. chitin-binding proteins in invertebrates and plants comprise a common chitin-binding structural motif. j. biol. chem. 275: 17929-17932, 2000. takaki y, seki n, kawabata s, iwanaga s, muta t. duplicated binding sites for (l→3)-β-d-glucan in the horseshoe crab coagulation factor g. j. biol. chem. 277: 14281-14287, 2002. tanaka s, aketagawa j, takahashi s, shibata y. activation of a limulus coagulation factor g by (l→3)-β-d-glucans. carbohydr. res. 218: 167-174, 1991. tennent ga, pepys mb. glycobiology of the pentraxins. biochem. soc. trans. 22: 74-79, 1994. theopold u, schmidt o, söderhäll k, dushay ms. coagulation in arthropod: defence, wound closure and healing. trends immunol. 25: 289-294, 2004. tokunaga t, yamada m, miyata t, ding y.-l, hiranaga-kawabata m, muta t, et al. limulus hemocyte transglutaminase: its purification and characterization, and identification of the intracellular substrates. j. biol. chem. 268: 252-261, 1993a. tokunaga t, muta t, iwanaga s, ichinose a, davie ew, kuma k, et al. limulus hemocyte transglutaminase: cdna cloning, amino acid sequence, and tissue localization. j. biol. chem. 268: 262-268, 1993b. trexler m, bányai l, patthy l. eur. j. biochem. the lccl module. 267: 5751-5757, 2000. vetvicka v, sima p. β-glucan in invertebrates. inv. surv. j. 1: 60-65, 2004. wang xw, tan ns, ho b, ding jl. evidence for the ancient origin of the nf-b/ib cascade: its archaic role in pathogen infection and immunity. proc. natl. acad. sci. usa 103: 4204-4209, 2006. zhu y, thangamani s, ho b, ding jl. the ancient origin of the complement system. embo j. 24: 382-394 , 2005. isj isj 6: s29-s34, 2009 issn 1824-307x review solitary ascidians embryos (chordata, tunicata) as model organisms for testing coastal pollutant toxicity g zega, r pennati, s candiani, m pestarino, f de bernardi department of biology, university of milan, milan, italy accepted march 13, 2009 abstract marine coastal communities are daily exposed to several chemical compounds commonly used in agriculture and industrial activities. therefore, toxicological studies evaluating the effects of these compounds on marine organisms are of primary importance for marine environment preservation. different model organisms are used to perform toxicity tests with potential pollutants, under laboratory conditions. in last decades, solitary ascidians have been selected as valuable model organisms to run bioassays with embryos and larvae. in fact, by in vitro fertilization, it is easy to obtain thousands of embryos, rapidly developing and therefore allowing a fast screen of pollutant toxicity. the aim of this review was to summarize results from toxicity tests, run with heavy metals, organometal and organic compounds, on solitary ascidian development and settlement to evidence that these animals offer several advantages as models to perform these kind of studies. first of all, they have a sensitiveness directly comparable to that of other marine model organisms. moreover, the effects of toxicants on exposed embryos and larvae could be studied using different approaches, from ultrastructure to genetic analysis. finally, since ascidians are chordates morphological and gene expression analyses could provide data for comparative studies with vertebrates. key words: heavy-metals; antifoulants; pesticides; development; tunicates; ascidians introduction marine environment pollution is a concrete risk along densely populated coastal regions, where urban and industrial development could facilitate the dispersal of several chemical agents. therefore, marine coastal ecosystems could be endangered by pollutants, such as heavy metals, pesticides and antifoulants that could be easily detected in the water column or in the sediment of harbours and estuaries (castillo et al., 2006; antizar-ladislao, 2008; bellas et al., 2008). these areas, often very rich in nutrients, host filter-feeders communities encompassing bivalves, serpulids and ascidians. marine mussels have been selected early for the study of coastal pollution impact on marine life. more recently, ascidians have been selected as potential model organisms for testing pollutants toxicity as they offer several advantages for these studies (mansueto et al., 1993; cooper et al., 1995; cima et al., 1996, 2008; bellas et al., 2003). ___________________________________________________________________________ correspondig author: fiorenza de bernardi department of biology university of milan via celoria 26, 20133 milan, italy e-mail: fiorenza.debernardi@unimi.it solitary ascidians (chordata, tunicata) are marine benthic filter-feeders that occur in dense populations along eutrophic coastal habitats, and therefore they could be easily sampled. they are hermaphrodite organisms that reproduce sexually by the simultaneous emission of eggs and sperm. fertilized eggs develop in the water column in about a day into a planktonic tadpole larva that shows some chordate characters, a dorsal hollow neural tube and a notochord flanked by muscle cells. adult solitary ascidians of ciona, phallusia and styela genus are world wide distributed and fertile almost all year round. gametes can be easily obtained by gonoduct dissection and, from in vitro fertilization, it is possible to obtain thousands of synchronously dividing embryos . under laboratory conditions, development is completed in about 16-24 hours in a range of decreasing temperature from 22 to 16°c. for these characteristics, solitary ascidians are valuable and reliable organisms to run toxicity tests on gametes and embryos, for the high number of specimen easy available every time and the rapid development. in last decades, several studies have been made to test the effect of different pollutants on ascidian development that is evaluating the percentage of s29 mailto:fiorenza.debernardi@unimi.it table 1 list of compounds whose toxicity has been tested on solitary ascidians embryos to determine median effective (ec50) concentration on development and settlement. when available the environmental concentration is also listed in bold, together with its reference compounds chemical classification ec50 (µm) embryos ec50 (µm) larval settlement environmental concentration (µm) hg heavy metal 0.22 0.39 0.002 bellas et al., 2004; ospar commission, 2000 cu heavy metal 0.58 1.61 5.67 ″ cd heavy metal 6.42 6.7 0.23 ″ cr heavy metal 226 289 ″ tbt tributyltin organometallic anti-foulant 0.02 0.01 bellas et al., 2005 zinc pyrithione (zpt) zinc 1-oxidopyridin-1-ium-2thiolate organometallic bactericide, antifoulant 0.23 0.11 bellas, 2005 lindane 1,2,3,4,5,6hexachlorocyclohexane organochloride insecticide 15.20 0.004 bellas et al., 2005; ospar commission, 2000 chlorpyrifos diethoxy-sulfanylidene-(3,5,6trichloropyridin-2yl)oxyphosphorane organophosphorus pesticide 15.70 ″ diuron 3-(3,4-dichlorophenyl)-1,1dimethylurea urea derived herbicide 17.80 ″ chlorothalonil 2,4,5,6-tetrachlorobenzene-1,3dicarbonitrile organochloride fungicide, antifoulant 0.12 0.16 0.005 bellas, 2006 sea-nine 211 (kathon 930) 4,5-dichloro-2-octyl-1,2-thiazol-3one organochloride anti-foulant 0.37 0.15 0.013 ″ ″ dichlofluanid n-(dichloro-fluoromethyl)sulfanyln-(dimethylsulfamoyl)aniline organochloride anti-foulant 0.85 0.39 0.017 ″ ″ tolylfluanid n-(dichloro-fluoromethyl)sulfanyln-(dimethylsulfamoyl)-4methylaniline organochloride anti-foulant 0.62 0.28 ″ irgarol 1051 n-tert-butyl-n'-cyclopropyl-6methylsulfanyl-1,3,5-triazine-2,4diamine anti-foulant 8.34 >25.60 0.016 ″ ″ imazalil 1-[2-(2,4-dichlorophenyl)-2-prop2-enoxyethyl]imidazole organochloride triazoleimidazole fungicide 0.67 0.47 pennati et al., 2006; fao, 2001 triadimefon 1-(4-chlorophenoxy)-3,3-dimethyl1-(1,2,4-triazol-1-yl)butan-2-one organochloride triazole fungicide 29.56 ″ fluconazole 2-(2,4-difluorophenyl)-1,3bis(1,2,4-triazol-1-yl)propan-2-ol organofluoride triazole fungicide 74.70 groppelli et al., 2007 s30 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=mesh&dopt=full&list_uids=67080469 normal larvae hatching from different treatments. in fact, larvae have a simple body plan that allows the rapid screening of malformed specimen. the tadpole larva body is formed by a trunk and a tail. the trunk bears main sensory organs: the three adhesive papillae or palps, situated at its anterior end, and one or two pigmented organs, situated in the sensory vesicle. the palps contain sensory neurons, through which the larva is able to choose a substratum where to settle, and mucus secreting cells to perform permanent attachment (groppelli et al., 2003, pennati et al., 2007). commonly, larvae possess two pigmented organs, the ocellus and the otolith respectively a photoand gravity-receptor, but some larvae could have only one, usually called the photolith, as it perceives both kind of stimuli. the sensory vesicle is the anterior portion of the central nervous system that continues towards the posterior end as a dorsal hollow tube, divided in three portions, the neck, the visceral ganglion and the tail nerve cord. the neck, devoid of neurons, connects the sensory vesicle to the visceral ganglion. this latter contains neurons with ascending projections and motor neurons with descending projections to tail muscle cells (imai and meinertzhagen, 2007). interestingly, tunicates are considered the vertebrate sister group (delsuc et al., 2006) and their embryos and larvae share basic homologies with vertebrates also at the level of the expression of developmental regulatory genes (meinertzaghen et al., 2004). in particular, the availability of ciona intestinalis genome sequences (dehal et al., 2002) could favour the study of toxicant effects on gene expression. for example, a chip for cdna microarray analysis has been developed to investigate gene expression profiles in tbt (table 1) exposed ascidians (azumi et al., 2004). in this review, results from toxicity tests run with heavy metals, organometals and organic compounds, such as pesticides and anti-foulants, on ascidian development will be reported. this overview has the aim of evidencing how it is possible to take advantage of solitary ascidians to perform toxicity studies, with several approaches. effects of heavy metals, organometallic and organic compounds on ascidian development and settlement among pollutants, heavy metals and organometallic compounds showed the highest toxic effects on ascidian development and settlement. exposure to hg, cu, cd and cr of c. intestinalis embryos for 20 h severely reduced percentage of hatching of normal larvae and of settlement. the ec50 (median effective concentration that determines larval malformation) values of hg, cu and cd were very low indicating that these metals could effectively impair development and consequently larval attachment (table 1). moreover, c. intestinalis sensitiveness to such pollutants resulted comparable to what previously reported for other marine organisms commonly used in toxicity test, such as the bivalve mytilus galloprovincialis and the sea-urchin paracentrotus lividus (bellas et al., 2004). in the group of tested organic compounds, organo-metallic ones resulted the most toxic for ascidians such as styela plicata and c. intestinalis. micromolar doses of organotin compounds (tbt, tpt, tcht), blocked development of s. plicata embryos in a stage-dependent manner. in fact, earliest developmental stages, 2-4 cells to gastrula, were more sensitive (cima et al., 1996) (table 2). the ultrastructural analysis of 1h exposed embryos of different stages revealed the presence of electron-dense precipitates in mithocondria, whose membrane were severely damaged. moreover, blastomere shape and adhesion were also affected most probably because organotin compounds could interfere with cytoskeletal proteins. similarly, c. intestinalis embryos exposed from neurula stage for 1 h showed malformed and disorganized blastomeres, lacking cytoskeletal elements. as a consequence, neurulation was blocked (dolcemascolo et al., 2005) (table 2). the effect of tbt was studied also on late developmental stage of c. intestinalis. pre-hatching and swimming larvae exposed for 1 h to 0.1µm tbt showed severe tail malformations. muscle cells had an abnormal distribution along the tail and irregularly shaped nuclei. moreover, the ultrastructure of sarcomeres and muscle mitochondria appeared completely compromised (gianguzza et al., 1996) (table 2). when c. intestinalis embryos were exposed to tbt throughout development (about 20h) development was blocked and ec50 was 0.022µm (bellas et al., 2005). another potent organometallic anti-foulant, zinc pyritione (zpt) showed similar effect on c. intestinalis development and settlement (table 1) (bellas, 2005). pesticides and anti-foulants are the last group of compounds whose toxicity was investigated on ascidian development (table 1). these substances have a broad-spectrum activity and their action on ascidian embryos were studied mainly evaluating dose-depending effects on development. for each compound the ec50 value was calculated (table 1). for some organic pesticides and anti-foulants, such as lindane, chlorpyrifos, diuron, irgarol 1051, triadimenfon and fluconazole, ec50 values were quite high in terms of toxicity, corresponding to micro-molar concentrations. organochloride antifoulants (chlorothalonil, sea-nine 211, dichlofluanid, tolylfluanid) instead resulted the most toxic substances, for their very low ec50 values. moreover, among fungicides, imazalil, that contains two chlorine atoms, showed a similar toxicity for ascidian embryos (bellas et al., 2005; bellas, 2006; pennati et al., 2006; groppelli et al., 2007). effects of the triazole fungicide was also evaluated in terms of teratogenicity as these substances induced specific malformations whose severity was dosedependent. therefore, larval phenotypes obtained after triazole exposure throughout development were classified using a dissection microscope and further characterized by means of histology and immunohistochemistry experiments. triazole exposed larvae showed typical malformation: the trunk appeared shortened, the palps were fused or not completely differentiated, and the sensory vesicle was reduced with displaced pigmented organs (fig. 1a, b). moreover, the anterior nervous s31 fig. 1 control larvae (a, c, e) and larvae developed from embryos exposed to 5 µm imazalil (b, d, f) of the solitary ascidian ciona intestinalis. control (a) and malformed larva (b) showing the typical imazalil induced phenotype. immunohistochemical localization of β-tubulin in control (c, e) and malformed (d, f) larvae, whose anterior nervous network appeared disorganized. bars = 100 µm. network was compromised, as evidenced by immunolocalization of β-tubulin (fig. 1c-f). in these studies, the teratogenic action of triazoles on ascidian development was directly compared with what known on vertebrate embryos, where these fungicides typically affect differentiation of the anterior structures, interfering with retinoic acid catabolism. the authors found evidences that also in ascidians the observed phenotypes could be due to an alteration of retinoic acid signalling (pennati et al., 2006; groppelli et al., 2007). conclusions coastal pollution could stress marine communities determining a decrease in biodiversity for the disappearing of more sensitive species (castillo et al., 2006; bellas et al., 2008). marine benthic invertebrates are easily exposed to toxic compounds commonly used in agriculture, industrial and harbour activities, and have been selected as model organisms to evaluate effects of these substances on life processes. some of the listed inorganic and organic compounds were proved to impair ascidian development at very low doses, ranging from nano to micro-molar concentrations. even if the average environmental concentration of these compounds is lower than their ec50 values on ascidian development (table 1), we believe that the chance of endangering coastal populations of sessile tunicates is a realistic risk. in fact, given the wide production of pesticides (tilman et al., 2001), the possibility of local accumulation by accidental spills must be also considered. similarly, the extensive uses of anti-foulants favour their accumulation in harbours shallow waters (bellas, 2006). moreover, among the substances considered in this review, cu, tbt and imazalil were detected in water or soils in concentrations directly comparable to their ec50 values on ascidian development (table 1). from the conspicuous studies reviewed here, it is clear that, among benthic coastal invertebrates, solitary ascidians are valuable organisms to be considered among models to test toxicity of potential or known pollutants on their development and settlement ability. in fact, it is possible to run toxicity tests on a high number of embryos and to rapidly (1 day) screen the effects. solitary ascidians embryos and larvae can be used in these laboratory studies with different approaches, from exposure bio-assays of different developmental stages to morphological analysis at different levels (ultrastructure, histology, immunohistochemistry). results summarized here evidenced that several common pollutants strongly impaired ascidians development and consequently their dispersal and recruitment phases. s32 table 2 effects of different compounds on the development and/or morphology of larvae of three different ascidian species effective concentrations (µm) developmental stage exposure time (h) target organs effects styela plicata tbt 0.1/1 tpt 0.1 tcht 0.1 2-4 cells, morula, gastrula 1 mitochondria, cytoskeleton block of cleavage cima et al., 1996 ciona intestinalis pre-hatching larvae tbt 0.1 swimming larvae 1 tail muscle cells, mitochondria, cytoskeleton block of hatching/swimming gianguzza et al., 1996 tbt 0.1/10 neurula 1 mitochondria, cytoskeleton block of neurulation dolcemascolo et al., 2005 phallusia mammillata imazalil 5 triadimefon 125 2 cells 10 palps, central nervous system pennati et al., 2006 fluconazole 125 2 cells 18 palps, central nervous system groppelli et al., 2007 recently, considering among other advantages, that ascidian have a sensitiveness, in terms of ec50, directly comparable to that of other model organisms, such as bivalves or sea-urchins, a standardized protocol for ascidian embryo-larval bioassays has been formulated (bellas et al., 2003). finally, as c. intestinalis genome has been released (dehal et al., 2002), it is also possible to investigate how expression of target genes could be altered by toxicants. for instance, azumi and colleagues (2004) found, through microarray analysis of gene expression in c. intestinalis exposed to tbt, strong differential expression of more than 200 genes concerned with stress response, detoxification, oxidoreduction reaction, biosynthesis and catabolism. considering basic vertebrate homologies of ascidians (meinertzaghen et al., 2004; delsuc et al., 2006), these genetic analysis could be powerful tools to forecast possible toxic effects on vertebrate organisms, including humans. references antizar-ladislao b. environmental levels, toxicity and human exposure to tributyltin (tbt)contaminated marine environment. a review. environ. inter. 34: 292-308, 2008. azumi k, fujie m, usami t, miki y, satoh n. a cdna microarray technique applied for analysis of global gene expression profiles in tributyltinexposed ascidians. mar. environ. res. 58: 543546, 2004. bellas j. toxicity assessment of the antifouling compound zinc pyrithione using early developmental stages of the ascidian ciona intestinalis. biofouling 21: 289-296, 2005. bellas j. comparative toxicity and of alternative antifouling biocides on embryos larvae of marine invertebrates. sci. total environ. 367: 573-585, 2006. bellas j, beiras r, marino-balsa j, fernandez n. toxicity of organic compounds to marine invertebrate embryos and larvae: a comparison between the sea urchin embryogenesis bioassay and alternative test species. ecotoxicology 14: 337-353, 2005. bellas j, beiras r, vazquez e. a standardisation of ciona intestinalis (chordata, ascidiacea) embryo-larval bioassay for ecotoxicological studies. water res. 37: 4613-4622, 2003. bellas j, beiras r, vazquez e. sublethal effects of trace metals (cd, cr, cu, hg) on embryogenesis and larval settlement of the ascidian ciona intestinalis. arch. environ. contam. toxicol. 46: 61-66, 2004. bellas j, fernandez n, lorenzo i, beiras r. integrative assessment of coastal pollution in a ria coastal system (galicia, nw spain): correspondence between sediment chemistry and toxicity. chemosphere 72: 826-835, 2008. castillo le, martinez e, ruepert c, savage c, gilek m, pinnock m, et al. water quality and macroinvertebrate community response following pesticide applications in a banana plantation, limon, costa rica. sci. total environ. 367: 418-432, 2006. cima f, ballarin l, bressa g, martinucci g, burighel p. toxicity of organotin compounds on embryos s33 of a marine invertebrate (styela plicata; tunicata). ecotoxicol. environ. saf. 35: 174182, 1996. cima f, bragadin m, ballarin l. toxic effects of new antifouling compounds on tunicate haemocytes i. sea-nine 211 (tm) and chlorothalonil. aquat. toxicol. 86: 299-312, 2008. cooper el, arizza v, cammarata m, pellerito l, parrinello n. tributyltin affects phagocytic activity of ciona intestinalis hemocytes. comp. biochem. physiol. 112c: 285-289, 1995. dehal p, satou y, campbell rk, chapman j, degnan b, de tomaso a, et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157-2167, 2002. delsuc f, brinkmann h, chourrout d, philippe h. tunicates and not cephalochordates are the closest living relatives of vertebrates. nature 439: 965-968, 2006. dolcemascolo g, gianguzza p, pellerito c, pellerito l, gianguzza m. effects of tri-n-butyltin(iv) chloride on neurulation of ciona intestinalis (tunicata, ascidiacea): an ultrastructural study. appl. organomet. chem. 19: 11-22, 2005. fao. 2001. fao specification and evaluations for plant protection products. imazalil. gianguzza m, dolcemascolo g, mansueto c, pellerito l. effects of tributyltin(iv) chloride exposure on larvae of ciona intestinalis (urochordata): an ultrastructural study. appl. organomet. chem. 10: 405-413, 1996. groppelli s, pennati r, scari g, sotgia c , de bernardi f. observations on the settlement of phallusia mammillata larvae: effects of different lithological substrata. ital. j. zool. 70: 321-326, 2003. groppelli s, zega g, biggiogero m, de bernardi f, sotgia c, pennati r. fluconazole induces teratogenic effects in the tunicate phallusia mammillata. environ. toxicol. pharmacol. 23: 265-271, 2007. imai jh, meinertzhagen ia. neurons of the ascidian larval nervous system in ciona intestinalis: i. central nervous system. j. comp. neurol. 501: 316-334, 2007. mansueto c, gianguzza m, dolcemascolo g, pellerito l. effects of tributiltin(iv) chloride exposure early embryonic stages of ciona intestinalis in-vivo and ultrastructural investigations. appl. organomet. chem. 7: 391399, 1993. meinertzhagen ia, lemaire p, okamura y. the neurobiology of the ascidian tadpole larva: recent developments in an ancient chordate. annu. rev. neurosci. 27: 453-485, 2004. ospar commission. 2000. quality status report. ospar commission, london. pennati r, groppelli s, zega g, biggiogero m, de bernardi f, sotgia c. toxic effects of two pesticides, imazalil and triadimefon, on the early development of the ascidian phallusia mammillata (chordata, ascidiacea). aquat. toxicol. 79: 205-212, 2006. pennati r, zega g, groppelli s, de bernardi f. immunohistochemical analysis of the adhesive papillae of botrylloides leachi (chordates, tunicata, ascidiacea): implications for their sensory function. ital. j. zool. 74: 325-329, 2007. tilman d, fargione j, wolff b, d’antonio c, dobson a, howarth r, et al. forecasting agriculturally driven global environmental change. science 292: 281-284, 2001. s34 http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=cima,%20f.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=bragadin,%20m.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=ballarin,%20l.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=cooper,%20e.%20l.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=arizza,%20v.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=cammarata,%20m.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=pellerito,%20l.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=parrinello,%20n.&searchfield=all&authorflag=1& phagocyotsis of apoptotic cells in invertebrates isj 3: 89-96, 2006 issn 1824-307x minireview mechanisms and roles of phagocytosis in drosophila and caenorhabditis elegans y nakanishi, a shiratsuchi graduate school of medical science, kanazawa university, kanazawa, ishikawa 920-1192, japan accepted september 19, 2006 abstract our understanding of the humoral immune response in both vertebrates and invertebrates has dramatically deepened in the past decade. in contrast, many of the mechanisms and roles of the cellular immune response remain to be elucidated. phagocytosis is at the center of the cellular responses in both innate and adaptive immunity. targets of phagocytosis are either invading microbes or altered self, that is, own cells that have become dispensable or harmful. the selective recognition and engulfment of target cells by phagocytes are achieved through the specific binding of receptors of phagocytes to ligands present on the surface of the target cells. however, these phagocytosis receptors and ligands are still being identified. the fundamental mechanism of phagocytosis appears to be the same in vertebrates and invertebrates, but whether or not genes are evolutionally conserved has yet to be determined. key words: apoptosis; caenorhabditis elegans; drosophila; innate immunity; microbial pathogen; phagocyte; phagocytosis introduction innate immunity is defined as a type of immune responses in which only products of genes already present in germ lines play roles, in contrast to adaptive immunity that uses both germ line genes and genes acquired by rearrangement of existing genes during development (janeway and medzhitov, 2002). simpler organisms like invertebrate animals have only innate immunity, while organisms more complex than jawed fish possess both innate and adaptive immune systems. innate immunity was once thought to be a prototype of the “more sophisticated” adaptive immunity, but it is now apparent that the two systems play individual roles and cooperate to protect against invaders and endogenous insults (hoebe et al., 2004). furthermore, the boundary between the two immune systems is becoming obscure (flajnik and pasquier, 2004). given that either form of immunity consists of humoral and cellular responses, there are two types of humoral and cellular reactions in vertebrates and only one type in invertebrates. ___________________________________________________________________________ corresponding author: yoshinobu nakanishi graduate school of medical science, kanazawa university, shizenken, kakuma-machi, kanazawa, ishikawa 920-1192, japan e-mail: nakanaka@kenroku.kanazawa-u.ac.jp this raises the possibility that the roles of innate immunity in vertebrates and invertebrates are somewhat distinct. if so, a better way to fully understand innate immunity would be to compare the mechanisms and roles of immune responses between vertebrates and invertebrates. previous studies have revealed that at least the mechanistic part of the innate immune response is well conserved between vertebrates and invertebrates although the players are not exactly the same (iwanaga, 2002; brennan and anderson, 2004). while the mechanisms and consequences of the humoral innate immune response have been intensively investigated particularly in insects and mammals (janeway and medzhitov, 2002), many issues still remain to be solved before we can gain a good understanding of the cellular response. provided that the fundamental mechanisms of the cellular innate immune response are almost the same in vertebrates and invertebrates, a smarter way to address these issues is to begin with the analysis of genetically tractable invertebrate animals such as drosophila and caenorhabditis elegans (mylonakis and aballay, 2005). in this minireview, we summarize what is known of phagocytosis, an event at the center of cellular responses, in drosophila and c. elegans, in an attempt to gain a deeper understanding of this phenomenon. 89 mailto:nakanaka@kenroku.kanazawa-u.ac.jp phagocytosis at a glance phagocytosis, a phenomenon whereby cells are engulfed and digested by other cells (aderem and underhill, 1999), is at the center of the cellular immune response in both innate and adaptive immunity; other cellular responses include cell-mediated killing in vertebrates, and cell-mediated killing and encapsulation in invertebrate animals. the cells in charge of phagocytosis are called phagocytes and consist of various types. phagocytes are classified into two groups, professional and amateur cells. professional phagocytes, macrophages as a representative, are full-time executors. in contrast, amateur phagocytes, which exert functions other than phagocytosis most of the time, exhibit phagocytic activity only when it is needed. there is another way to classify phagocytes; that is, phagocytes that circulate through the body and are responsible for phagocytosis in various places, and others that are localized to certain places and engaged in phagocytosis only there. the phagocytes of invertebrate animals have not been intensively characterized compared with those of vertebrates. in many invertebrates circulatory cells (either coelomocytes or hemocytes) exist, several types of which act as professional phagocytes. in the fruit fly drosophila, plasmatocytes are such phagocytes and responsible for the phagocytic elimination of invaders and altered self in many areas of the body (meister and lagueux, 2003). glia and some ectodermal cells also act as phagocytes in drosophila. in contrast, there are no mobile phagocytes in c. elegans; the cells that simply neighbor target cells seem to carry out phagocytosis (gravato-nobre and hodgkin, 2005). phagocytes selectively recognize and engulf cells that are foreign to our body or own cells that have become dispensable. the foreign cells are invading microbes, and the altered self includes cells that have become structurally and/or functionally spent, unwanted, aged, or harmful. removal of the former targets is accomplished to eliminate microbial pathogens that may cause infectious diseases, while that of the latter is necessary for morphogenesis, establishment of tissue functions, and maintenance of tissue homeostasis, i.e. tissue renewal, avoidance of excessive cellular actions, and extermination of pathogenic or noxious materials (stuart and ezekowitz, 2005). failure in the expeditious removal of such “unwanted” cells impairs normal development as well as increases the risk of infectious diseases, inflammation, or autoimmunity. phagocytosis is induced when receptors present on the surface of phagocytes are activated by target cells (aderem and underhill, 1999) (fig. 1). upon the binding of target cells, the intracellular portion of phagocytosis receptors activates a signaling pathway, which in most cases leads to rearrangement of the actin cytoskeleton. as a result, the plasma membrane of phagocytes locally extends and surrounds the target, which is then ingested as membrane vesicles called phagosomes. there is another mode of phagocytosis whereby target cells appear to “sink” into phagocytes without any extension of the plasma membrane. it is presumed fig. 1 recognition and engulfment of target cells by phagocytes. phagocytosis receptors of phagocytes bind to phagocytosis markers present at the surface of target cells. marker-bound receptors activate a signaling pathway that leads to reorganization of the actin cytoskeleton. portions of the plasma membrane of phagocytes then extend and surround targets. finally, target cells are incorporated into phagocytes as phagosomes. that the mode of engulfment varies depending on which receptors are responsible for the induction of phagocytosis as well as the size and shape of target cells (champion and mitragotri, 2006). target selectivity in phagocytosis is achieved through specific molecular recognition between phagocytosis receptors residing at the surface of phagocytes and their ligands or phagocytosis markers on the surface of target cells. the phagocytosis markers are either constituents of the surface of target cells or soluble molecules in body fluid that bind to the targets. the latter are called opsonins, being represented by immunoglobulin and complement of vertebrates that presumably do not exist in invertebrates: it is unclear if opsonin-dependent phagocytosis occurs in invertebrate animals (see below). cell surface constituents specific for microbes are exemplified by a group of molecules called the pathogen-associated molecular patterns that are recognized by a group of receptors of immune cells, the pattern recognition receptor (janeway, 2001). however, it is presumed that pattern recognition receptors and pathogen-associated molecular patterns, which are responsible for the induction of humoral innate immune responses, do not serve, at least in a direct way, as receptors and ligands, respectively, in phagocytosis. mammalian receptors responsible for the phagocytosis of microbes have been characterized, including mannose receptors, fc receptors, and complement receptors (aderem and underhill, 1999; taylor et al., 2005). mannose receptors directly recognize components of the bacterial cell wall, but fc receptors and complement receptors bind to the opsonins immunoglobulin and complement, respectively. several membrane proteins have recently been proposed to be receptors responsible for the phagocytosis of bacteria by phagocytes of drosophila, but the corresponding bacterial ligands have yet to be identified (see below). altered own cells are often induced to undergo apoptosis, a physiologic mode of cell death (wyllie et al., 1980; ellis et al., 1991), and become susceptible to phagocytosis (savill et al., 1993; savill and fadok, 2000). apoptotic cells 90 express phagocytosis ligands that do not exist at the surface of viable cells. these ligands are either endogenous molecules that move to the cell surface or pre-existing molecules at the surface whose structure or distribution changes during apoptosis (lauber et al., 2004). the apoptosis-dependent structural reorganization of the cell surface has been well studied with mammalian cells, and the membrane phospholipid phosphatidylserine is the best-characterized ligand for phagocytosis receptors (fadok et al., 1998; schlegel and williamson, 2001). apoptosis-dependent expression of phosphatidylserine at the cell surface is also observed in cells of drosophila and c. elegans. however, it is unclear whether phosphatidylserine serves as a phagocytosis ligand for apoptotic cells to be recognized by phagocytes of drosophila and c. elegans. to date, no molecule has been identified as a marker for phagocytosis of altered self in drosophila and c. elegans targets are incorporated into phagocytes as membrane vesicles called phagosomes, which are surrounded by the plasma membrane of phagocytes (aderem and underhill, 1999). the main fate of engulfed target cells is decomposition and digestion. phagosomes are processed so that the engulfed targets are killed and degraded mainly by reactive species such as reactive oxygen species and reactive nitrogen species (halliwell, 2006) and lysosomal enzymes, respectively (fig. 2). a key step in the production of reactive oxygen species is the activation of an enzyme called nadph oxidase (underhill and ozinsky, 2002; geiszt and leto, 2004). a prerequisite of the lysosomal degradation of engulfed target cells is the fusion of phagosomes with lysosomes that provide various enzymes for degrading components of engulfed cells. these processes are collectively called phagosome maturation and seem to be under a strict regulation. fig. 2 fate of engulfed cells in phagocytes. engulfed cells are killed and digested by a reactive oxygen-mediated mechanism and lysosomal enzymes, respectively. both reactions occur during structural and functional changes of phagosomes. contents of engulfed cells are sometimes used as antigens for the activation of t lymphocytes and thus for the induction of adaptive immunity. mhc, major histocompatibility complex. on the other hand, engulfed targets are sometimes not completely degraded but subjected to partial digestion. this occurs in a particular type of phagocyte, i.e. antigen-presenting cells such as dendritic cells in vertebrate animals, and processed components of engulfed cells are expressed at the surface together with the major histocompatibility complex for the activation of t lymphocytes (ackerman and cresswell, 2004). microbes, some types of bacteria in particular, possess the ability to resist the actions of phagocytes at various steps (ernst, 2000; coombes et al., 2004). yersinia inhibits phagocytosis itself through the actions of their own proteins that are delivered to phagocytes via the type iii secretion system. salmonella produce, after engulfment, proteins that inhibit the activation of nadph oxidase in phagosomes. listeria sneaks out of phagosomes by disrupting phagosome membranes using their own proteins. other bacteria including leginonella and chlamydia, and the protozoa leishmania also inhibit phagosome maturation. phagocytes of the host organisms counterattack such microbes, in at least two ways as follows. bacteria that have come out of phagosomes are surrounded again by membranes through a process called autophagy (shintani and klionsky, 2004), and phagocytes invaded by long-living microbes are often induced to undergo apoptosis and engulfed together with the microbes by other phagocytes. all these phenomena have been observed with mammalian phagocytes, and whether or not the same is true for phagocytes of invertebrate animals remains to be determined. phagocytosis of microbes in drosophila and c. elegans phagocytosis of bacteria by drosophila phagocytes in drosophila, most humoral immune responses are accomplished by cells of the fat body, a tissue equivalent to the mammalian liver, while blood cells called hemocytes are responsible for most cellular events (hoffmann and reichhart, 2002; mylonakis and aballay, 2005). there exist three cell lineages of drosophila hemocytes, which emerge at different stages of development and participate in particular types of cellular immune responses (meister and lagueux, 2003). two lineages, the plasmatocyte and the crystal cell, differentiate during the second half of embryogenesis. plasmatocytes account for no less than 90 % of circulating hemocytes and act primarily as phagocytes. therefore, these cells are considered to be responsible for the phagocytic elimination of invading microbes and altered self in drosophila. crystal cells are seemingly involved in humoral melanization mediated by phenoloxidase, but their role is not fully understood. the third hemocyte lineage emerges at the larval stage, and these cells called lamellocytes are responsible for the encapsulation of microbes, a cellular response often followed by phagocytosis. differently from mammalian phagocytes such as neutrophils and macrophages, how plasmatocytes act to phagocytose target cells has not been intensively studied. even the identity of phagocytosis receptors and corresponding microbial ligands has been elusive. it is presumed that most phagocytosis 91 receptors of drosophila phagocytes directly recognize molecules present at the surface of target microbes and apoptotic cells, because no molecules present in the hemolymph have been shown in vivo to serve as opsonin. several proteins have been proposed to be phagocytosis receptors (table 1) (cherry and silverman, 2006), although their ligands presumably present at the surface of microbes remain to be identified. the first receptor reported is known as a pattern recognition receptor. in drosophila, a family of proteins called peptidoglycan recognition proteins (pgrps) serves as pattern recognition receptors (brennan and anderson, 2004), as do toll-like receptors in mammals (akira et al., 2006). pgrp-lc, not pgrp-la or -ld, has been identified as a protein, a decrease in the expression of which leads to a decrease in the level of phagocytosis of gram-negative escherichia coli, but not gram-positive staphylococcus aureus, by s2 cells (rämet et al., 2002), a cell line established from hemocytes of drosophila embryos. this approach, namely, a genome-wide screen with rna interference-mediated inhibition of gene expression in phagocytes, was adopted by other investigators, and a group of proteins resembling a human protein called cd36 have been spotlighted. cd36 belongs to the class b scavenger receptor family (sr-b) that is responsible for the control of serum cholesterol levels as well as the removal of denatured serum proteins in mammals (peiser and gordon, 2001). besides these actions, two sr-b proteins, sr-bi of mammals (shiratsuchi et al., 1999) and croquemort of drosophila (franc et al., 1996), have been shown to be involved in the phagocytosis of apoptotic cells. the genome-wide screen revealed that the drosophila sr-b proteins peste and croquemort serve as receptors for the phagocytosis of bacteria by s2 cells. peste targets mycobacteria and lysteria but not e. coli and s. aureus (agaisse et al., 2005), but this specificity seems to change when peste is expressed in mammalian cells (philips et al., 2005). on the other hand, s. aureus is a preferred target for croquemort in phagocytosis by s2 cells (stuart et al., 2005). in addition, sr-ci, a class c scavenger receptor, of drosophila seems to have some role in the phagocytosis of bacteria (rämet et al., 2001; philips et al., 2005). all the aforementioned proteins remain as candidate phagocytosis receptors at present, because their role in vivo in the phagocytic removal of bacteria is yet to be shown. ezekowitz and colleagues extended their rna interference screen with s2 cells, in which the transcription factor serpent was found to be important for the phagocytosis of bacteria (rämet et al., 2002). they searched for gene products whose expression is regulated by serpent and examined their role in the phagocytosis of bacteria by s2 cells. eventually one protein named eater was found to bind to and engulf both e. coli and s. aureus (kocks et al., 2005). eater is a single-path membrane protein with epidermal growth factor (egf)-like repeats in its extracellular portion, and is expressed primarily in plasmatocytes. hemocytes prepared from mutant flies lacking the expression of eater showed a decreased level of the phagocytosis of both e. coli and s. aureus. furthermore, the phagocytosis of those bacteria injected into the adult mutant flies was significantly impaired. the final candidate for a phagocytosis receptor of drosophila is quite unique in that it possesses an immunoglobulin-like structure and is expressed as over one thousand isoforms through alternative splicing in hemocytes and the fat body (watson et al., 2005). this family of proteins, named dscam for immunoglobulin-superfamily receptor down syndrome cell adhesion molecule, bind to e. coli, and larval hemocytes prepared from mutant flies with a reduced level of the expression of dscam showed less activity to phagocytose e. coli than table 1 candidate receptors for the phagocytosis of bacteria by drosophila phagocytes receptor name domains e. coli s. aureus mycobacterium other targets in vivo evidence references___ pgrp-lc peptidoglycan binding yes no nd1 m.luteus2 nd2 rämet et al., 2002 peste scavenger receptor3 no no yes listeria nd philips et al., 2005 croquemort scavenger receptor3 no yes nd nd stuart et al., 2005 sr-ci scavenger receptor4 yes5 nd yes5 nd philips et al., 2005 eater egf-like repeat yes yes nd yes6 kocks et al., 2005 dscam immunoglobulin-like yes nd nd yes7 watson et al., 2005 1. not determined; 2. only viability of flies lacking pgrp-lc expression upon infection with bacteria was examined; 3. class b scavenger receptor family; 4. class c scavenger receptor family; 5. extent of contribution is small; 6. levels of phagocytosis of bacteria by larval hemocytes of flies lacking eater expression are reduced. levels of phagocytosis of bacteria injected into adult mutant flies are reduced; 7. levels of phagocytosis of bacteria by larval hemocytes of flies lacking dscam expression are reduced. 92 those from wild-type flies. in addition, its soluble form is present in the hemolymph. these findings evoke the possibility that dscam serves not only as a receptor but also as an immunoglobulin-like opsonin in the phagocytosis of bacteria. more recently, a family of secreted proteins, called teps for thioester-containing proteins, which serve as opsonins to mediate phagocytosis of microbes by s2 cells, was reported (stroschein-stevenson et al., 2006). of 6 teps tepii, tepiii, and tepvi have been suggested to be involved in the phagocytosis of e. coli, s. aureus, and candida albicans, respectively, though in vivo confirmation is required. teps resemble the complement c3, and the presence of other complement-like proteins has also been noted though their action as opsonins remains to be shown (lagueux et al., 2000; kocks et al., 2003). phagocytosis of bacteria by c. elegans phagocytes the nematode c. elegans maintained in laboratories propagates when fed with e. coli, and the lifespan of this worm is altered when the food source is changed to other bacteria. this suggests that c. elegans is immune to microbial pathogens. the study of innate immunity in c. elegans has begun with the analysis of the humoral immune response, and its mechanism and role have been shown to be basically the same as those in drosophila and mammals (gravato-nobre and hodgkin, 2005; kim and ausubel, 2005; mylonakis and aballay, 2005). infection with some types of bacteria causes apoptosis in germ lines, and a mutant line of the worm lacking the expression of ced-3 and ced-4 is more sensitive to infection. this means that c. elegans uses the apoptotic pathway for innate immune responses against invading microbes. in contrast, the role of cellular responses in the protection of the worm from infectious diseases has not yet been settled. there exists a type of cell that serves as a phagocyte in c. elegans, but the importance of the phagocytic elimination of pathogenic microbes is expected to be small. the phagocytosis of invading microbes by c. elegans phagocytes is inefficient, simply because those phagocytes are not mobile. further studies are needed to clarify the mechanisms and roles of the phagocytosis of microbes by c. elegans phagocytes. phagocytosis of apoptotic cells in drosophila and c. elegans phagocytosis of apoptotic cells by c. elegans phagocytes although the contribution of phagocytosis to defense against the invasion of pathogenic microbes is unclear, apoptotic cells are definitely eliminated by phagocytosis in c. elegans. there are no circulating “professional” phagocytes in this worm, and cells that neighbor dying cells are in charge of phagocytosis. the pioneer work done by horvitz and coworkers has revealed the existence of a set of genes responsible for the induction, execution, and regulation of programmed cell death or apoptosis in c. elegans (ellis et al., 1991; lettre and hengartner, 2006). such genes include those that play roles at the final stage of apoptosis, i.e. the engulfment and degradation of apoptotic cells (gumienny and hengartner, 2001; fig. 3 signaling pathways for the induction of phagocytosis of apoptotic cells. two partly overlapping signaling pathways for the induction of phagocytosis of apoptotic cells, which were genetically identified in c. elegans and are considered to be conserved beyond species, are schematically presented. shown in the parentheses are names of the drosophila counterparts of the c. elegans proteins. reddien and horvitz, 2004; mangahas and zhou, 2005). seemingly there are two partly overlapping signaling pathways, which involve signal mediators conserved beyond species, for the induction of the phagocytosis of apoptotic cells by c. elegans phagocytes (lettre and hengartner, 2006) (fig. 3), although a different opinion was recently provided (yu et al., 2006). the onset of engulfment should be the activation of receptors residing at the surface of phagocytes. this occurs most likely by the binding of marker molecules of phagocytosis present on the surface of target apoptotic cells. presumably there are two sets of phagocytosis receptor and ligand, but only one receptor has been identified to date. a single-path membrane protein named ced-1 has been genetically discovered and shown to serve as a phagocytosis receptor (zhou et al., 2001). there are counterparts of ced-1 in drosophila and human, which are respectively called draper (freeman et al., 2003) and megf10 (callebaut et al., 2003). draper seems to be responsible for the phagocytic removal of apoptotic cells by drosophila phagocytes (freeman et al., 2003; manaka et al., 2004) (see below), but whether or not megf10 plays roles in the clearance of apoptotic cells by mammalian phagocytes remains to be determined. ced-1 contains several structural domains, including egf-like repeats, in the extracellular region and two segments containing tyrosine residues, which are candidate domains for protein-protein interaction, in the intracellular region. the former domain could serve as a site for the binding of an as-yet unidentified phagocytosis ligand, and the latter could be, most likely after phosphorylation of the tyrosine residues, a site for the assembly of downstream signal mediators such as ced-6 (mangahas and zhou, 2005). there has been no information regarding the identity of the other phagocytosis receptor. in contrast to the fact that many molecules have been proposed to be phagocytosis markers in mammals (lauber et al., 2004), no molecules have 93 been shown to be ligands for phagocytosis receptors of c. elegans. c. elegans cells seem to express phosphatidylserine at their surface during apoptosis, but it is not known if externalized phosphatidylserine serves as a phagocytosis marker. the involvement of the c. elegans homologue of the mammalian phosphatidylserine receptor in the phagocytosis of apoptotic cells was reported, but the role for the mammalian protein itself as a phosphatidylserine-recognizing phagocytosis receptor is now doubted. it will be necessary to adopt an experimental strategy other than genetics for the identification of the other phagocytosis receptor and a couple of ligands, but the c. elegans system where mobile phagocytes and suitable cell lines are unavailable does not appear to be suitable for a rapid solution of these issues. phagocytosis of apoptotic cells by drosophila phagocytes in contrast to studies with c. elegans (see above) and mammals (lauber et al., 2004), mechanisms of the phagocytosis of apoptotic cells in drosophila have not been intensely investigated. three membrane proteins have so far been proposed as receptors responsible for the phagocytosis of apoptotic cells by drosophila phagocytes, but no molecules have been identified as phagocytosis markers presumably present on the surface of apoptotic cells. franc and coworkers were the first to identify a receptor responsible for the phagocytosis of apoptotic cells by drosophila phagocytes (franc et al., 1996, 1999). they searched for members of the c-type lectin family in larvae at the third instar stage and found a protein that belongs not to the c-type lectin family but to the sr-b family. this protein, named croquemort, standing for “catcher of death”, is a membrane protein (singleor double-path) and expressed in hemocytes (plasmatocytes/lamellocytes) of embryos and larvae. analyses of flies with a chromosomal deletion including the croquemort locus revealed that croquemort is responsible at least in part for the phagocytosis of apoptotic cells, but not of bacteria, in drosophila embryos, though the latter conclusion recently became controversial (stuart et al., 2005). it is still unknown what molecule at the surface of apoptotic cells croquemort binds to, and as to whether this receptor is contained in either one of the two signaling pathways (see fig. 3) is not clear. another molecule that has been shown to act as a receptor for the phagocytosis of apoptotic cells is draper, the drosophila homologue of the c. elegans phagocytosis receptor ced-1. draper, a single-path membrane protein with egf-like repeats, appears to serve as a receptor for the phagocytic elimination of apoptotic cells by both hemocytes and glia (freeman et al., 2003; manaka et al., 2004). moreover, draper is involved in the removal of axons by glia during metamorphosis for remodeling of the neural network and recovery from injury (awasaki et al., 2006; hoopfer et al., 2006; macdonald et al., 2006). a ligand(s) for draper remains to be identified for both apoptotic cells and degenerating axons, but phosphatidylserine is not likely to be the one (manaka et al., 2004). the externalization of phosphatidylserine occurs also in drosophila cells during apoptosis, but whether or not phosphatidylserine serves as a phagocytosis marker remains to be determined. the third candidate for a drosophila phagocytosis receptor is a protein named six microns under (simu) that is expressed in hemocytes and glia (kurant et al., 2006). the overall structure of simu resembles that of draper; both are single-path membrane proteins containing egf-like repeats in the extracellular region. a decrease in the expression level of simu in a hemocyte-derived cell line as well as in embryos leads to an increase in the number of apoptotic cells. two structurally similar phagocytosis receptors, draper and simu, are co-expressed in hemocytes and glia, and how they cooperate with each other needs to be investigated. perspectives in animals having both innate and adaptive immunity, cooperation between the two systems is necessary to maximize immune responses. for invertebrate animals lacking adaptive immunity, innate immune responses, either humoral or cellular, are more important than those in animals with both types of immunity. it is thus speculated that the role and mode of action of innate immune responses in invertebrate animals are somewhat different from those in vertebrate animals. phagocytosis is at the center of cellular immune responses, and thus clarification of its mechanisms and consequences in invertebrate animals should lead to a better understanding of immunity in general. there are many issues to be solved in order to achieve a full understanding of innate immunity in invertebrate animals. first, it is not known how these animals die upon being infected with some types of microbes. in mammals, septic shock is considered a consequence of excessive host responses to invading microbes, which are mostly mediated by proteins called cytokines secreted from immune cells. is this also true for invertebrates? probably the answer is yes, because cytokine-like proteins are secreted from drosophila hemocytes when insults such as infections with microbes occur (agaisse et al., 2003). the secreted proteins move via the hemolymph and stimulate the fat body, a drosophila tissue equivalent to the mammalian liver, to produce stress proteins. there could thus be septic shock in invertebrate animals, at least in drosophila. taking into consideration that drosophila produce immunoglobulin-like proteins (watson et al., 2005) and complement-like proteins (lagueux et al., 2000; kocks et al., 2003; stroschein-stevenson et al., 2006), the architecture and operation of immunity do not seem to differ between invertebrate and vertebrate animals. it needs to be confirmed in vivo if these proteins act as opsonins to mediate the phagocytosis of microbes and altered self by drosophila phagocytes. conversely, it is necessary to determine to what extent the opsonin-independent phagocytosis of microbes and microbe-infected cells contributes to the immune response against pathogenic microbes in vertebrate animals. another question to be answered is how invertebrates protect themselves against microbes other than bacteria, such as viruses and protozoa. this issue is also important for humans, because arthropods such as 94 insects can act as a vector for parasitic protozoa that cause severe infectious diseases. it is totally unclear how immune surveillance against invading plasmodia, the parasite responsible for malaria, is accomplished in mosquitoes. a study on innate immune responses to viral infections has just started with drosophila (cherry and silverman, 2006). the final general question is whether or not the contents of engulfed cells, microbes or altered self, are used to evoke further immune reactions in invertebrate animals. in mammals, cell contents are sometimes processed and presented as a complex with the major histocompatibility complex at the surface of specialized immune cells, antigen-presenting cells, for the activation of t lymphocytes (ackerman and cresswell, 2004). this seems unlikely to occur in invertebrates lacking adaptive immunity, but the presence of immunoglobulin-like molecules in drosophila catches our imagination. leaving the above-mentioned questions for a future task, issues to be immediately addressed are: 1) to identify marker molecules that exist at the surface of bacteria and are bound by phagocytosis receptors of drosophila phagocytes; 2) to identify a molecule that exists at the surface of apoptotic cells and is bound by c. elegans ced-1/drosophila draper; and 3) to identify the second phagocytosis receptor, after ced-1/draper, and its ligand for the phagocytosis of apoptotic cells by phagocytes of c. elegans and drosophila. acknowledgements we thank an anonymous reviewer for helping us to improve the manuscript. our studies cited in this paper were supported by the grant-in-aid for scientific research from jsps and mext, and also by institutional research grants from kanazawa university. references ackerman al, cresswell p. cellular mechanisms governing cross-presentation of exogenous antigens. nat. immunol. 5: 678–684, 2004. aderem a, underhill dm. mechanisms of phagocytosis in macrophages. annu. rev. immunol. 17: 593–623, 1999. agaisse h, burrack ls, philips ja, rubin ej, perrimon n, higgins de. genome-wide rnai screen for host factors required for intracellular bacterial infection. science 309: 1248–1251, 2005. agaisse h, petersen u-m, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell 5: 441–450, 2003. akira s, uematsu s, takeuchi o. pathogen recognition and innate immunity. cell 124: 783–801, 2006. awasaki t, tatsumi r, takahashi k, arai k, nakanishi y, ueda r, et al. essential role of the apoptotic cell engulfment genes draper and ced-6 in developmentally programmed axon pruning during drosophila metamorphosis. neuron 50: 855–867, 2006. brennan ca, anderson kv. drosophila: the genetics of innate immune recognition and response. annu. rev. immunol. 22: 457–483, 2004. callebaut i, mignotte v, souchet m, mornon j-p. emi domains are widespread and reveal the probable orthologs of the caenorhabditis elegans ced-1 protein. biochem. biophys. res. commun. 300: 619–623, 2003. champion ja, mitragotri s. role of target geometry in phagocytosis. proc. natl. acad. sci. usa 103: 4930–4934, 2006. cherry s, silverman n. host-pathogen interactions in drosophila: new tricks from an old friend. nat. immunol. 7: 911–917, 2006. coombes bk, valdez y, finlay bb. evasive maneuvers by secreted bacterial proteins to avoid innate immune responses. curr. biol. 14: r856–r867, 2004. ellis re, yuan j, horvitz hr. mechanisms and functions of cell death. annu. rev. cell biol. 7: 663–698, 1991. ernst jd. bacterial inhibition of phagocytosis. cell. microbiol. 2: 379–386, 2000. fadok va, bratton dl, frasch sc, warner ml, henson pm. the role of phosphatidylserine in recognition of apoptotic cells by phagocytes. cell death differ. 5: 551–562, 1998. flajnik mf, pasquier ld. evolution of innate and adaptive immunity: can we draw a line? trends immunol. 25: 640–644, 2004. franc nc, dimarcq j-l, lagueux m, hoffmann j, ezekowitz rab. croquemort, a novel drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. immunity 4:431–443, 1996. franc nc, heitzler p, ezekowitz rab, white k. requirement for croquemort in phagocytosis of apoptotic cells in drosophila. science 284: 1991–1994, 1999. freeman mr, delrow j, kim j, johnson e, doe cq. unwrapping glial biology: gcm target genes regulating glial development, diversification, and function. neuron 38: 567–580, 2003. geiszt m, leto tl. the nox family of nad(p)h oxidases: host defense and beyond. j. biol. chem. 279: 51715–51718, 2004. gravato-nobre mj, hodgkin j. caenorhabditis elegans as a model for innate immunity to pathogens. cell. microbiol. 7: 741–751, 2005. gumienny tl, hengartner mo. how the worm removes corpses: the nematode c. elegans as a model system to study engulfment. cell death differ. 8: 564–568, 2001. halliwell b. phagocyte-derived reactive species: salvation or suicide? trends biochem. sci. 31: 509–515, 2006. hoebe k, janssen e, beutler b. the interface between innate and adaptive immunity. nat. immunol. 5: 971–974, 2004. hoffmann ja, reichhart j-m. drosophila innate immunity: an evolutionary perspective. nat. immunol. 3: 121–126, 2002. hoopfer ed, mclaughlin t, watts rj, schuldiner o, o’leary ddm, luo l. wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning. neuron 50: 883–895, 2006. iwanaga s. the molecular basis of innate immunity in the horseshoe crab. curr. opin. immunol. 14: 87–95, 2002. janeway jr ca. how the immune system works to 95 protect the host from infection: a personal view. proc. natl. acad. sci. usa 98: 7461–7468, 2001. janeway jr ca, medzhitov r. innate immune recognition. annu. rev. immunol. 20: 197–216, 2002. kim dh, ausubel fm. evolutionary perspectives on innate immunity from the study of caenorhabditis elegans. curr. opin. immunol. 17: 4–10, 2005. kocks c, cho jh, nehme n, ulvila j, pearson am, meister m, et al. eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in drosophila. cell 123: 335–346, 2005. kocks c, maehr r, overkleeft hs, wang ew, iyer lk, lennon-duménil a-m, et al. functional proteomics of the active cysteine protease content in drosophila s2 cells. mol. cell. proteom. 2: 1188–1197, 2003. kurant e, leaman d, gaul u. the novel glial factor, six microns under (simu), functions in the phagocytosis of apoptotic particles. abstract in the 47th annual drosophila research conference, houston, tx, usa, 2006. lagueux m, perrodou e, levashina ea, capovilla m, hoffmann ja. constitutive expression of a complement-like protein toll and jak gain-of-function mutants of drosophila. proc. natl. acad. sci. usa 97: 11427–11432, 2000. lauber k, blumenthal sg, waibel m, wesselborg s. clearance of apoptotic cells: getting rid of the corpses. mol. cell 14: 277–287, 2004. lettre g, hengartner mo. developmental apoptosis in c. elegans: a complex cednario. nat. rev. mol. cell biol. 7: 97–108, 2006. macdonald jm, beach mg, porpiglia e, sheehan ae, watts rj, freeman mr. the drosophila cell corpse engulfment receptor draper mediates glial clearance of severed axons. neuron 50: 869–881, 2006. manaka j, kuraishi t, shiratsuchi a, nakai y, higashida h, henson p, et al. draper-mediated a n d p h o s p h a t i d y l s e r i n e i n d e p e n d e n t phagocytosis of apoptotic cells by drosophila hemocytes/macrophages. j. biol. chem. 279: 48466–48476, 2004. mangahas pm, zhou z. clearance of apoptotic cells in caenorhabditis elegans. sem. cell dev. biol. 16: 295–306, 2005. meister m, lagueux m. drosophila blood cells. cell. microbiol. 5: 573–580, 2003. mylonakis e, aballay a. worms and flies as genetically tractable animal models to study host-pathogen interactions. infect. immun. 73: 3833–3841, 2005. peiser l, godon s. the function of scavenger receptors expressed by macrophages and their role in the regulation of inflammation. microb. infect. 3: 149–159, 2001. philips ja, rubin ej, perrimon n. drosophila rnai screen reveals cd36 family member required for mycobacterial infection. science 309: 1251–1253. rämet m, pearson a, manfruelli p, li x, koziel h, gobel v, et al. drosophila scavenger receptor ci is a pattern recognition receptor for bacteria. immunity 15: 1027–1038, 2001. rämet m, manfruelli p, pearson a, mathey-prevot b, ezekowitz rab. functional genomic analysis of phagocytosis and identification of a drosophila receptor for e. coli. nature 416: 644–648, 2002. reddien pw, horvitz hr. the engulfment process of programmed cell death in caenorhabditis elegans. annu. rev. cell dev. biol. 20: 193–221, 2004. savill j, fadok v. corpse clearance defines the meaning of cell death. nature 407: 784–788, 2000. savill j, fadok v, henson p, haslett c. phagocyte recognition of cells undergoing apoptosis. immunol. today 14: 131–136, 1993. schlegel ra, williamson p. phosphatidylserine, a death knell. cell death differ. 8: 551–563, 2001. shintani t, klionsky dj. autophagy in health and disease: a double-edged sword. science 306: 990–995, 2004. shiratsuchi a, kawasaki y, ikemoto m, arai h, nakanishi y. role of class b scavenger receptor type i in phagocytosis of apoptotic rat spermatogenic cells by sertoli cells. j. biol. chem. 274: 5901–5908, 1999. stroschein-stevenson s, foley e, o’farrell ph, johnson ad. identification of drosophila gene products required for phagocytosis of candida albicans. plos biol. 4: e4, 2006. stuart lm, deng j, silver jm, takahashi k, tseng aa, hennessy ej, et al. response to staphylococcus aureus requires cd36-mediated phagocytosis triggered by the cooh-terminal cytoplasmic domain. j. cell biol. 170: 477–485, 2005. stuart lm, ezekowitz rab. phagocytosis: elegant complexity. immunity 22 : 539–550, 2005. taylor pr, martinez-pomares l, stacey m, lin h-h, brown gd, gordon s. macrophage receptors and immune recognition. annu. rev. immunol. 23: 901–944, 2005. underhill dm, ozinsky a. phagocytosis of microbes: complexity in action. annu. rev. immunol. 20: 825–852, 2002. watson fl, püttmann-holgado r, thomas f, lamar dl, hughes m, et al. extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 1874–1878, 2005. wyllie ah, kerr jfr, currie ar. cell death: the significance of apoptosis. int. rev. cytol. 68: 251–306, 1980. yu x, odera s, chuang c-h, lu n, zhou z. c. elegans dynamin mediates the signaling of phagocytic receptor ced-1 for the engulfment and degradation of apoptotic cells. dev. cell 10: 743–757, 2006. zhou z, hartwieg e, horvitz hr. ced-1 is a transmembrane receptor that mediates cell corpse engulfment in c. elegans. cell 104: 43–56, 2001. 96 mechanisms and roles of phagocytosis in drosophila and caenorhabditis elegans y nakanishi, a shiratsuchi graduate school of medical science, kanazawa university, kanazawa, ishikawa 920-1192, japan abstract introduction phagocytosis at a glance phagocytosis of microbes in drosophila and c. elegans phagocytosis of bacteria by drosophila phagocytes those from wild-type flies. in addition, its soluble form is present in the hemolymph. these findings evoke the possibility that dscam serves not only as a receptor but also as an immunoglobulin-like opsonin in the phagocytosis of bacteria. more recently, a family of secreted proteins, called teps for thioester-containing proteins, which serve as opsonins to mediate phagocytosis of microbes by s2 cells, was reported (stroschein-stevenson et al., 2006). of 6 teps tepii, tepiii, and tepvi have been suggested to be involved in the phagocytosis of e. coli, s. aureus, and candida albicans, respectively, though in vivo confirmation is required. teps resemble the complement c3, and the presence of other complement-like proteins has also been noted though their action as opsonins remains to be shown (lagueux et al., 2000; kocks et al., 2003). phagocytosis of bacteria by c. elegans phagocytes phagocytosis of apoptotic cells in drosophila and c. elegans phagocytosis of apoptotic cells by c. elegans phagocytes phagocytosis of apoptotic cells by drosophila phagocytes perspectives references tributiltyn-induced effects on mapk signaling in ascidian embryos isj 6: s87-s94, 2009 issn 1824-307x research report effects of tributyltin chloride in ascidian embryos: modulation of kinase-mediated signalling pathways f damiani, m gianguzza, g dolcemascolo dipartimento di biopatologia e metodologie biomediche, sez. di biologia e genetica, università di palermo, palermo, italy accepted march 13, 2009 abstract we studied the effects of various tbt concentrations by assaying the activity of erk 1/2 (p44/42) and phospho-erk1/2 (phospho-p44/42), proteins with a key role in ascidian development, and tyrosine kinase-dependent pathway. the effects of this xenobiotic and the role of some signalling mechanisms on ascidian embryos were examined by using western immunoblotting. the tyrosine phosphorylation pattern in the ascidians ciona intestinalis and phallusia mammillata development was examined and different levels of protein phosphorylation were found as a response to tbt at µm range. to determine whether another key signalling pathway was activated, the effects of tbt on the phosphorylation state of a component of tyrosine kinase-mediated signal transduction mapk, erk 1/2 (p44/42) were evaluated. embryos of ciona intestinalis exposed to 0.1, 0.25 and 0.5 µm tbt showed a slight decrement in the level of phosphorylated erk, while a remarkable decrement in level of phopshorylated erk were observed at higher tbt concentrations (0.5 µm to 10 µm). these data indicated that exposures to tbt induced changes in the total pattern of phosphotyrosine and in the phosphorylation levels of erk 1/2 but there were no changes on the overall level of total erk in ascidian embryos. key words: tributyltin-induced effect; tyrosine kinase signalling; mapk; erk (p44/42); ascidian embryos introduction among the class of organotin compounds, tributyltin (tbt) is well known. organotin compounds have many applications, which include use in pvc, as catalyst in chemical reactions, agricultural pesticides, glass coatings and food packaging materials (forsyth et al., 1993; ohno et al., 2002) and antifungal treatments for textile polymers (allsopp et al., 2000, 2001). in particular, tbt has been used in marine antifouling paints leading to the widespread distribution of tbt and its breakdown products in the marine sediments and biota (elgethun et al., 2000; connelly et al., 2001; de brito et al., 2002; lee et al., 2005). high levels of tbt dissolved in fresh and seawater impair reproduction, by inhibiting embryogenesis and larval development, in a variety of marine organisms (coelho ___________________________________________________________________________ corresponding author: francesca damiani dipartimento di biopatologia e metodologie biomediche sez. di biologia e genetica università di palermo via divisi 83, 90133 palermo, italy e-mail: francescadamiani@unipa.it et al., 2006; beiras et al., 2008). in invertebrates, symptoms of such a tbt exposure includes the development of male sexual characteristics as a penis and vas deferens by female (imposex) (miller et al., 1999; ten hallers-tjabbes et al., 2003; santos et al., 2004). tbt effects have been studied in many marine organisms including ascidians, in which immunotoxic effects such as decreasing of phagocytic activity and phenoloxidase activity have been observed (cooper et al., 1995; cima et al., 1998; cima and ballarin, 2000; tujula et al., 2001; arizza et al., 1995; cima et al., 1995). ascidians are an useful model for developmental biology, and they are also sensitive bio-indicators of environment degradation. previous light and electron microscope studies on ciona intestinalis embryos and larvae incubated in tbt chloride solutions showed morphological and fine structural changes in the embryos and larvae. in particular, changes in cytomembranes and mitochondria and anomalous blastomere arrangement during gastrulation were found (mansueto et al., 1993; gianguzza et al., 1996; dolcemascolo et al., 2005). s87 mailto:francescadamiani@unipa.it in ascidian embryos, a fibroblast growth factor (fgf)-like signal has been proposed to be involved in induction of notochord and mesoderm formation (nakatani and nishida, 1994; kim and nishida, 1999). a main pathway is a protein kinase transduction pathway, which include ras, raf, mek and mitogen-activated protein kinase (mapk). kim and nishida (2001) suggested that a mek-mapk signalling cascade is widely involved in embryonic induction in ascidians. mapks are serine/threonine kinases that transduce signals from the plasma-membrane to the nucleus (garrington and johnson, 1999; cobb and goldsmith, 2000) and they play a critical role in controlling cell survival, proliferation, and differentiation (chang and karin, 2001). three major subfamilies have been characterized, including the extracellular signal-regulated kinases (erks), the cjun n-terminal kinases (jnks), also known as stress-activated protein kinases (sapks) and the p38-mapks (kyriakis et al., 1996; seger et al., 1995; cohen, 1997). mapks have also a key role in responding to a wide variety of environmental stresses (storey and storey, 2001). canesi et al. (2004a) have shown that in hemocytes of the mussel mitylus galloprovincialis, 17β-estrediol (e2) induced shape changes, lysosomial membrane destabilization and release of hydrolytic enzymes by activation of the stress-activated p38 mapk and signal transducers and activators of transcription (stat). among signalling mechanisms, tyrosine kinasedependent pathways are triggered by citokines, growth factors and hormones and they are implicated in cell signalling, cell growth, differentiation and apoptosis (fischer, 1999). different stressors such as heavy metals, prooxidants and pollutants are known to stimulate tyrosine kinase signalling (rahman et al., 1993; nakashima et al., 1994; katano et al., 1995; burlando et al., 2006). therefore the possibility exists that both signal transduction pathways (tyrosine phosphorylation and mapk) could be used to study signalling mechanisms in organisms under enviromental stress. in the present paper, the effects of the tbt treatments on cell signalling, variation of protein tyrosine phosphorylation levels and erk module in embryos of ciona intestinalis and phallusia mammillata embryos were examined by the immunoblotting method. materials and methods animals adult specimens of ciona intestinalis and phallusia mammillata were collected from the gulf of palermo and termini imerese harbour (palermo). they were maintained in sea water and kept in aerated aquaria (200 liters). ascidians (about 100 for every experiments set) were held at 18-20 °c for no more than 7 days before use and were daily fed with various food types including freeze-dried rotifers, green unicellular algae and artificial diet. tbt chloride solutions concentrated stock solution of tributyltin (iv) chloride (schering bergkamen, germany) was obtained by dissolving the compound at 10 mm concentration in dimethylsulfoxide (dmso). working solutions were obtained by further diluitions of the stock in millipore-filtered seawater (fsw). these solutions were diluited at 0.1, 0.5, 1, 10, 50 and 100 µm final concentrations. experimental procedure in each experiment female and male gametes were removed from gonoducts and transferred into agar-coated syracuse dishes containing filtered sea water at 22 °c for cross-fertilization. ten min after fertilization, sperm excess was removed and sea water renewed. gastrulae were separated to form the following groups: (1) control a (ctrl): c. intestinalis or p. mammillata gastrulae in fsw (2) control b: c. intestinalis gastrulae in 0.1 % dmso (3) c. intestinalis gastrulae treated for 60 min with 0.1 µm, 0.5µm, 1µm, 10 µm tbt chloride solution (4) p. mammillata gastrulae treated for 60 min with 0.5µm, 1µm, 10 µm, 50 µm, 100 µm tbt chloride solution the final dmso concentration chosen to make up the experimental solution was 0.1 %; this represents a no-toxic concentration which is less than the one reported by bellas et al. (2005); in fact the controls treated with this concentration of dmso did not show significant differences with controls in fsw. electrophoresis and western blotting the levels of tyrosine phosphorylation and phosphorylated erk in whole cell extracts from ascidian embryos were determined using specific antibodies. different lots of ascidian embryos were incubated with tbt solutions for 60 min. then the sample were centrifugated and lysed in buffer (300 mm nacl, 50 mm tris-hcl ph 7.6, 0.1 % triton, 1 % protease inhibitor cocktail (sigma-aldrich), 4 mm edta, 2 mm sodium orthovanadate, 10 mm sodium pyrophosphate and 100 mm sodium fluoride) on ice for 120 min. the lysates were centrifuged at high speed (10.000xg) for 15 min and an aliquot of the supernatant was assayed to determine protein concentration by the bradford method (bradford, 1976). equal amounts of proteins (30 µg) were separated by 12 % sds-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (pvdf) membrane (immobilon-p, millipore, billerica, ma, usa) in 0.1m 3-(cyclo-hexylamino)-1propanesulfonic acid (caps, sigma-aldrich), ph 11; 10 % methanol, at 170 ma for 45 min. membranes were stained with ponceau s, incubated in block solution (3 % albumin from bovine serum, 10 % fetal bovine serum in phosphate buffer), and probed overnight with anti-phosphotyrosine antibody s88 fig. 1 effects of tbt chloride on protein tyrosine phosphorylation in c. intestinalis gastrulae. protein extracts from ascidian embryos were subjected to 12 % sds-page followed by western blot using anti-phosphotyrosine antibody. (a): representative blot obtained from embryos controls and tbt treatment (0.10.250.5 μm); (c): representative blot obtained from embryos control and tbt treatment (0.5110 μm). (b) and (d): densitometric analysis of phosphotyrosine bands; data plotted on bar charts represent the mean intensities (±sd, n = 3) obtained from all the bands of each lane. statistical evaluation of means were carried out using the student’s t test and statistically significant differences as compared to the control are indicated by asterisks. *p < 0.05, **p < 0.01, ***p< 0.001. (py20; santa cruz biotechnology, ca, usa). after 5 washes with washing solution (1x phosphate buffer, 0.1 % tween-20) the membranes were incubated with alkaline phosphatase-conjugated secondary antibody (promega corporation madison, usa). then the membranes were washed with alkaline phosphatase buffer (0.1 m tris hcl, 0.1 m nacl, 5 mm mgcl2, ph 9.5) and proteins were detected with 5-bromo-4chloro-3-indolyl phosphate/nitroblue tetrazolium liquid substrate system (bcip/nbt liquid substrate system sigma, saint louis, ms, usa). for p44/42 map kinase (erk 1/2) and phospho-p44/42 map kinase (p-erk 1/2) protein level evaluation, the samples were separated by sds-polyacrylamide gel electrophoresis, transferred to pvdf membrane and then the membranes were probed overnight with specific antibodies against erk 1/2 (cell signalling technology, beverly, ma, usa) and perk 1/2 (cell signalling technology), respectively. protein corresponding to erk 1/2 and p-erk 1/2 were indentified by using the detection protocol mentionated earlier. data analysis data from desitometric analyses of western blots are means±sd of three independent experiments. statistical evaluation of the data was performed with the student’s t test and p< 0.05 was assumed to be statistically significant. results effect of tbt chloride on tyrosine phosphorylation proteins pathway tyrosine phosphorylation as marker for signal transduction was examined. western blot analyses with a monoclonal antibody that specifically recognizes phosphorylated tyrosine residues allowed to detect different levels of phosphorylation in various proteins obtained by treating different embryos of c. intestinalis and p. mammillata with tbt. s89 fig. 2 effects of tbt chloride on protein tyrosine phosphorylation in p. mammillata gastrulae. protein extracts from ascidian embryos were subjected to 12 % sds-page followed by western blot using anti-phosphotyrosine antibody. (a): representative western blot showing variations in protein phosphorylation of p. mammillata gastrulae after tbt treatment (0.511050100 μm). (b): densitometric analysis of phosphotyrosine bands; data plotted on bar charts represent the mean intensities (±sd, n= 3) obtained from all the bands of each lane. statistical evaluation of means were carried out using the student’s t test and statistically significant differences as compared to the control are indicated by asterisks. **p < 0.01, ***p < 0.001. exposure of c. intestinalis embryos at gastrula stage to 0.1µm, 0.25µm or 0.5 µm tbt for 60 min induced only a slight increase of phosphorylated proteins around 116 kda and 180 kda. (figs 1a, b). when embryos of c. intestinalis were treated with 0.5 µm, 1 µm and 10 µm tbt solution, they showed an increase of phosphotyrosine levels, reaching, at 10 µm tbt exposure, a significant variation of about 1-fold over the control (p<0.001) (figs 1c, d). in particular, phosphorylated proteins of 36 kda, 48 kda, 58 kda, 90 kda and 180 kda were mainly identified. densitometry analysis values of the protein patterns were presented as mean density of the total bands in each sds-page lane. the same experiments were carried out in p. mammillata gastrula stage by using a 0.5-100 µm tbt. at 10 µm tbt an increase of phosphorylation level of proteins with molecular size nearby 48 kda and 55 kda was found. the bands nearby 36 kda, showed a slight increase at 0.5 µm e 1 µm and then decrease until the control levels at 10 µm (fig. 2a). because variations of the densitometric analysis were evaluated as a mean of the total pattern of tyrosine protein phosphorylation, the decrease of a single band gives an unimportant contribution respect to the density increase of the other proteins. as shown in fig. 2b, the highest increase in tyrosine phosphorylation was found at 10 µm tbt (0.18-fold above the control; p< 0.001); exposure of embryos to 50 and 100 µm tbt resulted in a remarkable decrement in tyrosine phosphorylation levels of total pattern (0.6and 0.7fold beneath the control respectively; p < 0.01). effect of tbt chloride on p44/42 and phospho p44/42 levels the erk module responds primarily to growth factors and mitogens and stimulates transcriptional responses in the nucleus. generally, activation of an erk signalling pathway has a role in mediating cell division, migration and survival. to determine whether erk 1/2 signalling pathway was activated in response to tbt treatments, whole embryos extracts were prepared and immunoblotted with antibodies specifically recognizing the phosphorylated and active forms of erk 1/2 (p44 and p42). at first a putative protein was identified in ciona genomic database (blast genomic database and swiss prot, embl) showing about 90 % of sequence similarity with mammalian erk1 and erk2. exposure of c. intestinalis embryos to 0.1 µm, 0.25 µm, and 0.5 µm tbt induced only a slight decrease in the level of phosphorylated erk (fig. 3a, top panel); at 0.5 µm of tbt exposure the average decrement was 0,4-fold beneath the control (p< 0.05) (fig. 3b), whereas the overall p44/42 levels remained did not change as shown by representative experiment in fig. 3a (bottom panel). as shown in fig. 4 a (top panel), when higher tbt concentration, from 0.5 µm to 10 µm, a remarkable dose-dependent erk phosphorylation inhibition was evident, and an average decrease of 0.85-fold beneath the control at 10 µm tbt (p < 0.001) (fig. 4b) was reached. there were no changes about endogenous levels of total erk due to tbt treatment (fig. 4a, bottom panel). high concentrations of tbt inhibit erk signalling as shown by above mentioned results. s90 fig. 3 effects of tbt chloride on erk 1/2 (p44/42) and phospho-erk1/2 (phosphop44/42) in c. intestinalis gastrulae. (a, top panel): representative experiment of phopho-erk 1/2 levels obtained from embryos of control (ctrl) and embryos treated with tbt (0.10.250.5 μm). (a, bottom panel): representative experiment of erk 1/2 levels at the same concentration above-mentionated. (b): densitometric analysis of phospho-p44/42 bands. results are means for three independent experiments (mean±sd). statistical evaluation of means were carried out using the student’s t test and statistically significant differences (p < 0.05) as compared to the control are indicated by an asterisk. discussion ascidians represent an intriguing candidate experimental system for studying the effects of environmental stress. in fact, these organisms are able to survival in a wide range of marine pollutions and some species are abundant in highly transformed and altered environments such as harbors and industrial areas. for this reason, numerous studies have outlined the importance of this group as pollution bio-indicators (papadopouolu et al., 1972; papadopouolu and kanias, 1977; bell et al., 1982; galletly et al., 2007). among the various signal transduction pathways involved in response to environmental stress, both tyrosine kinase signalling and mapks appear to play a significant role (burlando et al., 2006; schaffer and weber, 1999; widmann et al., 1999; kyriakis and avruch, 2001). to further elucidate molecular mechanisms affected by tbt exposure we studied the two signal transduction pathways above mentioned. they have an important role in the response of marine organisms to pollutant, and are considered as biomarkers. different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects produced by pollutants on different organisms. at the beginning, we examined the effects of tbt exposure on tyrosine kinase signalling in ascidian embryos by western immunoblotting. to evaluate whether the effects of the xenobiotic might be associated with a change in tyrosine phosphorylation levels, embryos of c. intestinalis at gastrula stage, were exposed for 60 min to µm range of tbt solution. the treatments with 1 µm and 10 µm tbt, showed an enhanced phosphorylation level for proteins ranging from 36 and about 180 kda. the fold increase in phosphotyrosine levels, reached a significant variation of about 1-fold over the control (p < 0.001) at 10 µm tbt, compared to the control. in p. mammillata embryos at gastrula stage, the exposure to 10 µm tbt, induced a significant increase in phosphotyrosine levels (p< 0.001). at higher tbt concentrations (50 µm and 100 µm) a remarkable decrease in the phosphorylated protein pattern was observed. the possibility exists that such a decrease can be linked to apoptotic or necrotic events caused by high tbt concentrations. taking in account total pattern, the highest phosphorylation values were found after treatment with 10 µm tbt. it has been suggested that distinct upstream mechanisms exist leading to apoptosis and necrosis by different concentrations of an organotin compounds (gunasekar et al., 2001; jurkiewicz et al., 2004). although further research are needed to elucidate the role of tyrosine kinase signalling and mechanism of tbt-induced apoptosis, the here reported results suggest that protein tyrosine phosphorylation may represent a key element in the signal transduction of ascidian embyos exposed to marine pollution. in this respect these embryos could be used for detecting the involvement of cell signalling in organisms exposed to pollutant or stressors. mapk cascades are important amplifying modules that can transduce stress signals into cellular responses (kultz and avila, 2001; poonam et al., 2002; ranganna et al., 2002). studies on the characterization and function of mapk modules have been carried out by using mammalian models and non mammalian experimental systems, for i.e. xenopus laevis, drosophila melanoganster and caenorhabditis elegans (widmann et al., 1999), and recently mussel hemocytes or isolated digestive gland cells (canesi et al., 2001, 2002). in the present study the stimulation of mapk signal transduction pathways by tbt treatment was examined in ascidian embryos. in particular, we observed the response of proteins tyrosine phosphorylation and erk 1/2 signalling pathway to tbt exposure. the erk signalling pathway responds mainly to growth factors and mitogens, stimulating s91 fig. 4 effects of tbt chloride on erk 1/2 (p44/42) and phospho-erk1/2 (phosphop44/42) in c. intestinalis gastrulae. (a, top panel): representative experiment of phopho-erk 1/2 levels obtained from embryos of control (ctrl) and embryos treated with tbt (0.51and 10 μm). (a, bottom panel): representative experiment of erk 1/2 levels at the same concentration above-mentionated. (b): densitometric analysis of phospho-p44/42 bands. results are means for three independent experiments (mean ±sd). statistical evaluation of means were carried out using the student’s t test and statistically significant differences as compared to the control are indicated by asterisks. *p < 0.05, **p < 0.01, ***p < 0.001. transcriptional responses and its activation has a role in mediating cell division, migration and survival (garrington and johnson, 1999; cobb and goldsmith, 2000). as regards the role of erk 1/2 signalling pathway, the results of our studies revealed that 0.1 µm, 0.25 µm and 0.5 µm tbt induced only a slight decrement in the level of phopshorylated erk, while the levels of total erk were unvaried. higher tbt concentration up to 10 µm leads to a remarkable decrement in the level of phopshorylated erk, but no effects on endogenous levels of total erk were found. these results indicate that these tbt exposures caused an inhibition in the phosphorylation of erk 1/2 while not changing their overall levels. it could be suggested that tbt, a strong toxic agent, could interfere with normal mechanisms of signal transduction involved in ascidians embryo development. there are indications that the mapk signalling cascade, mediated via the erk family, plays a role in the onset of apoptosis during the embryogenesis (kling et al., 2002; yao et al., 2003) and that mapk plays a pro-apoptotic role during the ascidian metamorphosis. in fact, chambon et al. (2002) have demonstrated that the activation of the c. intestinalis mapk/erk (ci-erk) in tail cells precedes the wave of apoptosis, suggesting that the phosphorylated form of ci-erk transduces the death-activating signal in tail tissues during metamorphosis, whereas the inhibition of ci-erk blocks metamorphosis. moreover it has been proposed that mitogenactivated protein kinase (mapk) signalling might be involved in the regulation of hsp expression in blue mussels (anestis et al., 2007). batel et al. (1993) have reported that in response to tbt, the sponge cells of geodia cydonium not only undergoes apoptosis but also expresses the stress protein, hsp70, to a higher degree. in preliminary experiments we also investigated the possible modulation of the heat shock protein-70 (hsp70) by tbt treatment. considering the tbt exposure cause hsp70 induction in ascidian embryos (data not shown), it could be interesting to verify if there is a link between mapk activation and hsp 70 expression. acknowledgements this work was supported by grant from università di palermo (ex 60% miur). references allsopp a, santillo d, johnston p. hazardous chemicals in carpets. greenpeace research laboratories, technical note 01/2001 (published under cover title “poison underfoot: hazardous chemicals in pvc flooring and hazardous chemicals in carpets, isbn 9073361-68-0), 2001. allsopp a, santillo d, johnston p. hazardous chemicals in pvc flooring. greenpeace research laboratories, technical note 14/00 (published under cover title “poison underfoot: hazardous chemicals in pvc flooring and hazardous chemicals in carpets, isbn 9073361-68-0), 2000. anestis a, lazou a, portner ho, michaelidis b. behavioral, metabolic, and molecular stress responses of marine bivalve mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. am. j. physiol. regul. integr. comp. physiol. 293: 911-921, 2007. arizza v, cammarata m, tomasino mc, parrinello n. phenoloxidase characterization in vacuolar hemocytes from the solitary ascidian styela plicata. j. invertebr. pathol. 66: 297-302, 1995. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri. dev. comp. immunol. 22: 479-92, 1998. batel r, bihari n, rinkevic b, dapper j, schacke h, schroder hc, et al.. modulation of organotininduced apoptosis by the water pollutant methyl mercury in a human lymphoblastoid tumor cell s92 line and a marine sponge. mar. ecol. prog. ser. 93: 245-51, 1993. beiras r, bellas j. inhibition of embryo development of the mytilus galloprovincialis marine mussel by organic pollutants; assessment of risk for its extensive culture in the galician rias. acquaculture 277: 208-212, 2008. bell mv, pirie vj, mcphail db, goodman ba, falkpetersen b. contents of vanadium and sulphur in the blood cells of ascidia mentula and ascidiella aspersa. j. mar. biol. ass. uk 62: 709-716, 1982. bellas j, beiras r, marino-balsa jc, fernandez n. toxicity of organic compounds to marine invertebrate embryos and larvae: a comparison between the sea urchin embryogenesis bioassay and alternative test species. ecotoxicology 14: 337-353, 2005. bradford m m. a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254, 1976. burlando b, berti e, viarengo a. effects of seawater pollutants on protein tyrosine phosphorilation in mussel tissue. aquat. toxicol. 78s: s79-s85, 2006. canesi l, ciacci c, betti m, lorusso lc, marchi b, burattini s, falcieri e, gallo g. rapid effect of 17β-estradiol on cell signaling and function of mytilus hemocytes. gen. comp. endocrinol. 136: 58-71, 2004a. canesi l, betti m, ciacci c, scarpato a, citterio b, pruzzo c, et al. signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 map kinases. dev. comp. immunol. 26: 325-34, 2002. canesi l, betti m, ciacci c, gallo g. insulin effect of zinc in mytilus digestive gland cells: modulation of tyrosine kinase-mediated cell signaling. gen. comp. endocrinol. 122: 60-66, 2001. chambon jp, soule j, pomies p, fort p, sahuquet a, alexandre d, et al. tail regression in ciona intestinalis (prochordate) involves a caspasedependent apoptosis event associated with erk activation. development 129: 3105-14, 2002. chang l, and karin m. mammalian map kinase signalling cascades. nature 410: 37-40, 2001. cima f, ballarin l. tributyltin induces cytoskeletal alterations in the colonial ascidian botryllus schlosseri phagocytes via interaction with calmodulin. aquat. toxicol. 48: 419-429, 2000. cima f, ballarin l, bressa g, burighel p. cytoskeleton alterations by tributyltin (tbt) in tunicate phagocytes. ecotoxicol. environ. saf. 40: 160-165, 1998. cima f, ballarin l, bressa g, sabbadin a. immunotoxicity of butyltins in tunicates. appl. organomet. chem. 9: 567-72, 1995. cobb mh, goldsmith ej. dimerization in map-kinase signalling. trends biochem. sci. 25: 7-9, 2000. coelho mr, langston wj, bebianno mj. effect of tbt on ruditapes decussatus juveniles. chemosphere 63: 1499-1505. 2006. cohen p. the search for physiological substrates of map and sap kinases in mammalian cells. trends cell biol. 7: 353-361, 1997. connelly dp, readman jw, knap ah, davies j. contamination of the coastal waters of bermuda by organotins and the triazine herbicide irgarol 1051. mar. pollut. bull. 42: 409-414, 2001. cooper el, arizza v, cammarata m, pellerito l, parrinello n. tributyltin affects phagocytic activity of ciona intestinalis hemocytes. comp. biochem. physiol. 112c: 285-9, 1995. cowan kj, storey kb. mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress. j. exper. biol. 206: 1107-1115, 2003. de brito ap, takahashi s, ueno d, iwata h, tanabe s, kubodera t. organochlorine and butyltin residues in deep-sea organisms collected from the western north pacific, off-tohoku, japan. mar. pollut. bull. 45: 348-361, 2002. dolcemascolo g, gianguzza p, pellerito c, pellerito l, gianguzza m. effects of trybutiltin (iv) chloride on neurulation of ciona intestinalis (tunicata, ascidiacea): an ultrstructural study. appl. organomet. chem. 19: 11-22, 2005. elgethun k, neumann c, blake p. butyltins in shellfish, finfish, water and sediment from the coos bay estuary (oregon, usa). chemosphere 41: 953-964, 2000. fischer eh. cell signalling by protein tyrosine phosphorylation. adv. enzyme regul. 39: 359369, 1999. forsyth ds, dabeka r, sun wf, dalglish k. speciation of organotins in poly(vinyl chloride) products. food addit. contam. 10: 531-540, 1993. galletly bc, blows mw, marshall dj. genetic mechanisms of pollution resistance in a marine invertebrate. ecol. appl. 17: 2290-2297, 2007. garrido c, gurbuxani s, ravagnan l, kroemer g. heat shock proteins: endogenous modulators of apoptotic cell death. biochem. biophys. res. commun. 286: 433-442, 2001. garrington tp , johnson, g. organization and regulation of mitogen-activated protein kinase signalling pathways. curr. opin. cell biol. 11: 211-218, 1999. gianguzza m, dolcemascolo g, mansueto c, pellerito l. effects of trybutiltin (iv) chloride exposure on larvae of ciona intestinalis (urochordata): an ultrstructural study. appl. organomet. chem. 10: 405-413, 1996. gunasekar p, li l, prabhakaran k, ebyl v, borowitz jl, isom ge. mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells. toxicol. sci. 64 : 83-89, 2001. jurkiewicz m, averill-bates da, marion m, denizeau f. involvment of mitochondrial and death receptor pathways in tributylin-induced apoptosis in rat epatocytes. biochim. biophys. acta 1963: 15-27, 2004. katano y, pu my, akhand aa, hamaguchi m, koga y, isobe k, et al. evidence of redox-linked signalling for producing a giant signal complex. j. cell biochem. 57: 432-439, 1995. s93 kim gj, nishida h. role of fgf and mek signalling pathway in the ascidian embryo. dev. growth differ. 43: 521-533, 2001. kim gj, nishida h. suppression of muscle fate by cellular interaction is required for mesenchyme formation during ascidian embryogenesis. dev. biol. 214: 9-22, 1999. kyriakis jm, avruch j. protein kinase cascades activated by stress and inflammation. j. biol. chem. 271: 24313-24316, 1996 kultz d, avila k. mitogen-activated protein kinases are in vivo transducers of osmosensory signals in fish gill cells. comp. biochem. physiol. 129b: 821-829, 2001. kyriakis jm, avruch j. mammalian mitogenactivated protein kinase signal transduction pathways activated by stress and inflammation. physiol. rev. 81: 807-869, 2001. lee cc, wang t, hsieh cy, tien cj. organotin contamination in fishes with different living patterns and ist implications for human health risk in taiwan. environ. pollut. 137: 198-208, 2005. mansueto c, gianguzza m, dolcemascolo g and pellerito l. effects of trybutiltin (iv) chloride exposure on early embryonic stages of ciona intestinalis: in vivo and ultrstructural investigations. appl. organomet. chem. 7: 391399, 1993. miller kl, fernandes tf, read pa. the recovery of populations of dogwhelks suffering from imposex in the firth of forth 1987-1997/98. environ. pollut. 106: 183-192, 1999. nakashima i, pu my, nishizaki a, rosila i, ma l, katano y, et al. redox mechanisms as alternative to ligand binding for receptor activation delivering disregulated cellular signals. j. immunol. 152: 1064-1071, 1994. nakatani y, nishida h. induction of notochord during ascidian embryogenesis. dev. biol. 166: 289-299, 1994. ohno h, suzuki m, nakashima s, aoyama t, mitani k. determination of organotin compounds in plastic products by gc/ms after ethyl derivatization with sodium tetraethylborate. shokuhin eiseigaku zasshi 43: 208-214, 2002. papadopuolou c, kanias g. tunicate species as marine pollution indicators. mar. pollut. bull 8: 229-331, 1977. papadopuolou c, hadzistelios i, grimanis ap. trace element uptake by cynthia claudicans (savigny). limnol. oceanogr. 11: 651-663, 1972. poonam ak, wuster rd, lucchesi pa. mitogenactivted protein kinase activation and oxidant signalling in astrocytoma cells. j. neurol. oncol. 56: 109-117, 2002. rahman sm, pu my, hamaguchi m, iwamoto t, isobe k, nakashima i. redox-linked ligandindependent cell surface triggering for extensive protein tyrosine phosphorylation. febs lett. 317: 35-85, 1993. ranganna k, yousefipour z, nasif r, yatsu fm, milton sg, hayes be. acrolein activates mitogen-activated protein kinase signal transduction pathways in rat vascular smooth muscle cells. mol. cell. biochem. 240: 83-98, 2002. robinson mj, cobb mh. motogen-activated protein kinase pathways. curr. opin. cell biol. 9: 180186, 1997. santos mm, vieira n, reis-henriques ma, santos am, gomez-ariza jl, giraldez i, et al. imposex and butyltin contamination off the oporto coast (nw portugal): a possible effect of the discharge of dredged material. environ. int. 30: 793-798, 2004. schaeffer ht, weber mj. mitogen-activated protein kinases: specific messages from ubiquitous messengers. mol. cel.l biol. 19: 2435 -2444, 1999. seger r, krebs eg. the mapk signaling cascade. fadeb j. 9: 726-735, 1995. storey kb and storey jm. signal transduction and gene expression in the regulation of natural freezing survival. rev. cell mol. responses to stress. 2: 1-19, 2001. sugisawa n, matsuoka m, okuno t, igisu h. suppression of cadmium-induced jnk/p38 activation and hsp70 family gene expression by ll-z1640-2 in nih3t3 cells. toxicol. appl. pharmacol. 196: 206-214, 2004. takashi u, masayuki k, shinji t, yasunobu o, yasuyuki n. possible involvment of p38 map kinase in hsp70 expression induced by hipoxia in rat primary astrocytes. brain res. 823: 226230, 1999. takayama s, reed j, homma s. heat-shock proteins as regulators of apoptosis. oncogene 22: 9041-9047, 2003. ten hallers-tjabbes cc, wegener jw, van hattum ba, kemp jf, ten hallers e, reitsemae tj, et al. imposex and organotin concentrations in buccinum undatum and neptunea antiqua from the north sea: relationship to shipping density and hydrographical conditions. mar. environ. res. 55: 203-233, 2003. tujula n, radford j, nair sv, raftos da. effects of tributyltin and other metals on the phenoloxidase activating system of the tunicate, styela plicata. aquat. toxicol. 55:191201, 2001. zhang x, beuron f, freemont ps. machinery of protein folding and unfolding. curr. opin. struct. biol. 12: 231-238, 2002. widmann c, gibson s, jarpe mb, johnson gl. mitogen-activated protein kinase: conservation of a three kinase module from yeast to human. physiol. rev. 79:143-180, 1999. s94 19 isj 15: 19-30, 2018 issn 1824-307x research report sequence features, expression profiles and biochemical characteristics of a sigma class glutathione s-transferase gene (aigstσ) from bay scallop argopecten irradians m wang1, 3, b wang1, m liu1, k jiang1, l wang1, 2 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3research platform for marine molecular biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china accepted december 5, 2017 abstract glutathione s-transferases (gsts) are a class of enzymes that facilitate the detoxification of xenobiotics and also play important roles in innate immunity. in the present study, a novel sigma class gst gene (designated as aigstσ) was cloned from the bay scallop argopecten irradians via rapid amplification of cdna ends (race) technique. the complete cdna sequence of aigstσ consisted of a 5’ untranslated regions (utr) of 48 bp, a 3’ utr of 113 bp with a poly a tail and an open reading frame (orf) of 618 bp. the orf encoded a polypeptide of 205 amino acid residues with a calculated molecular mass of approximately 23.11 kda and a theoretical isoelectric point of 5.354. the deduced amino acid sequence of aigstσ contained a gst_n domain and a gst_c domain, and exhibited high similarity with other reported sigma class gsts. in the phylogenetic tree, aigstσ was located in the sigma class gsts sub-branch. the aigstσ mrna transcripts were constitutively expressed in the tissues of hemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hemocytes, and the mrna expression levels of aigstσ were significantly up-regulated in hemocytes after various pathogen associated molecular patterns (pamps) stimulation. the purified recombinant aigstσ protein exhibited catalytic activity against the common substrate 1-chloro-2, 4-dinitrobenzene (cdnb) with low thermal stability and narrow optimum ph spectrum. all these results indicated that aigstσ was a fragile but efficient antioxidant enzyme and was potentially involved in the innate immune responses of scallop. key words: argopecten irradians; glutathione s-transferase; innate immunity introduction the innate immunity is almost the solo defence mechanism for invertebrates that protects hosts against microbial invaders (song et al., 2015). in the innate immune defence mechanism, hemocytes can phagocytize and kill the microbial pathogens (lu et al., 2013; chen et al., 2014; wang et al., 2014). when the host is attacked by microbial invaders, phagocytosis is activated with high oxygen consumption named the respiratory burst and followed by mass reactive oxygen intermediates (roi) and reactive oxygen species (ros) production ___________________________________________________________________________ corresponding author: lei w ang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn (shao et al., 2017). therefore, organisms employ the antioxidant system to maintain roi and ros at the normal physiological levels (zhang et al., 2017a, b). as an essential kind of antioxidant enzymes, glutathione s-transferases (gsts, ec 2.5.1.18) are a superfamily of multifunctional phase ii enzymes primarily catalyzing reduced glutathione to both endogenous and exogenous electrophiles (sheehan et al., 2001). gsts have been identified from the cytosol, mitochondria and microsomes of all the prokaryotic and eukaryotic organisms that have been studied (raza et al., 2002). generally, based on their primary and tertiary structures, substrate and inhibitor specificity, and immunological cross reactivity, gsts could be grouped into at least fifteen classes, which were termed as alpha (α), beta (β), delta (δ), epsilon (ε), kappa (κ), lambda (λ), mu (μ), omega (ω), phi (φ), pi (π), sigma (σ), tau (τ), theta (θ), zeta (ζ) and rho (ρ) (wilce and parker, 1994). 20 table 1 primers used in the present study primer sequence (5`-3`) brief information adaptor primer ggccacgcgtcgactagtac anchor primer for 3` race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) for cdna synthetize aiactin-qrt-f caaacagcagcctcctcgtcat internal control for real-time pcr aiactin-qrt-r ctgggcacctgaacctttcgtt internal control for real-time pcr aigstσ-cds-f atgccttcctacaaacttatctac gene specific primer for cds aigstσ-cds-r ttagatcacgctctcgggacgcga gene specific primer for cds aigstσ-qrt-f ctgatccgtctcgctttcgct gene specific primer for real-time pcr aigstσ-qrt-r gctgtttcccgtccacttcca gene specific primer for real-time pcr aigstσ-race-f1 cccaaatttgccgaaatc gene specific primer for race aigstσ-race-f2 agttgaacccagattgtttgaagg gene specific primer for race m13-47 cgccagggttttcccagtcacgac vector primer for sequencing rv-m gagcggataacaatttcacacagg vector primer for sequencing t7 acatccactttgcctttctc vector primer for sequencing t7-ter tgctagttattgctcagcgg vector primer for sequencing among all the gsts classes, sigma class gst (gstσ) comprises one of the largest gst subfamilies identified from invertebrates to vertebrates, which was believed to evolve from ancestral gst genes and exhibit high levels of enzymatic activity toward the common substrate 1-chloro-2, 4-dinitrobenzene (cdnb) (flanagan and smythe, 2011). recently, several sigma class gsts were identified and investigated in marine invertebrates (boutet et al., 2004; lee et al., 2007; wan et al., 2008; ren et al., 2009; li et al., 2012; umasuthan et al., 2012; yang et al., 2012; zhang et al., 2012a, b; wang et al., 2013a; li et al., 2015). among these sigma class gsts, the abgstsigma gene from abalone haliotis diversicolor was significantly expressed in the hemocytes, gill, mantle and digestive gland of bacteria-challenged abalone (ren et al., 2009). bacterial challenge could significantly up-regulate the mrna expression of both vpgst-1 and vpgst-2 from manila clam ruditapes (venerupis) philippinarum (li et al., 2012). the mrna expression level of sggst-s1 in hemocytes was significantly up-regulated after razor clam solen grandis was stimulated by peptidoglycan (pgn) or β-1, 3-glucan (glucan) (yang et al., 2012). while after bacterial challenge, the mrna expression levels of sigma class gsts in hemocytes were all significantly higher than those of the control group in mussels mytilus galloprovincialis (wang et al., 2013a). all these research achievements revealed that sigma class gsts from marine invertebrates were functional diversity and might not only serve as an antioxidant enzyme involving in the detoxification but also play important roles in the modulation of innate immune responses. bay scallop argopecten irradians was introduced from usa in 1982 and has become one of the most important aquaculture species in china, due to its high economic value, fast growth rate and adaptation ability to different regions for aquaculture (li et al., 2007). and a. irradians was also considered as an attractive model to study immunology because of its relatively simple innate immune system and its propensity to undergo various manipulations, which allows researchers to study the effects of both biological and non-biological factors on the innate immune responses (matozzo, 2016). by now, several antioxidant enzyme genes have been identified and characterized in a. irradians, such as metallothionein (mt) (wang et al., 2009), peroxiredoxin (prx) (li et al., 2011) and superoxide dismutase (sod) (bao et al., 2008, 2009a, b, 2010), however, no information about gst genes was available in bay scallop till now. to bridge this gap, the main objectives of the present study were (1) to clone the full-length cdna of sigma class gst from a. irradians (designated as aigstσ), (2) to investigate the tissue distribution of aigstσ mrna transcripts and their temporal expression after different pathogen associated molecular patterns (pamps) stimulation, and (3) to validate the activities of recombinant aigstσ protein under different treatments. materials and methods scallops, immune stimulation and sample collection the bay scallops used in the present study were obtained from a local farm in qingdao, china, and all the experiments were conducted in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (nih). the study protocol and all the experimental design were conducted with approval from experimental animal ethics committee of institute of oceanology, chinese academy of sciences. approximately 200 scallops with an average 50 mm in shell length were employed for the pamps stimulation treatment. the scallops were randomly divided into 6 groups and each group contained about 30 40 individuals. the scallops were received an injection of 50 μl phosphate buffered saline (pbs, 0.14 mol l-1 sodium chloride, 3 mmol l-1 potassium chloride, 8 mmol l-1 disodium hydrogen phosphate dodecahydrate, 1.5 mmol l-1 21 table 2 information of gst proteins used in phylogenetic analysis class species accession number omega chlamys farreri adf32018 craassostrea gigas xp_011429380 danio rerio np_001002621 haliotis discus discus abo26600 haliotis madaka alu63761 perna viridis agn03944 sigma argopecten irradians ang56313 chlamys farreri acf25904 chlamys farreri adf32019 hyriopsis cumingii agu68336 pinctada fucata jas04242 ruditapes philippinarum aew46325 rho chlamys farreri acf25903 cyprinus carpio bas29983 ruditapes philippinarum aew46331 sebastes schlegelii anw83217 siniperca chuatsi aci32418 solea senegalensis bag12568 zeta chlamys farreri add82544 cyprinus carpio bas29981 oplegnathus fasciatus ady80028 xenopus laevis xp_018084636 microsomal chlamys farreri adf45336 gallus gallus np_001129022 microtus ochrogaster xp_005364596 osmerus mordax aco10098 sinonovacula constricta alc77324 xenopus tropicalis np_001011245 potassium phosphate monobasic, ph 7.4), lipopolysaccharides from escherichia coli 0111:b4 (lps, l2630, sigma-aldrich, usa, 0.5 mg ml-1 in pbs), pgn from staphylococcus aureus (77140, sigma-aldrich, usa, 0.5 mg ml-1 in pbs), glucan from baker’s yeast saccharomyces cerevisiae (g5011, sigma-aldrich, usa, 0.5 mg ml-1 in pbs) or polyinosinic-polycytidylic acid (poly ic, p1530, sigma-aldrich, usa, 0.5 mg ml-1 in pbs), respectively. the injected scallops were returned to seawater tanks immediately and five individuals were randomly sampled from each stimulated and unstimulated group at 3, 6, 12, 24 and 48 h post injection. the hemolymphs were collected and centrifuged at 800 g, 4 °c for 10 min to harvest the hemocytes for rna preparation. hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops were collected to determine mrna transcripts of aigstσ. rna isolation and cdna synthesis total rna was isolated from the hemocytes of scallops with rnaiso plus reagent (9108, takara, japan). the first-strand synthesis was carried out using the dnaseⅰ (rq1, m6101, promega, usa) treated raw rna as template and adaptor primer-oligo (dt) as primer (table 1). the reaction were performed at 42 °c for 1 h, terminated by heating at 95 °c for 5 min, and then stored at -80 °c till use. est analysis and cloning of full-length aigstσ cdna an est (ai_f00346) from bay scallop cdna library in national center for biotechnology information (ncbi) homologous to previously identified sigma class gst genes was selected for further cloning the cdna of aigstσ. two gene-specific primers, aigstσ-race-f1/f2 (table 22 1), were designed to clone the 3’ sequence of aigstσ cdna by rapid amplification of cdna ends (race) technique. and the coding sequence (cds) of aigstσ was amplified and confirmed using another two gene-specific primers, aigstσ-cds-f/r. all pcr amplification was performed in an a300 fast thermal cycler (longgene, china), and the pcr products were purified using monarch dna gel extraction kit (t1020s, neb, usa) and cloned into the pmd18-t simple vector (d103a, takara, japan). after being transformed into the competent cells escherichia coli strain dh5α (cb101, tiangen, china), the positive recombinants were identified via anti-ampicillin selection and verified by pcr screening using m13-47 and rv-m primers (table 1). three of the positive clones were sequenced using a prism 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatical analysis of cdna and protein sequences the protein sequences information for homologous and phylogenetic analysis was listed in table 2. the search for protein sequence similarity was conducted with blastp 2.6.0. the deduced protein sequences were analyzed by the editseq module in lasergene program suite 14.0.0.88. the function domains were predicted using simple modular architecture research tool (smart) 7.0. multiple sequence alignments were performed with clustal omega 1.2.4 and visualized by multiple alignment show module in sequence manipulation suite 2.0. a neighbor-joining (nj) phylogenic tree was constructed with mega 7.0.26. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1,000 times. expression patterns analysis via quantitative real-time pcr the mrna transcripts of aigstσ in different tissues or their temporal expression patterns in hemocytes of scallops stimulated with various pamps were investigated by quantitative real-time pcr (qrt-pcr). all qrt-pcr reactions were performed with the sybr premix extaq (tli rnaseh plus) (rr420, takara, japan) using 100 ng cdna template in a linegene k fqd-48a (a4) fluorescence quantitative pcr detection system (bioer, china). all the primers using in qrt-pcr were listed in table 1. the mrna expression levels of aigstσ were normalized to those of β-actin for each sample. the relative mrna expression levels of aigstσ were generated using comparative ct method (2-δδct method) (schmittgen and livak, 2008). all the data were subjected to one-way analysis of variance (anova) followed by a multiple comparison using costat 6.400, and the p values less than 0.05 were considered statistically significant. recombinant and purification of aigstσ in e. coli the cds of aigstσ was amplified using two gene-specific primers, aigstσ-cds-f/r (table 1), and ligated to the expression vector peasy-blunt e1 (ce111, transgen, china). the recombinant plasmid, peasy-blunt e1/aigstσ, was isolated by monarch plasmid miniprep kit (t1010s, neb, usa) and then transformed into e. coli strain bl21 (de3) (cd601, transgen, china). the positive transformants, e. coli bl21 (de3)/peasy-blunt e1/aigstσ, were incubated in artmedia protein expression auto-inducing medium (cp101, transgen, china) containing 100 mg l-1 ampicillin (gg101, transgen, china) at 28 °c with shaking at 220 rpm for 24 h. the recombinant protein (designated as raigstσ) was purified using a his-tag protein purification kit (p2226, beyotime, china) under natural condition. the resultant protein was separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and visualized with protein stains h (c510041, sangon, china). analysis of enzymatic activity of raigstσ the specific activities of raigstσ were measured as described in previous reports (habig et al., 1974 wan et al., 2008; umasuthan et al., 2012). briefly, the reaction was carried out in a 1 ml mixture containing 100 mm pbs, 10 mm gsh (s0073, beyotime, china), and an appropriate amount of raigstσ. the enzyme mixture was incubated at 25 °c for 5 min before the reaction was initiated by adding 1 mm cdnb (703318, cayman chemical, usa) and absorbance was monitored for 5 min at 340 nm while the reaction was maintained at 25 °c. the changes in absorbance per minute were converted into amounts of substrate conjugated per min per mg enzyme by using the molar extinction coefficient for cdnb ε340 = 9.6 mm-1 cm-1. to characterize the raigstσ, enzymatic activity was evaluated at different temperature and ph. to determine the optimal temperature, protein samples were treated at 10 °c intervals between 10 °c and 90 °c for 1 h. to investigate the optimal ph, protein samples were treated between ph 3.5 and 10.5 at 1.0 ph intervals using different buffers for 1 h. acetate, phosphate and glycine-naoh buffers were used to obtain the ph ranges of 3.5-5.5, 6.5-7.5 and 8.5-10.5, respectively, according to previously reports (wang et al., 2013b, 2015). results sequence features of aigstσ a sigma class gst gene, aigstσ, was identified from the bay scallop est database, and its full-length cdna sequence was obtained via race technique and deposited into genbank under the accession number ku301768. the full-length cdna sequence of aigstσ comprised 779 bp, containing a 5’ untranslated regions (utr) of 48 bp, a 3’ utr of 113 bp with a poly a tail and an open reading frame (orf) of 618 bp. the orf encoded a polypeptide of 205 amino acid residues with a calculated molecular mass of approximately 23.11 kda and a theoretical isoelectric point of 5.354. no signal peptide was revealed in the deduced amino acid sequence of aigstσ by signalp program. a gst_n domain (from y4 to r73) and a gst_c domain (from i92 to n190) were found in the deduced amino acid sequence of aigstσ (fig. 1). 23 fig. 1 nucleotide and deduced amino acid sequences of aigstσ. the nucleotides and amino acids were numbered along the left margin. the function domain was in shade. the asterisks indicated the stop codon. two single typical polyadenylation signal sequences (aataaa aataaa) was underlined. phylogenetic ananlysis of aigstσ the deduced protein sequence of aigstσ exhibited high similarity with other previously identified sigma class gsts, such as 78 % identity with that of sigma class gst 2 from chlamys farreri (adf32019). the nj phylogenetic tree based on protein sequences from multiple gst genes was positioned separately into five main branches, and aigstσ were clustered with sigma class gst 2 from c. farreri and located in the sigma class gsts sub-branch (fig. 2). tissue distribution of aigstσ mrna transcripts the qrt-pcr technique was employed to detect the distribution of aigstσ mrna transcripts in different tissues with β-actin gene as internal control (fig. 3). the highest mrna expression level of aigstσ was found in hemocytes, which was 21.30-fold (p < 0.05) of that in muscle, while that in hepatopancreas was 13.28-fold (p < 0.05) of that in muscle. expression profiles of aigstσ mrna transcripts the temporal mrna expression profiles of aigstσ in hemocytes after various pamps stimulation were also examined via qrt-pcr (fig. 4a-d). the mrna transcripts of aigstσ all increased for the first time at 3-6 h and reached the peak at 12 h post different pamps stimulation. the mrna transcripts of aigstσ significantly increased at 3 h after lps stimulation (2.79-fold compared with the origin level, p < 0.05, fig. 4a), with the highest level observed at 12 h (17.74-fold, p < 0.05, fig. 4a). the mrna expression level of aigstσ was up-regulated at 6 h post pgn stimulation (3.13-fold, p < 0.05, fig. 4b) and then up-regulated to the highest level at 12 h (6.82-fold, p < 0.05, fig. 4b), and finally down-regulated to the normal level at 48 h. in the glucan stimulation group, after a significant increase at 3 h post stimulation (3.18-fold, p < 0.05, fig. 4c), the mrna transcripts of aigstσ increased to the peak at 12 h (12.15-fold, p < 0.05, fig. 4c), and finally decreased to the original level at 48 h. 24 fig. 2 consensus neighbor-joining phylogenetic tree based on the amino acid sequences of gsts from different organisms. the evolutionary history was inferred using the neighbor-joining method. the bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed. all positions containing gaps and missing data were eliminated. the numbers at the forks indicated the bootstrap value. the sequence information has been listed in table 2. the mrna transcripts of aigstσ significantly increased at 6 h post poly ic stimulation (6.71-fold, p < 0.05, fig. 4d), reached the peak at 12 h (9.07-fold, p < 0.05, fig. 4d), and then decreased to the normal level at 24 h. in the normal group, no significant change of aigstσ mrna expression level was observed during the whole experiment, while after pbs injection, a slight but significant increase was observed at 6 h (2.93-fold, p < 0.05, fig. 4a-d). purification of recombinant aigstσ protein to investigate the potential activities of aigstσ, the recombinant plasmid peasyblunt e1/aigstσ was transformed into e. coli strain bl21 (de3). after auto-induction, the whole-cell lysate was separated by sds-page, and a distinct band of raigstσ was revealed (fig. 5). biochemical characteristics of recombinant aigstσ protein according to the method previously described, the activity of raigstσ was measured for five times, and raigstσ exhibited detectable activity towards cdnb, which was 3.28 ± 0.03 μmol min-1 mg-1. to investigate the stability of aigstσ, the enzymatic activities of raigstσ were measured at different ph and temperature. for the optimal ph assay, raigstσ could maintain more than 50 % of its activity at a ph range from 7.5 to 9.5, but lost more than 60 % of its activity when the ph was lower than 6.5 or at 10.5 (fig. 6a). while when the temperature increased from 10 °c to 20 °c, raigstσ exhibited stable enzymatic activities, but lost more than 60 % of its enzymatic activity over 30 °c and almost devitalized at 50 °c (fig. 6b). 25 fig. 3 tissue distribution of aigstσ mrna transcripts detected by qrt-pcr. the β-actin gene was used as an internal control to calibrate the cdna template for each sample. the mrna expression level of aigstσ in hemocytes, muscle, mantle, gill, hepatopancreas and gonad of five adult scallops was normalized to that of muscle. vertical bars represented mean ± sd (n = 5), and bars with different characters indicated significantly different (p < 0.05). discussion sigma class gsts are a large sub-family of gsts (flanagan and smythe, 2011), and accumulating research achievements revealed that sigma class gsts from marine invertebrates were functional diversity and might not only serve as an antioxidant enzyme involving in the detoxification but also play important roles in the modulation of innate immune responses (boutet et al., 2004; lee et al., 2007; wan et al., 2008; ren et al., 2009; li et al., 2012; yang et al., 2012; umasuthan et al., 2012; zhang et al., 2012a, b; wang et al., 2013a; li et al., 2015). in the present study, the full-length cdna sequence of aigstσ was obtained from bay scallop a. irradians. the deduced polypeptide of aigstσ consisted of 205 amino acids, and its calculated molecular weight was 23.11 kda, which was very close to gsts of vertebrate and invertebrate. the amino acid sequence of aigstσ shared as high as 78 % identity with the previously identified sigma class gst 2 from c. farreri. in the phylogenetic tree, aigstσ was located in the sigma class gsts sub-branch. its sequence characteristics, high similarity with other known sigma class gsts and the phylogenetic relationship collectively suggested that aigstσ is a novel member of invertebrate sigma class gst family and may have similar function with sigma class gsts from other marine invertebrates. sigma class gst acts as the principal scavenger of xenobiotics (flanagan and smythe, 2011), and it was reported to be ubiquitously distributed in multiple tissues in marine invertebrates (boutet et al., 2004; lee et al., 2007; wan et al., 2008; ren et al., 2009; li et al., 2012; yang et al., 2012; umasuthan et al., 2012; zhang et al., 2012a, b; wang et al., 2013a; li et al., 2015). in the present study, the tissue distribution of aigstσ mrna transcripts was detected by qrt-pcr to investigate its possible function, and the ubiquity of aigstσ transcripts indicated that it could be involved in many important physiological processes of scallops. similar to the observation in sigma class gsts from m. galloprovincialis, s. grandis and v. philippinarum (yang et al., 2012; zhang et al., 2012a; wang et al., 2013a), the highest mrna expression level of aigstσ was observed in hemocytes, followed by hepatopancreas. the variable tissue distribution of aigstσ mrna transcripts was speculated to be related with tissue dependent oxidative load. the hemocytes have been considered to play pivotal roles 26 fig. 4 temporal mrna expression profiles of aigstσ detected by qrt-pcr in hemocytes at 3, 6, 12, 24 and 48 h post different pamps stimulation (a: lps, b: pgn, c: glucan, d: poly ic). the β-actin gene was used as an internal control to calibrate the cdna template for each sample. each values was shown as mean ± sd (n = 5), and bars with different characters indicated significantly different (p < 0.05). in the innate immune response in invertebrates mainly via phagocytosis, which was usually companied with oxidative stress, while the hepatopancreas is regarded as the main organ where multiple oxidative reactions and antioxidant defenses occur with high metabolic activity (song et al., 2015). additionally, hemocytes and hepatopancreas were also considered as the main immune related organs in scallops (song et al., 2015), the high mrna expression level of aigstσ in these two organs indicated that it could be involved in the innate immunity of scallop. it has been reported that sigma class gsts could rapidly respond to various foreign particles or invading microbes in mrna levels. for examples, a sigma class gst gene from h. diversicolor could be significantly induced in the hemocytes, gill, mantle and digestive gland of bacteria-challenged abalone (ren et al., 2009). bacterial challenge could significantly induce the mrna expression of two sigma class gsts from v. philippinarum (li et al., 2012). the mrna expression of a sigma class gst in hemocytes was significantly up-regulated after razor clam was stimulated by pgn or glucan (yang et al., 2012). while after bacterial challenge, the mrna expression levels of sigma class gsts in hemocytes were all significantly up-regulated in m. galloprovincialis (wang et al., 2013a). in the present study, the mrna transcripts of aigstσ could be significantly induced by the stimulation of four typical pamps, confirming the hypothesis that it could be involved in the innate immune response of scallops. additionally, a slight but significant increase of aigstσ mrna transcripts was also observed at 6 h after pbs injection, indicating aigstσ might be also involved in the responses to injury in scallop. to further investigate the potential role of aigstσ in bay scallop, the catalytic activity of its recombinant protein was determined in vitro using 27 fig. 5 sds-page analysis of the raigstσ protein in e. coli strain bl21 (de3). line m was the unstained protein marker (26610 thermo fisher scientific, usa). line u was the supernatant of non-induced bacteria lysate. line i was the supernatant of auto-induced bacteria lysate. line p was the purified recombinant protein. cdnb as substrate. in a previous research, the recombinant hdgsts1 and hdgsts2 proteins in h. discus discus exhibited catalytic activities of 0.17 ± 0.01μmol min-1 mg-1 and 1.06 ± 0.02 μmol min-1 mg-1, respectively, with relatively broad optimum ph spectrum and temperature range (wan et al., 2008). while rrpgstσ from r. philippinarum demonstrated a high catalytic ability toward cdnb of 4.64 ± 0.17 μmol-1 min-1 mg-1, but xhibited narrow optimal ph spectrum and temperature range (umasuthan et al., 2012). similarly, in the present study, aigstσ exhibited a high enzymatic activity of 3.28 ± 0.03 μmol min-1 mg-1, but lost more than 60% of its activity when the ph was lower than 6.5 or when the temperature was over 30 °c. it has been reported that both sea surface temperature rise and ocean acidification affect survival and reproduction of marine organisms negatively, including scallop (zhang et al., 2014; lagos et al., 2016). so, the lower active stability of raigstσ, especially susceptible to low ph or high temperature, might provide valuable insights into a possible mechanism of large scale mortalities of cultured bay scallops in summer. in conclusion, the full-length cdna encoding a sigma class gst was identified from bay scallop a. irradians. it was constitutively expressed in all the tested tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, and the mrna expression levels of aigstσ were all up-regulated in hemocytes after various pamps stimulaton. the purified raigstσ protein exhibited relatively high catalytic activity against cdnb with low thermal stability and narrow optimum spectrum of ph. all these results indicated that it was a fragile but efficient antioxidant enzyme and was potentially involved in the innate immune responses of scallop. this study would enrich the understanding of the scallop innate immunity. 28 fig. 6 the enzymatic activities of raigstσ under different treatment (a: ph, b: temperature). each values was shown as mean ± sd (n = 5), and bars with different characters were significantly different (p < 0.05). 29 acknowledgement this research was supported by the key research program of the chinese academy of sciences (kfzd-sw-106). we would like to thank all the three expert reviewers for their constructive suggestions and enlightening comments during the revision. references bao yb, li l, wu q, zhang gf. cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop argopecten irradians. fish shellfish immunol. 27: 17-25, 2009a. bao yb, li l, xu f, zhang gf. intracellular copper/zinc superoxide dismutase from bay scallop argopecten irradians: its gene structure, mrna expression and recombinant protein. fish shellfish immunol. 27: 210-220, 2009b. bao yb, li l, zhang gf. the manganese superoxide dismutase gene in bay scallop argopecten irradians: cloning, 3d modelling and mrna expression. fish shellfish immunol. 25: 425-432. 2008. bao yb, li l, zhang gf, polymorphism of the superoxide dismutase gene family in the bay scallop (argopecten irradians) and its association with resistance/susceptibility to vibrio anguillarum. dev. comp. immunol. 34: 553-561, 2010. boutet i, tanguy a, moraga d. characterisation and expression of four mrna sequences encoding glutathione s-transferases pi, mu, omega and sigma classes in the pacific oyster crassostrea gigas exposed to hydrocarbons and pesticides. mar. biol. 146: 53-64, 2004. chen x, zhang r, li ch, bao yb. mercury exposure modulates antioxidant enzymes in gill tissue and hemocytes of venerupis philippinarum. inv. surv. j. 11: 298-308, 2014. flanagan ju, smythe ml. sigma-class glutathione transferases. drug metab. rev. 43: 194-214, 2011. habig wh, pabst mj, jakoby wb. glutathione s-transferases. the first enzymatic step in mercapturic acid formation. j. biol. chem. 249: 7130-7139, 1974. lagos na, benitez s, duarte c, lardies ma, broitman br, tapia c, et al. effects of temperature and ocean acidification on shell characteristics of argopecten purpuratus: implications for scallop aquaculture in an upwelling-influenced area. aquacult. env. interac. 8: 357-370, 2016. lee ym, lee kw, park h, park hg, raisuddin s, ahn iy, et al. sequence, biochemical characteristics and expression of a novel sigma-class of glutathione s-transferase from the intertidal copepod, tigriopus japonicus with a possible role in antioxidant defense. chemosphere 69: 893-902, 2007. li ch, su xr, li y, li tw, sun cj, zhou tt, et al. two classes of glutathione s-transferase genes with different response profiles to bacterial challenge in venerupis philippinarum. fish shellfish immunol. 32: 219-222, 2012. li f, wu hf, wang q, li xh, zhao jm, glutathione s-transferase (gst) gene expression profiles in two marine bivalves exposed to bde-47 and their potential molecular mechanisms. chin. j. oceanol. limnol. 33: 705-713, 2015. li hj, liu x, hu jj, bao zm, zhang gf. a set of polymorphic microsatellite loci for the bay scallop, argopecten irradians. mol. ecol. notes 7: 422-424, 2007. li j, li l, zhang sd, zhang gf. cloning, genomic structure, and expression analysis of peroxiredoxin v from bay scallop argopecten irradians. fish shellfish immunol. 30: 309-316, 2011. lu yl, zhang ag, li ch, zhang p, su xr, li y, et al. the link between selenium binding protein from sinonovacula constricta and environmental pollutions exposure. fish shellfish immunol. 35: 271-277, 2013. matozzo v. aspects of eco-immunology in molluscs. inv. surv. j. 13: 116-121, 2016. raza h, robin ma, fang jk, avadhani ng. multiple isoforms of mitochondrial glutathione s-transferases and their differential induction under oxidative stress. biochem. j. 366: 45-55, 2002. ren hl, xu dd, gopalakrishnan s, qiao k, huang wb, wang kj. gene cloning of a sigma class glutathione s-transferase from abalone (haliotis diversicolor) and expression analysis upon bacterial challenge. dev. comp. immunol. 33: 980-990. 2009. shao yn, lv zm, li ch, zhang ww, duan xm, qiu qf, et al. molecular cloning and functional characterization of theta class glutathione s-transferase from apostichopus japonicus. fish shellfish immunol. 63: 31-39, 2017. schmittgen td, livak kj. analyzing real-time pcr data by the comparative ct method. nat. protoc. 3: 1101-1108, 2008. sheehan d, meade g, foley vm, dowd ca. structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. biochem. j. 360: 1-16, 2001. song ls, wang ll, zhang h, wang mq, the immune system and its modulation mechanism in scallop. fish shellfish immunol. 46: 65-78, 2015. umasuthan n, revathy ks, lee y, whang i, choi cy, lee j. a novel molluscan sigma-like glutathione s-transferase from manila clam, ruditapes philippinarum: cloning, characterization and transcriptional profiling. comp. biochem. physiol. 155c: 539-550, 2012. wan q, whang i, lee j. molecular cloning and characterization of three sigma glutathione s-transferases from disk abalone (haliotis discus discus). comp. biochem. physiol. 151b: 257-267, 2008. wang cy, zhao jm, mu ck, wang q, wu hf, wang cl. cdna cloning and mrna expression of four glutathione s-transferase (gst) genes from mytilus galloprovincialis. fish shellfish immunol. 34: 697-703, 2013a. wang ll, song ls, ni dj, zhang h, liu wq. alteration of metallothionein mrna in bay 30 scallop argopecten irradians under cadmium exposure and bacteria challenge. comp. biochem. physiol. 149c: 50-57, 2009. wang mq, wang ll, guo y, zhou z, yi ql, zhang dx, et al. a high mobility group box 1 (hmgb1) gene from chlamys farreri and the dna-binding ability and pro-inflammatory activity of its recombinant protein. fish shellfish immunol. 36: 393-400, 2014. wang mq, wang ll, yi ql, gai yc, song ls. molecular cloning and characterization of a cytoplasmic manganese superoxide dismutase and a mitochondrial manganese superoxide dismutase from chinese mitten crab eriocheir sinensis. fish shellfish immunol. 47: 407-417, 2015. wang mq, wang ll, zhou z, gao y, wang ll, shi xw, et al. the molecular characterization of a catalase from chinese mitten crab eriocheir sinensis. int. j. immunogenet. 40: 230-240, 2013b. wilce mcj, parker mw. structure and function of glutathione s-transferases. bba-protein struct. m. 1205: 1-18, 1994. yang jl, wei xm, xu j, yang dl, liu xq, yang jm, et al. a sigma-class glutathione s-transferase from solen grandis that responded to microorganism glycan and organic contaminants. fish shellfish immunol. 32: 1198-1204, 2012. zhang h, zhou z, yue f, wang ll, yang cy, wang mq, et al. the modulation of catecholamines on immune response of scallop chlamys farreri under heat stress. gen. comp. endocrinol. 195: 116-124, 2014. zhang lb, qiu lh, wu hf, liu xl, you lp, pei d, et al. expression profiles of seven glutathione s-transferase (gst) genes from venerupis philippinarum exposed to heavy metals and benzo[a]pyrene. comp. biochem. physiol. 155c: 517-527, 2012a. zhang lb, wu hf, liu xl, chen ll, wang q, zhao jm, et al. molecular cloning and differential expression patterns of sigma and omega glutathione s-transferases from venerupis philippinarum to heavy metals and benzo[a]pyrene exposure. chin. j. oceanol. limnol. 30: 413-423, 2012b. zhang z, lv zm, shao yn, qiu qf, zhang ww, duan xm, et al. microsomal glutathione transferase 1 attenuated ros-induced lipid peroxidation in apostichopus japonicus. dev. comp. immunol. 73: 79-87, 2017a. zhang z, lv zm, wei zx, li ch, shao yn, zhang ww, et al. microsomal glutathione transferase 2 modulates ltc4 synthesis and ros production in apostichopus japonicus. mol. immunol. 91: 114-122. 2017b. isj 5: 179-yyy, 2008 issn 1824-307x isj 5: 190-191, 2008 issn 1824-307x erratum to: lipase and invertase activities in midgut and salivary glands of chilo suppressalis (walker) (lepidoptera, pyralidae), rice striped stem borer [5: 180-189, 2008] a zibaee, ar bandani, s ramzi plant protection department, faculty of agriculture, university of tehran, karaj 31584, iran in the above article table 2 was reproduced incorrectly, the table and caption should have appeared as below: table 2 relative activity of c. suppressalis invertase towards different compounds compounds concentration (mmol/l) relative activity (midgut) relative activity (salivary gland) control 100 100 nacl 5 83.33 152.63* 10 63.88* 121.05* 20 41.66* 94.7* 40 10* 52.63* cacl2 5 25.83* 78.94* 10 66.66* 131.57* 20 113.88 157.89* 40 125* 194.73* kcl 5 161.11* 273.68* 10 105.55 252.63* 20 69.44* 194.73* 40 50* 89.47* mgcl2 5 97.22 168.42* 10 61.11* 152.63* 20 44.44* 84.21* 190 40 21.38* 27.36* edta 0.5 144.44* 168.42* 1 116.66 131.57* 2 97.22* 105.28 4 52.77* 73.68* sds 2 111.11 173.68* 4 91.66 136.84* 6 61.11* 121.05 8 38.88* 73.68* urea 1000 83.33* 173.68* 2000 63.88* 147.36* 4000 50* 115.78 5000 33.33* 73.68* 6000 25* 47.36* plant extract 10 % 82* 91* 15 % 64* 74* 25% 21* 52* *p < 0.05 vs control ___________________________________________________________________________ corresponding author: ali reza bandani plant protection department college of agriculture and natural resources university of tehran, karaj, 31584 iran e-mail: abandani@ut.ac.ir 191 isj111.pdf 4 isj 3: 4-17, 2006 issn 1824-307x review proteomics and insect immunity l shi, sm paskewitz department of entomology, university of wisconsin, madison, wisconsin, usa accepted january 24, 2006 abstract insect innate immunity is both a model for vertebrate immunity as well as a key system that impacts medically important pathogens that are transmitted by insects. recent developments in proteomics and protein identification techniques combined with the completion of genome sequences for anopheles gambiae and drosophila melanogaster provided the tools for examining insect immunity at a new level of molecular detail. application of proteomics to insect immunity resulted in predictions of new roles in immunity for proteins already known in other contexts (e.g. ferritin, transferrin, chi-lectins) and helped to target specific members of multi-gene families that respond to different pathogens (e.g. serine proteases, thioester proteins). in addition, proteomics studies verify that post-translational modifications play a key role in insect immunity since many of the identified proteins are modified in some way. these studies complement recent work on insect transcriptomes and provide new directions for further investigation of innate immunity. key words: phagocytosis; antimicrobial peptides; melanization; drosophila melanogaster; anopheles gambiae; 2dpage; hemolymph introduction innate immunity refers to the first-line host defense against the early phases of microbial infection and is an evolutionarily ancient defense mechanism. insects and vertebrates display considerable overlap in the intracellular signaling pathways that regulate innate immune responses (salzet, 2001; giot et al., 2003; hultmark, 2003) and in some of the effector mechanisms used against microbes (e.g. phagocytosis, fluid lysozymes). thus, discoveries made through research in the fruit fly, drosophila melanogaster may be applicable to innate immunity in humans (fallon et al., 2001). corresponding author: susan m paskewitz department of entomology, 237 russell labs, 1630 linden drive, university of wisconsin, madison, wisconsin 53706, usa email: paskewit@entomology.wisc.edu the study of innate immunity in insects has also garnered increasing attention because of the role of many insects in transmission of human disease agents. understanding how the insect immune system interacts with pathogens may contribute to development of new strategies to block transmission of disease agents (christophides, 2005). for example, cecropin is a protein originally identified for its antibacterial activity in lepidopteran insects but eventually shown to reduce malaria parasite development (gwadz et al., 1989). as a result, transgenic mosquitoes were developed to overexpress cecropin in the midgut, resulting in significant decreases in the number of developing malaria parasites following infection (kim et al., 2004). the sequencing of the genomes coupled with est projects for two dipteran species, d. melanogaster and the african malaria mosquito, anopheles gambiae, provided new opportunities for studying immunity. new genes with candidate immune functions were quickly identified (christophides et al., 2002) and microarrays were applied to survey transcriptome changes after bacterial, fungal or parasite infections (degregorio et al., 5 2001; irving et al., 2001; dimopoulos et al., 2002; roxstrom-lindquist et al., 2004). these studies have been fruitful in identifying a set of genes that can be tested for functional involvement in insect immune responses. however, mrna-based approaches suffer some limitations. first, they can be misleading in estimating how much protein is present. although changes in mrna levels sometimes accurately serve as surrogates for changes in the respective protein levels, several studies have shown that this is not the case about 40-50 % of the time (gygy et al., 1999; ideker et al., 2001). a specific example of this type of problem can be seen in mosquito immunity where the antimicrobial peptide defensin displayed high levels of transcript that did not correlate with detectable peptide (bartholomay et al., 2004). second, mrna analyses tell us nothing about whether the encoded proteins are active or not as the functions of proteins are often regulated by post-translational modification. for example, several proteolytic cascades are involved in insect responses to pathogens, with cleavage of a series of proteins necessary for activation of the end product. these functional modifications cannot be directly determined from dna sequence information or mrna levels. third, mrna cannot be used to profile changes in secreted proteins that occur in the hemolymph when the body cavity is invaded by pathogens or to identify components of extracellular reactions such as melanization or coagulation. thus, a complementary approach for investigating immunity in greater detail is to focus on the proteins themselves. proteomics is a tool for detecting changes in protein expression and modification in whole organisms and in specific cells, tissues, and fluids. this review will focus on the methods and applications of proteomics to insect immunity. methodologies definition of proteomics the term “proteome" was coined in 1994 and defined as the entire protein complement expressed by a sample. proteomics encompasses a broad set of disciplines aimed at understanding and monitoring proteins. this includes work correlating genetic sequence with three-dimensional protein structure and 3d structure with protein function, development of protein separation and protein profiling techniques, and investigation of protein-protein interactions. recent studies of insect immunity concentrate on “profiling/expression" and “functional" proteomics (table 1). profiling or expression proteomics focuses on the description of the whole proteome in a given tissue, body fluid, cell type, or organelle, and differential measurement of protein expression levels in samples collected under different conditions (choudhary and grant, 2004). functional proteomics includes research approaches that directly analyze a subset of proteins, such as a family of sequenceor function-related proteins (kocks et al., 2003), as well as those that characterize the protein’s biological functions, proteinprotein or protein-dna/rna interactions, or posttranslational modifications (cai et al., 2004). the tools of proteomics have been developing over the past three decades, but it was not until mass spectrometry began to be used for the identification of proteins in complex mixtures that the field really started to take off (karas and hillenkamp, 1988; fenn et al., 1989). a summary of methodologies used for insect immunity studies will be presented next, with notes concerning limitations of the various procedures. protein separation all proteomic technologies rely on the ability to separate a complex mixture so that individual proteins are more easily processed with other techniques. twodimensional gel electrophoresis (2-de) is still the most widely used protein separation technology for insect immunity (o’farrell 1975; table 1; fig. 1). in this approach, proteins are separated in the first dimension by isoelectric focusing using immobilized ph gradient strips. then, these proteins are again separated, this time by molecular weight in standard polyacrylamide gel electrophoresis, resulting in a 2-dimensional display of proteins. the advantage of this method is that a large number (3,000 to 10,000) of proteins can be visually separated and those spots exhibiting changes between treatments can then be singled out for further exploration. electrophoresis is followed by excision of specific spots, digestion, and protein identification (fig. 1). a number of problems have to be confronted when using 2-de as a protein separation and expression profiling technique. for example, proteins present at low concentrations, those of very high or very low molecular weight, or membrane proteins are generally not detected on the gels. detection of some of these proteins can be improved by pre-separation fractionation and processing of tissue and cell extracts or by altering the conditions of the 2-de (fig. 1b; chevallet et al., 1998), but pre-separation requires additional manipulation to make quantification possible. insect tissues such as the fat body that have high lipid contents may also require pre-separation fractionation (e.g. stadler and hales, 2002). perhaps most frustrating, the reproducibility of 2-de experiments can be poor, requiring many replicates to ensure confidence in the results. however, several tools have reduced some of the variability associated with 2-de. immobilized ph gradients are commercially available and replace the unstandardized tube gels used in older protocols. 2ddige (see below) provides internal standards and reduces the non-biological variability associated with standard 2-de. companies have introduced software that greatly facilitates 2-d gel image analysis. such programs generally automate the alignment of spots on one gel with corresponding spots on another, facilitating analysis even when gel distortions occur. an alternative to 2-de that is likely to become more widely used in the future is mudpit (multidimensional protein identification technology), which couples twodimensional chromatography of peptides in mass 5 table 1. summary of proteomics studies in insect immunity insect samples treatment(s) methods ref profiling proteomics drosophila melanogaster 3th instar larval hemolymph lps, m.luteus, s. cerevisiae 2d-dige vierstraete et al., 2005 3th instar larval hemolymph no 2de, malditof vierstraete et al., 2003 3th instar larval hemolymph m. luteus, s. cerevisiae, lps. 2d-dige, ms vierstraete et al., 2004a, b 3th instar larval hemolymph m. luteus, e. coli, b. bassiana 2de, malditof levy et al., 2004a, b 3th instar larval hemolymph d. pneumoniae, n. catarrhalis, s. aureus, k. pneumoniae, h. influenza, s. pyogenes 2de, malditof guedes et al., 2005 3th instar larval hemolymph hemolymph clot 2de, malditof karlsson et al., 2004 mbn-2 cell line lps 2de, malditof loseva and engstrom, 2004 anopheles gambiae adult 4a rr and l3-5 strains, hemolymph bacteria (m. luteus and e. coli) sephadex beads 2de, ms paskewitz and shi, 2005 chun et al., 2000 adult g3 strain female salivary gland blood meal sds-page, lc-ms/ms kalume et al., 2005 g3 strain male and female midgut sugar-fed or blood meal 2de prevot et al., 2003 anopheles stephensi female midgut from mosquitoes of different susceptibility to p. falciparum sugar-fed or blood meal 2de prevot et al., 1998 aedes aegypti larval tissues v. culicis 2de, malditof biron et al, 2005 fat body eclosion or blood meal 2de shih and fallon, 2001 rkf, lvp, rlvp strain female thoracic tissue sucrose meal, blood meal or b. malayi sds-page, 2de wattam and christensen, 1992 bombyx mori 5th instar larvae hemolymph, midgut and fatbody lps 2de, malditof wang et al., 2004 functional proteomics drosophila melanogaster drosophila s2 cell line dcg-o4 sds-page ms kocks et al., 2003 anopheles gambiae adult female midgut, 4a-3a cell line purified p. berghei ookinetes bind annexins maldi-tof, 2de kotsyfakis et al., 2005 spectrometry-compatible solutions directly to tandem mass spectrometry (2d-lc-ms/ms), allowing for the identification of proteins from highly complex mixtures (fig. 1c). a useful aspect of this technique is the ability to analyze proteins when the amount of starting material is too small for 2-de. this technique has been used by levy et al. (2004b) to investigate small peptide (1-11 kda) profiles in hemolymph from immune challenged and naïve drosophila adults and by florens et al. (2002) to investigate mosquito-stage proteins of the malaria parasite, plasmodium falciparum. protein modification almost all proteins are modified following translation. specialized methods have been developed to study phosporylation (phosphoproteomics; salih, 2005) and glycosylation (glycoproteomics; hirabayashi et al., 2002) but these have not yet been applied to studies of insect immunity. however, changes due to glycosylation and protein spot was quantified by its staining intensity. in this approach, protein mixtures are often prepared from two different samples and resolved by separate 2d gels for subsequent comparison of protein expression or changes in protein modification. gels can be compared by eye but are now usually digitized and analyzed using imaging software. for reliable densitometric analysis, image acquisition needs to be done by calibrated gel scanners with a wide dynamic range. several software packages that can analyze this input exist and have greatly facilitated spot quantification and comparison. 6 5 fig. 1 strategies for proteome analysis. (a) analysis of whole proteomes by two-dimensional gel electrophoresis (2dpage). in this approach, a protein extract is prepared from two different samples and the proteins are resolved by 2d gel electrophoresis. proteins that differ by some variable then are selected for identification by mass spectrometry (ms). although this method allows for the selection of relevant proteins, few low-copy proteins can be identified. (b) analysis of whole proteomes by ms. in this approach, all cellular proteins are converted to peptides. the peptides are resolved by liquid chromatography and analyzed by ms. this method allows for the identification of low-abundance proteins but, since there is no selection for relevant proteins, all proteins must be analyzed by ms. (c) analysis of sub-proteomes. in this approach, a protein extract is separated into individual sub-proteomes by fractionation or specialized affinity chromatography and proteins are resolved by 1d or 2d-page. this allows for the enrichment of low-copy proteins and their selection for further analysis (graves and haystead, 2003). 7 5 protein quantification traditionally, visualization of spots in a 2d gel was by coomassie or silver staining. proteomics analysis utilizing 2-de protein separation is frequently criticized as being lowthroughput, in part due to the time-consuming process of image analysis that is necessary to determine differential protein expression. this process can be laborious due to gel-to-gel variations that confound the analysis process. through the use of fluorescent dyes to label protein samples prior to 2-de, the dige (difference gel electrophoresis) technique allows multiple samples to be co-separated and visualized on one 2d gel (tonge et al., 2001; fig. 2). the protein extracts, for example one control and one treated, are labeled with different fluorescent dyes (e.g. cy2, cy3), then combined and separated by 2-de. in this example, two images of the gel would be captured – using the cy2 and cy3 excitation wavelengths. the images are then merged, and differences between them can be determined using image analysis software. the method minimizes the gelto-gel variation implicit in 2-de but cannot eliminate variation due to differences in the extraction or labeling steps. the dyes are reported to produce a linear response to protein concentration over five orders of magnitude, have enhanced sensitivity in comparison with other commonly used protein stains, and are compatible with ms analysis. the 2d-dige method was used successfully by veirstraete and colleagues (2004a,b; 2005) to profile changes in the larval hemolymph of d. melanogaster following immune challenge. protein identification protein identification is now an essential part of almost every proteomics experiment. some of the studies of insect immunity incorporated n-terminal sequencing or sequencing of proteolytic fragments from spots by edman degradation to generate sequences for database searches (chun et al., 2000). currently, however, the most commonly used identification approach is ms. ms can rapidly, and with high sensitivity, determine masses and structures of peptides. software is available to use the two different types of data generated by mass spectrometers to search sequence databases. protein identification using peptide mass fingerprinting is an effective technique when studying organisms with completed genomes. using the programs mascot (www.matrixsciences.com), profound (http://prowl. rockefeller.edu/) and peptldent (www.expasy.org/tools/ peptident.html), one can analyze the peptide mass profiles produced by ms. findmod (www.expasy.org/ tools/findmod/) is used to find modifications for analysis of unmatched peptide masses. a second method for protein identification is based on the use of sequence data created by tandem mass spectrometers. this information can be used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (est) sequences. the ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but for which a large amount of expressed gene sequence is available. data analysis to make the most of the wealth of proteomics data being produced around the world, it is important to establish standards for storing and reporting proteomic data enable comparisons across platforms and research groups. the universal protein resource, or uniprot, (http://www.pir.uniprot.org/) was recently established by nih as a centralized database of protein information such as function, classification and cross-reference. uniprot combines the resources from the major annotated protein databases swissprot and trembl from the european bioinformatics institute (ebi) and the swiss institite for bioinformatics (sib) as well as the protein sequence database (psd) from the protein information resource (pir) (http://pir.georgetown.edu/) (apweiler et al., 2004). for drosophila melanogaster or anopheles gambiae, gene identity and predicted protein function are found at the flybase report (http://flybase.bio.indiana.edu/) or anobase (www.anobase.org/anobase/genes/ano-xcel) (loseva and engstrom, 2004; ribeiro et al., 2004). application of proteomics to the study of insect immunity mechanisms of insect immunity in this section, we will briefly introduce proteins that have known or likely functions in insect immunity to place them in context when discussing the results of proteomics studies. insect innate immunity is based on the recognition of microbial molecules, such as lps, peptidoglycans, or β-1,3-glucans, by specific receptors with the subsequent activation of immune effector responses. non-microbial surfaces (e.g. sephadex beads) also elicit responses. proteins that are considered to have a recognition or opsonizing function include gnbp, tep and pgrp. recognition leads to activation of cellular and/or humoral effector mechanisms. these include phagocytosis by hemocytes, encapsulation or nodulation of pathogens by hemocytes, activation of proteolytic cascades leading to localized melanization and hemolymph clotting, and synthesis of a battery of amps. the latter process can occur in most insect tissues including the fat body, hemocytes, respiratory system, cuticular and midgut epithelia, malpighian tubules, and male and female genital tracts (tzou et al., 2000). the activation of amp synthesis is the best described of these effector processes. in drosophila, there are several groups of amps that act mainly against either gram positive bacteria, gram negative bacteria or fungi. two distinct signaling pathways, toll and imd (immune deficiency), control their expression. the toll pathway is activated by an extracellular proteolytic cascade, where serine proteases are important. serine proteases can be regulated by inhibitors, including serpins and kunitz types. 8 4 fig. 2 schematic representation of the 2d-dige (differential in gel electrophoresis) method. two samples for comparison are individually labeled with distinct fluorochromes. the two samples are mixed and separated on the same 2d gel to reduced gel-to-gel variation. the resulting gel is imaged twice using the two different wavelengths for the two fluors. image analysis software is used to detect spots in each image, overlay gels, and quantify differences. 9 4 2002) and, with a few exceptions, specific targets have not been identified for them. some members of a subgroup called the clip-domain serine proteases, as well as their inhibitors, are important in activating the toll pathway and in localized melanization responses. mosquitoes are known to melanize malaria parasites, nematode worms, yeast, microsporidial spores, bacteria and sephadex beads. the melanin pathway depends on the activity of phenoloxidase, which exists in a zymogen form prior to cleavage by an activating serine protease. phenoloxidases and serine proteases are also involved in hemolymph clotting (karlsson et al., 2004). interestingly, a serpin mutant suggests that toll activation is linked to the melanization pathway in drosophila (green et al., 2000; ligoxygakis et al., 2002). expression profiling proteomics protein expression profiling of insect immune responses has been initiated for several insect species and tissues (table 1) and many differentially expressed proteins have been identified by ms. most of these studies have been carried out in d. melanogaster (uttenweiler-joseph et al. 1998; guedes et al., 2003; sabatier et al. 2003; vierstraete et al. 2003, 2004a,b, 2005; engstrom et al., 2004; levy et al., 2004a,b; guedes et al., 2005). other taxa where immunity has been investigated by proteomic methods include the mosquitoes, a. gambiae (chun et al. 2000; paskewitz and shi 2005) and aedes aegypti (wattam and christensen, 1992; biron et al., 2005), the silkworm, bombyx mori (wang et al., 2004), and the locust, odaleus australis (stadler and hales 2002). overall, we are lacking in genomic and proteomic information for hemimetabolous insects as well as broad coverage of the holometabolous orders. there are significant and extensive methodological differences between the above mentioned proteomics studies, including types of challenge agents (single species or mixes of living bacteria, lps, bacterial lysates, yeast, filamentous fungi, microsporidia, picorna-like virus, sephadex beads) as well as the method of introduction of the agent (feeding, external exposure, injection), the length of incubation time after exposure, the tissue examined (hemolymph, hemocyte-like cells in culture, fat body, midgut, thorax, whole insect larvae), the method of tissue collection, and the stage of the insect (larvae vs adult). standardizing these aspects would provide better ability to compare innate immunity across taxonomic groups. nevertheless, taken together these studies identify some patterns in proteins that are affected by immune challenges and provide a framework for future investigations. hemolymph profiles hemolymph is a critical immune fluid in insects. it contains hemocytes and is a transport tissue for effector molecules like the fat body-produced amps. most proteomic data are from this fluid. several studies provide reference maps of identified hemolymph proteins that are not immune responsive but may be useful for other studies (guedes et al., 2003; vierstraete et al., 2003; karlsson et al., 2004; paskewitz and shi, 2005). the hemolymph reference maps contain constitutively expressed proteins from several groups: storage and transport proteins, metabolic proteins, cytoskeletal proteins, defense/immune proteins, antioxidant and stress proteins, and novel proteins. there are relatively few proteins in common between some of the drosophila studies, reflecting the methodological issues described above. many of the identified proteins in the reference maps of drosophila hemolymph are intracellular metabolic proteins while more than half of those identified in a. gambiae are related to immunity and appear to be secreted proteins. cellular proteins occur in these samples because hemocytes and other contaminants are generally not removed (but see karlsson et al., 2004 for serum versus plasma maps). in a. gambiae, fat body can be a major hemolymph contaminant, so care must be taken in attributing changes in cellular proteins to the hemocytes. in addition, some of the hemocyte types are known to be quite labile (e.g. crystal cells in drosophila, oenocytoids in anopheles) meaning that they break down and quickly release their contents when their environment changes. we attributed finding phenoloxidases in hemolymph and plasma to this feature, since oenocytoids produce pos and they do not contain signal peptides (paskewitz and shi 2005). other cellular proteins that are abundant in labile hemocytes might also exhibit this pattern. cellular protein profiles may also be affected by changes in hemocyte behavior after infection. microbial challenge can result in mobilization of sessile hemocytes, so the relative cellular content of immune-challenged versus unchallenged hemolymph may not be equal. some considerations as to controls controls for immune-induced hemolymph samples must be carefully chosen since the manner of introduction of the immune challenge is often traumatic. pricking an insect with a needle dipped in a pellet of bacteria is the most common challenge, although some studies use feeding or external exposure to simulate more natural conditions. we found that several of the proteins we had labeled as “wound-induced” proteins in a. gambiae were probably a specific result of damage to the thoracic musculature during aseptic wounding, rather than a generalized response to damaging the cuticle that would include hemolymph clotting and wound healing. comparison with mosquitoes wounded in the abdomen did not produce the same group of proteins and the thoracic wound samples contained some proteins of obvious muscle origin (paskewitz and shi, 2005). damage to tissue in vertebrates can also lead to the release of cellular proteins into circulation (alleyne et al., 2001; renz et al., 2001) if defined numbers of microbes or aliquots of surface molecules are to be injected, it is critical to use a sterile solution of the suspension buffer for the controls. vierstraete et al. (2004a) report that they injected lps in a solution that contained 1 % ethanol but did not use ethanol in the controls. this may explain the fact that the strongest induction they observed was for alcohol dehydrogenase in the lps-injected samples. 10 5 fig. 3 two dimensional gel profiles of anopheles gambiae hemolymph incubated in vitro in the absence (a) or presence (b) of heat-killed bacteria (m. luteus and e. coli). black arrows indicate the chi-lectins br1 and br2 in the bacterially-induced sample. red arrows indicate some of the other proteins altered after exposure to bacteria in this sample. guedes et al. (2005) tried to overcome the problems of injection by feeding bacterial lysates to larvae. they combined lysates from six different bacterial species and incorporated them into the feeding medium. this procedure might result in relatively low activation of the immune system, since only the digestive and exoskeletal systems would be directly exposed to bacterial products and bacteria are a normal part of the larval feeding environment. the controls for this experiment were not exposed to a change in their normal feeding medium. it is possible that a change in food quality could induce stress or cause metabolic shifts in an insect as it does in vertebrate animals. indeed, most of the induced proteins identified were metabolic or stress-related. amp production could be a useful marker for verifying immune induction under these conditions. peptidomic studies of hemolymph most proteomic studies are not designed to capture low molecular weight proteins. unfortunately, many of the amps fall into this category. uttenweiler-joseph et al. (1998) and levy et al. (2004a) report results of using hplc and maldi-tof for analysis of bacteriallyinduced peptides (1-15 kda) in adult drosophila hemolymph. of the 28 induced peptides that were characterized, ten were amps exhibiting posttranslational modifications and one was a kunitz type serine protease inhibitor; many of the others are hypothesized to have a chemokine function (levy et al., 2004a). additional kunitz type inhibitors were described in the reference maps of larval hemolymph (vierstraete et al., 2003). the targets of these inhibitors are not known. similar methodology has been applied to peptides induced in the hemolymph of a. aegypti after bacterial challenge (lowenberger, 2001). several amps (defensins and cecropin) were identified as well as novel peptides. the identification of a large number of peptides with unknown functions in drosophila and aedes indicates that much remains to be done to fully characterize even this narrow aspect of immunity. proteomic studies of hemolymph approximately 130 larger proteins that are described as immune-induced have been documented in larval or adult hemolymph from d. melanogaster by 2de and protein identification methods (levy et al. 2004a, b; vierstraete et al. 2004a,b; guedes et al. 2005). in general, the studies identify the following groups of proteins as regulated by the immune treatments: i) a b 11 5 immune-responsive proteins that have functional roles that are somewhat or well defined; ii) immuneresponsive proteins that have hypothesized functions that can be tested; iii) cellular proteins that are involved in stress responses; iv) proteins that appear following injury; v) metabolic proteins; and vi) proteins that are not similar to any other proteins in databases and as yet have no hypothesized functions. catalogs of these proteins can be found in the studies listed above and not all of these groups will be discussed in detail in this review. here we will first consider some of the patterns suggested by these studies. an example of a 2d-page separation of hemolymph proteins is provided in fig. 3. in studies of adult hemolymph following bacterial challenge, a similar number of proteins were affected in drosophila and anopheles. for example, 50 of 350 (14 %) silver-stained spots were upor down-regulated in drosophila (levy et al., 2004a, b) while 14 of 280 (5 %) silver-stained spots were upregulated in a. gambiae (paskewitz and shi, 2005). by comparison, a much larger number of spots were said to be specifically regulated in adult fruit flies by 72 h after fungal exposure (levy et al., 2004a, b). comparison of a subset of 42 proteins regulated following fungal exposure showed that twelve of the proteins were also affected by bacterial infection. three of the twelve were upregulated following both types of challenge (αamylase distal; hsp20-like chaperone; fructose 1,6, bisphosphate aldolase) while nine were regulated oppositely (propo-ae cg16705; dnase ii cg7780; aldehyde dehydrogenase; glyceraldehyde 3 phsophate dehydrogenase; enolase; cathepsin l; transferrin; ferritin; obp99c). in larval drosophila hemolymph, proteins that were immune-regulated were compared at 25 min after injection of micrococcus luteus or saccharomyces cerevisiae. thirteen were upregulated with either challenge agent. three additional proteins were regulated only following saccharomyces and two were regulated only following micrococcus inoculation. some proteins were present as multiple spots, and these were often differentially regulated between challenges. for example, several different ferritin heavy chain spots were identified, each upregulated after a different type of challenge agent (vierstraete et al., 2004b). in drosophila larval hemolymph, 131 of approximately 289 (45 %) silver-stained spots were altered following exposure to a diet that included bacterial lysates (guedes et al., 2005). the majority of the 71 proteins that were identified were cellular proteins involved in metabolism and stress responses. since only 20 % of the immune-regulated proteins in a. gambiae and 14 % of those in drosophila adult hemolymph were identified, we don’t yet have a complete picture of the changes that are occurring at the protein level at any time after infection of adults. however, one study did identify 94 % of the proteins found to be upregulated in larval fruit fly hemolymph by 25 min after immune challenge (vierstraete et al., 2004b). this study provides a good baseline for examining the overall profile of the types of immune proteins that are quickly secreted or processed following infection, and that would not be detectable by transcript analysis. teps are complement-like proteins characterized by a conserved thioester motif that enables covalent binding to target surfaces. these proteins appear to be fundamental to a number of immune processes in insects. reverse genetics clearly demonstrate that a. gambiae tep1 regulates phagocytosis of bacteria and killing of malaria parasites in this mosquito (levashina et al., 2001; blandin et al., 2004). among the proteins that increase in drosophila hemolymph upon infection are other members of the family, including tep2 following m. luteus or lps injection and tep4 after beauvaria bassiana exposure (levy et al., 2004b; vierstraete et al., 2004a,b). tep2 was especially sensitive to m. luteus injection; this was the strongest upregulation seen in this study of larval proteins. the rapid appearance of tep2 (within 25 min) means that other recognition processes occur to trigger its release or cleavage in the hemolymph. all of the tep proteins identified in the proteomics studies were smaller than predicted based on gene sequences and probably represent cleaved forms. the only other recognition/opsonizing protein identified by proteomics is a protein related to gram negative binding proteins. this was the most strongly upregulated protein seen in samples taken following fungal infection of drosophila adults (gnbp3; levy et al., 2004b). serine proteases and serine protease inhibitors play important roles in modulating and amplifying the toll signaling and melanization activation pathways. four different clip-domain serine proteases were identified following fungal/yeast infection in larval or adult drosophila. one, cg5390, was found within 25 min of challenge with saccharomyces cerevisiae but not after m. luteus or lps inoculation (vierstraete et al. 2004b). three others (cg1102, cg16705 and cg9372) were induced 72 h after b. bassiana exposure (levy et al., 2004a, b). two clip-domain serine proteases were identified in the a. gambiae proteomic studies but neither was reliably immune-induced (chun et al., 2000; paskewitz and shi, 2005). finally, three other serine proteases (not clip domain sps) were upregulated in hemolymph when high doses of bacterial lysates were fed to drosophila larvae (guedes et al., 2005). the significance of this observation is not clear but some serine proteases have direct antibacterial activity (tsuji et al., 1998). inhibitors of serine proteases called serpins are also important in immunomodulation. two serpins (srpn2 and srpn15) were identified in a. gambiae, both as constitutively expressed proteins (paskewitz and shi, 2005). two drosophila serpins (cg1857, cg6687) were significantly upregulated in adult flies on fungal but not bacterial infection. one of them (nec, cg1857) had previously been shown to regulate the induction of the toll pathway through inhibition of the activation of spaetzle, the toll ligand (levy et al., 2004a, b). a serpin was also found in hemolymph of injected silkworm larvae at 24 h post inoculation (wang et al., 2004). serpins were not found in the studies of the fruitfly larval hemolymph taken at 25 min after challenge (vierstraete 12 6 et al., 2004a,b). in addition to recognition factors and immunomodulators, some of the known effector proteins have also been identified. as noted above, many of the amps are small molecules and need to be examined by special methods. these studies have shown that posttranslational modifications are frequent and biochemical analysis indicates that the modifications affect the antimicrobial activity of the peptides. phenoloxidases are another effector category. in a. gambiae there are nine po genes, while drosophila has only three. the contribution of the individual po gene products to immunity has not yet been examined. proteomics pinpointed the po6 protein as strongly upregulated at one timepoint (24 h) following bacterial injection in a. gambiae (paskewitz and shi, 2005). one of the drosophila pos was down-regulated upon feeding of bacterial lysates. both of these observations could be reconciled by considering processing of ppo. that is, the down-regulated spot might represent ppo and the upregulated spot might be activated po6. po2 was identified as a constitutive protein in a. gambiae hemolymph that was not altered on bacterial injection. the proteomics studies pointed to post-translational modifications of proteins involved in iron metabolism as a potentially fruitful area for investigation. paskewitz and shi (2005) found decreases in both ferritin subunits at 6 and 24 h after a bacterial challenge. levy et al. (2004b) reported that ferritin was down-regulated in fungally challenged adults but unchanged after bacterial infection, although a more complex pattern is indicated in levy et al. (2004a). vierstraete et al. (2004b) found a large increase in the amount of a spot corresponding to the ferritin heavy chain homologue in larval hemolymph 25 minutes after m. luteus challenge. the spot is described as “shifted” indicating that post-translational modifications occurred. additional information on multiple forms of the light and heavy chains of ferritin can be found in levy et al. (2004a) and vierstraete et al. (2004b). the biology of ferritin in relation to immunity is not at all clear but it might serve to sequester iron from invading microorganisms (yoshiga et al., 1997). tsf is an iron transport protein that also occurs in the hemolymph. the tsf protein was found to be upregulated upon treatment of mosquito cells with bacteria (yoshiga et al., 1997) and during encapsulation of filarial worms in a. aegypti (beerntsen et al., 1994). proteomics studies also identified differences in tsf during fungal infections (levy et al., 2004a). in addition to an increase in tsf production, fungal infection induced proteolytic cleavage of tsf. in vertebrates tsf fragments have been linked to immunity, acting directly as antimicrobial peptides or as inducers of nitric oxide production by macrophages. a role in sequestering iron is also possible (yoshiga et al., 1997). another group of proteins ripe for investigation belong to a group called chi-lectins. we identified two of these proteins (br1 and br2) by proteomic analysis of a. gambiae as they were very strongly induced by bacterial infection (shi and paskewitz, 2004). the two new spots represented c-terminal peptides and we found that both proteins are converted to smaller forms in hemolymph in vivo or in vitro on exposure to bacteria. we could identify this processing as early as 5 min after incubation of hemolymph with bacteria in vitro. a related protein, drosophila ds47 behaves similarly (shi and paskewitz, 2004). br2 and ds47 also can be processed on exposure to peptidoglycan alone but not lps (shi and paskewitz, 2004). other members of this group are called imaginal disc growth factor proteins (idgfs) in drosophila, because of a role in stimulating proliferation of an imaginal disc cell line in conjunction with insulin (kawamura et al., 1999). vierstraete and colleagues (2004b) reported that two spots identified as chain a of idgf2 were significantly regulated by 25 min after either yeast or bacterial challenge, while ds47 was down-regulated after bacteria only. levy et al. (2004a) found that spots corresponding to idgf2 and idgf3 were down-regulated after fungal or bacterial infections, respectively. again, differences in direction of regulation likely reflect processing, with down-regulated spots corresponding to the full-length proteins and upregulated spots representing the processed forms. this group of proteins is particularly interesting because vertebrates have members of the family that are also immune responsive but not yet well-understood (houston et al., 2003). some act as growth factors while others promote migration of immune cells (owhashi et al., 2000; chang et al., 2001; hung et al., 2002; recklies et al., 2002). a related molecule in manduca sexta, haip, inhibits aggregation of hemocytes in vitro (kanost et al., 1994). finally, the proteomics studies have identified several types of proteins that belong to categories not previously known to have immune functions. for example, an odorant binding protein (obp99c) was upregulated after fungal infection of drosophila adults (levy et al., 2004a). odorant binding proteins (obp) bind small hydrophobic molecules and function in chemoreception. obps are found in the antennae but some members of the group appear in the hemolymph (paskewitz and shi, 2005). two pherokines, proteins related to the obp family, were also induced by viral or bacterial infections (sabatier et al., 2003). another group of proteins that was found in to be immuneinduced in both larval and adult drosophila contains members with a phosphatidylethanolamine binding domain. the peb family is widespread but little is known about the function of this group. one possibility is in coordinating regulation of the various signaling pathways but see levy et al. (2004a) for other possible functions. hemocytes (blood cells) hemocytes play important roles in immunity in all insects but there is a great deal of variation in cell types and number between taxa. in general, an insect will have several different types of hemocytes, each of which has a specialized function. some hemocytes are capable of phagocytosis of foreign targets, including latex beads, bacteria, and malaria sporozoites. larger targets can elicit encapsulation responses, where layers of hemocytes adhere to the target surface and enclose it. 13 7 this process resembles some types of granuloma formation in vertebrates. hemocytes also produce some of the effector molecules for humoral immunity, including components of the po cascade and some of the amps. cellular responses were examined in many of the hemolymph studies, since neither hemocytes nor other cellular contaminants were separated from the plasma component. cellular responses were also examined by using a drosophila cell line called mbn-2 for analysis of cytoplasmic and nuclear proteins that changed following exposure to lps (loseva and engstrom, 2004). the mbn-2 line is hemocyte-like and retains its ability to phagocytose bacteria and the signaling systems necessary to activate the genes coding for amps. in this proteomic profiling study, 24 intracellular proteins were identified as regulated (up or down) or modified in response to immune challenge. at 30 min following lps administration, proteins that regulate post-translational modifications and traffic over the nuclear membrane were up-regulated including lamin dm (lam), nuclear porin p62 (nup62), calmodulin (cam), and the receptor of activated protein kinase c1 (rack1). after 6 h of lps treatment, proteins that are directly involved in effecting an immune response proteins were differentially regulated. these included actin-binding/cytoskeletal remodeling proteins and lysosomal proteases (cathepsin l, d, k), (loseva and engstrom, 2004). examination of immune-challenged hemolymph resulted in the identification of a few of the same proteins as well as others likely to be involved in the same processes (vierstraete et al., 2004a,b; guedes et al., 2005). changes in cytoskeletal proteins and their regulators are likely markers of activation of cells in preparation for phagocytosis. lysosomal cathepsins are also related to the phagocytic function since they localize to the phagolysosome in mammals and in drosophila. cathepsin d (cg1548) was found in seven isoforms and lps treatment of cells resulted in the disappearance of four of these (loseva and engstrom, 2004) while lps treatment of larvae resulted in upregulation of a cathepsin d spot (vierstraete et al., 2004b). cathepsins were further characterized in drosophila s2 cells using a functional proteomics technique whereby these enzymes are labeled covalently in an activity dependent manner. chemical tagging allowed the investigators to follow increases in cathepsin activity within the phagolysosome after phagocytosis of latex beads (kocks et al., 2003). in addition to the direct immune responses described above, proteomic studies reveal that infection causes shifts in homeostasis that alter proteins involved in metabolic and redox processes. oxidative stress results when the production of reactive oxygen species exceeds the ability of the animal to remove them. both transcript and proteome analyses indicate that oxidative stress occurs in insects during infection. members of the peroxiredoxin family (cg12405 and cg1633) were regulated in mbn-2 cells (loseva and engstrom, 2004) and in drosophila hemolymph following bacterial and fungal challenge (vierstraete et al., 2004b) or after feeding on bacterial lysates (guedes et al. 2005). glutathione s-transferase was also upregulated in all cases of infection and may have a protective role against oxidative stress (vierstraete et al., 2004b; guedes et al., 2005). two forms of gst were also identified as upregulated in a. gambiae following wounding (paskewitz and shi, 2005) and gst activity increased in hemolymph sampled following immune challenge (data not shown). a large number of cellular proteins involved in carbohydrate and amino acid metabolism were also described as immune-responsive in hemolymph and cell line studies. possible involvement of these proteins in responses to oxidative damage or in shifts in energy/biosynthetic pathways is discussed in the relevant articles (levy et al., 2004a; vierstraete et al., 2004b; guedes et al., 2005). other tissues because of their important role as developmental sites for vector-borne disease agents, mosquito midguts, salivary glands, and thoracic tissues are also potential targets for studying immune responses to parasites. work is in progress on identification of midgut proteins that are altered on malaria parasite infection. other proteomic studies of the mosquito midgut have been undertaken on strains of anopheles stephensi that exhibited different susceptibility to the human parasite, plasmodium falciparum (prevot et al., 1998). whether susceptibility is governed by immune factors is not known but 29 differences in spot patterns were identified following blood feeding in the susceptible line. additional work was done to describe spots that differed between male and female mosquitoes and following blood feeding (prevot et al., 2003). nematode worms such as brugia malayi occupy a different developmental location in mosquitoes, traveling from the midgut to the thoracic musculature. wattam and christensen (1992) compared different strains of a. aegypti and reported that thoracic muscle of refractory strains (rlvp and rkf) produced seven polypeptides in response to a blood meal, whereas a susceptible strain (lvp) did not exhibit this pattern. subsequent work identified mosquito transferrin as a protein altered in this tissue upon worm infection (yoshiga et al., 1997). the proteome of a. aegypti larvae was examined following infection by a microsporidian parasite, vavraia culicis (biron et al., 2005). samples contained the head, thorax and part of the abdomen of larvae that were sampled at either 5 or 15 days following infection. fifteen proteins that were upregulated following infection were identified by peptide mass fingerprinting. among others, these included heat shock protein co-chaperone cdc37, proteins involved in protein biosynthesis, an obp, a nitrophorin, and a gst. additional proteins that were suppressed included proteins involved in ornithine metabolism, nitric-oxide synthase, gsts, and an obp. future perspectives as an important complement to genomics, proteo 14 8 mics allows for the examination of the entire complement of proteins in an organism, tissue, or celltype. current proteomics technologies not only identify protein expression, but also post-translation modifications and interactions of immune response proteins in insect. successful protein profiling with identifications has only recently been applied to insect immunity and only the two dipterans with completed genomes have been used as models. clearly, many milestones have yet to be reached. in the future, increased use of lc-ms/ms will increase the sensitivity, resolution, dynamic range and throughput of proteomics. future studies will take advantage of the availability of new genomes and better identification technologies so that we will be able to compare proteomic results across taxonomic groups and life stages, leading to advances in our understanding of the evolutionary history of innate immunity. within model organisms, reverse genetics will allow us to inactivate key immune regulators through rna interference and to examine the effects on proteome profiles. identification of post-translational modifications suggests experiments designed to identify activators or changes in efficacy of the modified molecules. protein-protein interactions and subcellular locations will provide more precise information about the functions of the unknown proteins that are induced by infection. in short, we predict satisfying application of the methods of proteomics to questions of insect immunity on all fronts within the coming decades. abbreviations 2-de: 2 dimensional electrophoresis; 2d-dige 2: dimensional differential in gel electrophoresis; amp: antimicrobial peptide; br1: bacterially responsive protein 1; br2: bacterially responsive protein 2; est: expressed sequence tags; gnbp: gram negative binding protein; gst: glutathione s transferase; hplc: high performance liquid chromatography; hsp20: heat shock protein 20; idgf: imaginal disc growth factor; imd: immune deficiency protein; lps: lipopolysaccharide; ms: mass spectrometry; maldi-tof: matrix-assisted laser desorption/ionization time-of-flight; nec: necrotic; obp: odorant binding protein; peb: phosphatidylethanolamine binding protein; pgrp: peptidoglycan recognition protein; ppo: prophenoloxidase; po: phenoloxidase; srpn: serpin; tep: thioester protein; tsf: transferring. references alleyne t, joseph j, sampson v. cytochrome c detection. a diagnostic marker for myocardial infarction. appl. biochem. biotechnol. 90: 97-105, 2001. apweiler r, bairoch a, wu ch, barker wc, boeckmann b, ferro s, gasteiger e, huang h, lopez r, magrane m, martin mj, natale da, o'donovan c, redaschi n, yeh ls. uniprot: the universal protein knowledgebase. nucleic acids res. 32: d115-119, 2004. bartholomay lc, fuchs jf, cheng ll, beck et, vizioli j, lowenberger c, christensen bm. reassessing the role of defensin in the innate immune response of the mosquito, aedes aegypti. insect mol. biol. 13: 125-132, 2004. beerntsen bt, severson dw, christensen bm. aedes aegypti: characterization of a hemolymph polypeptide expressed during melanotic encapsulation of filarial worms. exp. parasitol. 79: 312-321, 1994. biron dg, agnew p, marche l, renault l, sidobre c, michalakis y. proteome of aedes aegypti larvae in response to infection by the intracellular parasite vavraia culicis. int. j. parasitol. 5 : 1-13, 2005. blandin s, shiao s-h, moita lf, janse cj, waters ap, kafatos fc, levashina ea. complement-like protein tep1 is a determinant of vectorial capacity in the malaria vector anopheles gambiae. cell 116: 661-670, 2004. cai z, chiu jf, he qy. application of proteomics in the study of tumor metastasis. gen. proteom. bioinf. 2: 152-166, 2004. chang nca, hung s-i, hwa k-y, kato i, chen j-e, liu c-h, chang ac. a macrophage protein, ym1, transiently expressed during inflammation is a novel mammalian lectin. j. biol. chem. 276: 17497-17506, 2001. chevallet m, santoni v, poinas a, rouquie d, fuchs a, kieffer s, rossignol m, lunardi j, garin j, rabilloud t. new zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. electrophoresis 19: 1901-1909, 1998. choudhary j, grant sg. proteomics in postgenomic neuroscience: the end of the beginning. nat. neurosci. 7: 440-445, 2004. christophides gk. transgenic mosquitoes and malaria transmission. cell. microbiol. 7: 325-333, 2005. christophides gk, zdobnov e, barillas c, blandin s, blass c, brey pt, et al. immunity related genes and gene families in anopheles gambiae: a comparative genomic analysis. science 298: 159-165, 2002. chun j, mcmaster j, han ys, schwartz a, paskewitz sm. twodimensional gel analysis of hemolymph proteins from plasmodium-melanizing and nonmelanizing strains of anopheles gambiae. insect mol. biol. 9: 39-45, 2000. de gregorio e, spellman pt, rubin gm, lemaitre b. genomewide analysis of the drosophila immune response by using oligonucleotide microarrays. proc. natl. acad. sci. usa 98: 12590-12595, 2001. dimopoulos g, christophides gk, meister s, schultz j, white kp, barillas-mury c, kafatos fc. genome expression analysis of anopheles gambiae: responses to injury, bacterial challenge, and malaria infection. proc. natl. acad. sci. usa 99: 8814-8819, 2002. engstrom y, loseva o, theopold u. proteomics of the drosophila immune response. trend. biotech. 22: 600-605, 2004. fallon p, allen r, rich t. primitive toll signaling: bugs, flies, worms, and man. trend. immunol. 22: 63-66, 2001. fenn jb, mann m, meng ck, wong sf, whitehouse cm. electrospray ionization for mass spectrometry of large biomolecules. science 246: 64-71, 1989. florens l, washburn mp, raine jd, et al. a proteomic view of the plasmodium falciparum life cycle. nature 419: 520526, 2002. giot l, bader js, brouwer c, chaudhuri a, kuang b, li y et al. a protein interaction map of drosophila melanogaster. science 302:1727-1736, 2003. graves pr, haystead ta. a functional proteomics approach to signal transduction. recent prog. horm. res. 58:1-24, 2003. green c, levashina e, mckimmie c, dafforn t, reichhart j-m, gubb d. the necrotic gene in drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43a1.2. genetics 156: 1117-1127, 2000. guedes sm, vitorino r, tomer k, domingues mrm, correia ajf, amada f, domingues p. drosophila melanogaster larval hemolymph protein mapping. biochem. biophys. res. comm. 312: 545-554, 2003. 15 9 guedes sdm, vitorino r, domingues r, tomer k, correia aj, amado f, domingues p. proteomics of immunechallenged drosophila melanogaster larvae hemolymph. biochem. biophys. res. commun. 328: 106-115, 2005. gwadz rw, kaslow k, lee jy, maloy wl, zasloff m, miller lh. effects of magainins and cecropins on the sporogonic development of malaria parasites in mosquitoes. infect. immunol. 57: 26287-2633, 1989. gygy sp, rochon y, franza br, aebersold r. correlation between protein and mrna abundance in yeast. mol. cell. biol. 19: 1720-1730, 1999. hirabayashi j, hashidate t, kasai k. glyco-catch method: a lectin affinity technique for glycoproteomics. j. biomol. techn. 13: 205-218, 2002. houston dr, recklies ad, krupa jc, aalten dmf. structure and ligand-induced conformational change of the 39-kda glycoprotein from human articular chondrocytes. j. biol. chem. 278: 30206-30212, 2003. hultmark d. drosophila immunity: paths and patterns. curr. opin. immunol. 15: 12-19, 2003. hung s-i, chang ac, kato k, chang nca. transient expression of ym1, a heparin-binding lectin, during developmental hematopoiesis and inflammation. j. leuk. biol. 72: 72-82, 2002. ideker t., thorsson v., ranish j.a., christmas r., buhler j., eng j.k., bumgarner r., goodlett d.r., aebersold r., and hood l. integrated genomic and proteomic analysis of a systematically perturbed metabolic network. science 292: 929-934. 2001. irving p, troxler l, heruer ts, belvin m, kopczynski c, reichhart j-m, hoffmann ja, hetru c. a genome-wide analysis of immune responses in drosophila. proc. natl. acad. sci. usa 98: 15119-15124, 2001. kalume de, okulate m, zhong j, reddy r, suresh s, deshpande n, kumar n, pandey a. a proteomic analysis of salivary glands of female anopheles gambiae mosquito. proteomics 5: 3765-3777, 2005. kanost mr, zepp mk, ladendorff ne, andersson la. isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of manduca sexta larvae. arch. insect biochem. physiol. 27: 123-136, 1994. karas m, hillenkamp f. laser desorption ionization of proteins with molecular masses exceeding 10,000 dalton. anal. chem. 60: 2299-2301, 1988. karlsson c, korayem am, scherfer c, loseva o, dushay ms, theopold u. proteomic analysis of the drosophila larval hemolymph clot. j. biol. chem. 279: 52033-52041, 2004. kawamura k, shibata t, saget o, peel d, bryant pj. a new family of growth factors produced by the fat body and active on drosophila imaginal disc cells. development 126: 211-219, 1999. kim w, koo h, richman am, seeley d, vizioli j, klocko ad, o’brochta da. ectopic expression of a cecropin transgene in the human malaria vector mosquito anopheles gambiae (diptera: culicidae): effects on susceptibility to plasmodium. j. med. entomol. 41:447-455. 2004. kocks c, maehr r, overkleeft hs, wang ew, iyer lk, lennondumenil am, ploegh hl, kessler bm. functional proteomics of the active cysteine protease content in drosophila s2 cells. mol. cell proteomics 2: 1188-1197, 2003. kotsyfakis m, ehret-sabatier l, siden-kiamos i, mendoza j, sinden re, louis c. plasmodium berghei ookinetes bind to anopheles gambiae and drosophila melanogaster annexins. mol. microbiol. 57: 171-179, 2005. levashina ea, moita lf, blandin s, vriend g, lagueux m, kafatos fc. conserved role of a complement-like protein in phagocytosis revealed by dsrna knockout in cultured cells of the mosquito, anopheles gambiae. cell 104: 709718, 2001. levy f, bulet p, ehret-sabatier l. proteomic analysis of the systemic immune response of drosophila. mol. cell proteomics 3: 156-166, 2004a. levy f, rabel d, charlet m, bulet p, hoffmann ja, ehretsabatier l. peptidomic and proteomic analyses of the systemic immune response of drosophila. biochimie 86: 607-616, 2004b. ligoxygakis p, pelte n, ji c, leclerc v, duvic b, belvin m, jiang h, hoffmann ja, reichhart jm. a serpin mutant links toll activation to melanization in the host defence of drosophila. embo j. 21: 6330-6337, 2002. loseva o, engstrom y. analysis of signal-dependent changes in the proteome of drosophila blood cells during an immune response. mol. cell. proteomics 3: 796-808, 2004. lowenberger c. innate immune response of aedes aegypti. insect biochem. mol. biol. 31: 219-229, 2001. o'farrell ph high resolution two-dimensional electrophoresis of proteins. j. biol. chem. 250: 4007-4021, 1975. owhashi m, arita h, hayai n. identification of a novel eosinophil chemotactic cytokine (ecf-l) as a chitinase family protein. j. biol. chem. 275: 1279-1286, 2000. paskewitz sm, shi l. the hemolymph proteome of anopheles gambiae. insect biochem. mol. biol. 35: 815-824, 2005. prevot gi, laurent-winter c, feldmann am, rodhain f, bourgouin c. two-dimensional gel analysis of midgut proteins of anopheles stephensi lines with different susceptibility to plasmodium falciparum infection. insect mol. biol. 7: 375-383, 1998. prevot gi, laurent-winter c, rodhain f, bourgouin c. sexspecific and blood meal-induced proteins of anopheles gambiae midguts: analysis by two-dimensional gel electrophoresis. malaria j. 2: 1-7, 2003. recklies ad, white c, ling h. the chitinase 3-like protein human cartilage glycoprotein 39 (hc-gp39) stimulates proliferation of human connective-tissue cells and activates both extracellular signal-regulated kinaseand protein kinase b-mediated signaling pathways. biochem. j. 365: 119-126, 2002. renz a, berdel we, kreuter m, belka c, schulze-osthoff k, los m. rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo. blood 98: 1542-1548, 2001. ribeiro jm, topalis p, louis c. anoxcel: an anopheles gambiae protein database. insect mol. biol. 13: 449-457, 2004. roxstrom-lindquist k, terenius o, faye i. parasite-specific immune response in adult drosophila melanogaster: a genomic study. embo rep. 5: 201-212, 2004. sabatier l, jouanguy e, dostert c, zachary d, dimarcq j-l, bulet p, imler j-l. pherokine-2 and -3. two drosophila molecules related to pheromone/odor-binding proteins induced by viral and bacterial infections. eur. j. biochem. 270: 3398-3407, 2003. salih e. phosphoproteomics by mass spectrometry and classical protein chemistry approaches. mass spectrom. rev. 24: 828-46, 2005 salzet m. vertebrate innate immunity resembles a mosaic of invertebrate immune responses. trend. immunol. 22: 285288, 2001. shi l, paskewitz sm. identification and molecular characterization of two immune-responsive chitinase-like proteins from anopheles gambiae. insect mol. biol. 13: 387-398, 2004. shih km, fallon am. two-dimensional electrophoretic analysis of aedes aegypti mosquito fat body proteins during a gonotropic cycle. am. j. trop. med. hyg. 65: 42-46, 2001. stadler f, hales d. highly-resolving two-dimensional electrophoresis for the study of insect proteins. proteomics 2: 1347-1353, 2002. 16 10 tonge r, shaw j, middleton b, et al. validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. proteomics 1: 377-396, 2001. tsuji y, nakajima y, homma k, natori s. antibacterial activity of a novel 26-kda serine protease in the yellow body of sarcophaga peregrina (flesh fly) pupae. febs lett. 425: 131-3, 1998. tzou p, ohresser s, ferrandon d, capovilla m, reichhart j-m, lemaitre b, hoffmann ja, imler j-l. tissue-specific inducible expression of antimicrobial peptide genes in drosophila surface epithelia. immunity 13: 737-748, 2000. uttenweiler-joseph s, moniatte m, lagueux m, van dorsselaer a, hoffmann ja, bulet p. differential display of peptides induced during the immune response of drosophila: a matrix-assisted laser desorption ionization time-of-flight mass spectrometry study. proc. natl. acad. sci. usa 95: 11342-11347, 1998. vierstraete e, cerstiaens a, baggerman g, van den bergh g, de loof a, schoofs l. proteomics in drosophila melanogaster: first 2d database of larval hemolymph proteins. biochem. biophys. res. commun. 304: 831-838, 2003. vierstraete e, verleyen p, baggerman g, d'hertog w, van den bergh g, arckens l, de loof a, schoofs l. a proteomic approach for the analysis of instantly released wound and immune proteins in drosophila melanogaster hemolymph. proc. natl. acad. sci. usa 101: 470-475, 2004a. vierstraete e, verleyen p, sas f, van den bergh g, de loof a, arckens l, schoofs l. the instantly released drosophila immune proteome is infection-specific. biochem. biophys. res. commun. 317: 1052-1060, 2004b. vierstraete e, verleyen p, de loof a, schoofs l. differential proteomics for studying drosophila immunity. ann. n.y. acad. sci. 1040: 504-507, 2005. wang y, zhang p, fujii h, banno y, yamamoto k, aso y. proteomic studies of lipopolysaccharide-induced polypeptides in the silkworm, bombyx mori. biosci. biotechnol. biochem. 68: 1821-1823, 2004. wattam ar, christensen bm. induced polypeptides associated with filarial worm refractoriness in aedes aegypti. proc. natl. acad. sci. usa 89: 6502-6505, 1992. yoshiga t, hernandez vp, fallon am, law jh. mosquito transferrin, an acute-phase protein that is up-regulated upon infection. proc. natl. acad. sci. usa 94: 1233712342, 1997. 17 56 isj 14: 56-62, 2017 issn 1824-307x research report gene expression of hsp90 and hsp70 in four silkworm hybrids (bombyx mori l.) in response to severe thermal shock sf mousavi1, sh hosseini moghaddam2, n ghavi hossein-zadeh1, sz mirhosseini2 1department of animal science, faculty of agricultural sciences, university of guilan, rasht, iran 2department of animal science and department of sericulture, faculty of agricultural sciences, university of guilan, rasht, iran accepted march 6, 2017 abstract usually silkworm egg producers provide several silkworm hybrids with different qualities of productivity and viability traits. this study was conducted to compare the expression of hsp70 and hsp90 genes among different silkworm hybrids. in the fourth day of fifth larval instar of two japanese maternal parents (103×104 and 107×110) and two chinese maternal parents (110×107 and 104×103), silkworm larval fat body was sampled from heat shock (45 °c for 35 min) and non-heat shock groups. sampling was done at 0, 2, 4 and 24 h after heat shock. gene expression of hsp70 and hsp90 (target genes) were measured by qrt-pcr using rpl27a as reference gene. the prolonged heat shock (8 h at 39 °c) was utilized to examine the thermal tolerance of larvae in comparison with control group (24°c). the results showed both hsp70 and hsp90 have been up-regulated in the treated larvae. the hybrids with japanese female parents (107×110 and 103×104) were more sensitive than two others; moreover in these hybrids, hsp90 and hsp70 were expressed significantly more than others after heat exposure. the maximum expression was occurred in the time zero, then a decreasing trend was observed over time. comparison of larval mortality among four hybrids revealed that (103×104) and (110×107) hybrids had the highest and the lowest mortality rate. key words: gene expression; heat shock proteins; real-time pcr; silkworm hybrids; thermotolerance introduction sericulture has an important role in village economy in countries like iran where sustainable rural development is very important. high sensitivity of commercial hybrids to high temperature is the main barrier in its expansion in hot and dry climates. therefore, factors affecting silkworm tolerance should be considered in the silkworm breeding and introduction of new hybrids (hosseini moghaddam, 2005). moreover, substantial studies have been carried out by researcher to evaluate the effect of heat stress on various organisms due to probability of sudden and rapid climate change when the earth is getting warmer. when insects expose to extreme temperature, they respond to this temperature change by arising heat shock proteins (hsps) as molecular chaperones to protect of protein folding process. hsps family is divided according to their molecular __________________________________________ corresponding author: seyed hossein hosseini moghaddam department of sericulture, faculty of agriculture sciences university of guilan, rasht, iran e-mail: hosseini@guilan.ac.ir weight, function and sequence homology into several groups including hsp100, hsp90, hsp70, hsp60, hsp40, small hsp (shsps) and hsp10(zhao and jones, 2012). among these hsps, high molecular weight hsp70s as a housekeeping molecule has different responsibilities in insects such as developmental processes and fecundity (jensen et al., 2014), development (huang et al., 2009), diapause (rinehart et al., 2007), longevity (choi et al., 2014), and metamorphosis (zheng et al., 2010). hsp70s genes were up-regulated in response to cold or heat stress in a variety of insects, including coleoptera (mahroof et al., 2005), diptera (gray, 2013) and lepidoptera (jiang et al., 2012; choi et al., 2014; shen et al., 2014). in a study on flash fly (sarcophaga crassipalpis) in response to hypoxia, hsp90 showed little response, however, hsp70 was the most responsive and increased several hundred fold in the cells (michaud et al., 2011). results of previous studies on the chilo suppressalis, liriomyza trifolii, and pteromalus puparum showed that hsp70 can be related to thermotolerance and survivability (cui et al., 2010; zheng et al., 2010). in drosophila melanogaster, mailto:hosseini@guilan.ac.ir 57 family of hsp70’s could not make thermotolerance against severe thermal shock (bettencourt et al., 2008). the well-defined role of hsps in acquired thermotolerance in the silkworm and other insects is not clearly known yet (manjunatha et al., 2010). hsp90 that forms about 1 2 % of cellular proteins is one of the most abundant proteins in the living cells. when insects are under normal conditions, the hsp90 expressed at low levels (wegele et al., 2004). hsp90 acted as a protective against thermal stress in some insects including plutella xylostella and liriomyza huidobrensis, (sonoda et al., 2006; huang and kang, 2007). zhang et al. (2009) reported that among different tissues of silkworm, sometimes the hsp90 gene expression was up-regulated and sometimes was down-regulated. the commercial silkworms are usually twoway cross hybrids (direct and reciprocal crosses) which are synthetized by two parents including japanese (e.g. 103, 107) and chinese (e.g. 104, 110) strains. according to previous report (hosseini moghaddam, 2005) the productivity traits (cocoon weight and cocoon shell weight) of japanese maternal parents (103×104 and 107×110) were higher than chinese maternal parents (110×107 and 104×103) and conversely for viability traits. the purpose of this research is the comparison of gene expression of two high molecular weight hsps in four commercial silkworm hybrids (including two maternal chinese and two maternal japanese parents), besides the relationship of gene expression with thermal tolerance of silkworm larvae. materials and methods silkworm rearing, thermal treatment and sampling two iranian silkworm hybrids: 110×107 and 104×103 (first parent is female and a chinese cocoon shape strain) and their reciprocal 103×104 and 107×110 (first parent is female and a japanese cocoon shape strain) were reared in the iran silk research center. the 103 and 104 silkworm strains are originally from japan and the strains 107 and 110 were isolated from a korean hybrid under fao/undp tcp project (1992-1997). fifth instar larvae were transferred to the silkworm laboratory, faculty of agricultural sciences, university of guilan to continue rearing and heat shock treatments. sampling was done from fat body. this tissue is under larval skin that can easily affected by heat exposure. fat body as a site for energy storage tissue in the silkworm, synthetize many biological proteins like heat shock proteins that these substances are expected to be active in the fat body (kajiwara et al., 2006). while all process was performed on ice, the fat body free of muscle was collected in the fourth day of fifth larval instar from both heat shock (45 °c for 35 min) and non-heat shock larvae at 0, 2, 4 and 24 h after heat treatment. before collection of fat body, the surface of dissected larva was washed by icecold insect physiological salt solution (0.7 % nacl) (chavadi et al., 2006). three independent fat body samples were mixed together to minimize variation and to get enough amount of samples. they were store immediately at -80 °c for further use. rna extraction, cdna synthesis and real time-pcr rna extraction procedure was carried out by trizol reagent (invitrogen, usa) based on the supplier’s instructions. the quality and quantity of extracted rna was evaluted using nanodrop-2000 spectrophotometer (thermo scientific) and the quality of agarose gel electrophoresis bands. dnase i treatment (takara, japan) was applied to avoid possible contamination of genomic dna. the cdna of all samples were made using cdna synthesis kit (thermo scientific) based on manufacturer protocol. specific primers of hsp70 and hsp90 were designed using online software primer 3 (table 1). real time-pcr was carried out using maxima sybr green/rox qpcr master mix (thermo scientific). data was normalized using livak (2001) formula (2-δδct). a factorial experiment (4×4) with completely randomized design was utilized to evaluate the effects of different hybrids (110×107, 104×103, 103×104, 107×110) and the times after heat shock (0, 2, 4, 24 h), as fixed effects, and their interactions on gene expression data. statistical analysis of data was performed using the glm procedure of sas 9.0. least squares means were used to identify the main effects. table 1 specific primer sequences for real-time pcr gene ncbi reference primer sequence pcr product length annealing temperature rpl27a nm_001044057 right : tgacaggttgtttggggag left: cagacgaggctgaagtatgc 144 60 hsp70 nm_001043931.1 right: gtgcttcatgtcctgctgaa left: tcgccttgaaccctaacaac 100 60 hsp90 nm_001043411.1 right: aggccttcgaacttcacctt left: atggcaagacccttgtatcg 103 54.7 58 table 2 least-squares means of hsp70 and hsp90 gene expression in the studied silkworm hybrids at four times after thermal exposure expression of hsp90 expression of hsp70 times hybrids 2183.49a 1549.54a 0 107×110 595.96b 646.83b 2 107×110 83.54c 1.002e 4 107×110 1.85c 0.01e 24 107×110 32.78c 277.47c 0 110×107 7.26c 7.51e 2 110×107 0.13c 0.06e 4 110×107 0.04c 0.007e 24 110×107 278.11a 1598.42a 0 103×104 49.90c 26.07 d 2 103×104 2.15c 21.11 d 4 103×104 0.43c 0.09 e 24 103×104 26.99c 97.57 d 0 104×103 20.87c 85.62 d 2 104×103 10.06c 2.28 e 4 104×103 0.01c 80.86* 0.07 e 27.78* 24 104×103 se a-e different superscripts within a column indicate significant differences (p < 0.0001) * standard error for all least squares means of gene expression evaluation of silkworm viability and thermal tolerance in order to measure thermotolerance of different silkworm hybrids, larvae were exposed to 39 °c for 8 h (9 am to 5 pm) from second to seventh day of fifth instar as long-term heat stress; in other word six days from one day after forth molting to one day before larval cocooning. while larval mortality was recorded daily, mortality rate was measured after two days (lo1) and also at the end of heat shock period (lo2). because, it is expected that sensitive genotypes will respond more quickly to the initial thermal shock and die; therefore, the first time measurement of mortality rate (lo1) was considered as a thermotolerance criterion. in the same time, larval mortality was recorded for nonheat stress groups. lo2 is not a good criterion to rank the silkworm hybrids; since long-term heat exposure may cause to be killed most of larvae in each tray. average larval mortality in response to heat shock was reported as log-transformed values to assure normality. a factorial experiment (4×2) with completely randomized design was utilized to evaluate the effects of different silkworm hybrids (110×107, 104×103, 103×104, 107×110) and heat exposure (with or without thermal shock), as fixed effects, and their interactions on larval mortality (lo1 and lo2). statistical analysis of data was carried out using the glm procedure of sas 9.0. least squares means were used for identifying the main effects. results the results showed that gene expression of hsp90 and hsp70 in hybrids with japanese maternal parents) (103×104 and 107×110) were significantly (p < 0.0001) higher than hybrids with chinese maternal parents (104×103 and 110×107). table 2 shows that the fluctuation of hsp expression for both genes is almost the same. in other word, the expression of hsp90 was in good accordance with hsp70. least-squares means of hsp70 and hsp90 gene expression (table 2) indicated that there was a high significant difference(p < 0.0001) in the hsp70 and hsp90 expression between two reciprocal hybrids (110×107 vs. 107×110 and 103×104 vs. 104×103) immediately after heat exposure (fig. 1). expression of both genes immediately after heat shock (time zero) was significantly higher than other times (p < 0.0001) in all genotypes, afterwards, gradually decreased to the lowest level after 24 h (table 2). in this time down-regulation was observed in almost all genotypes, in fact, hsp70 and hsp90 expression reduced over time. four h after thermal exposure, while hsp70 expression in the three hybrids declined but still 103×104 had higher expression. the hybrid 104×103 had significantly lower expression among hybrids (p < 0.0001) which proposed that this hybrid need less hsp70 and hsp90 in thermal shock conditions. in another experiment a prolonged heat shock (8 h at 39 ◦c for six days) was implemented to examine the thermotolerance of larvae. the mortality rate (in %) was considered as a measure of thermotolerance. mortality of all treatment groups was higher than control groups. the hybrid 110×107 and 103×104 had the lowest and the highest mortality rates, respectively. in fact, 103×104 as a sensitive hybrid had the highest losses and 110×107 hybrid as a resistant hybrid had the lowest 59 fig. 1 hsp70 and hsp90 gene expression immediately after heat exposure (time zero) in the studied silkworm hybrids losses (fig. 2). the same result was obtained control group; however differences was not significant (table 3). 110×107 and 107×110, two silkworm hybrids that their parents were originally from korea, were more resistant than hybrids 103×104 and 104×103 with japanese parents. among four genotypes, the sensitive hybrids (103×104 and 107×110) had the highest gene expression immediately after heat exposure (zero time) implying that these genotypes need more hsp70 and hsp90 under severe thermal conditions. in fact, hybrids with japanese maternal parents (107×110 and 103×104) were sensitive and hybrids with chinese maternal parents (110×107 and 104×103) were relatively resistant. therefore, sensitive hybrids immediately after heat exposure need both hsp70 and hsp90 proteins to reduce thermal effects. discussion a large difference was observed between the genotypes in terms of hsp expression. generally, the results showed that the hybrids with japanese maternal parents had more hsps expression than chinese maternal parents. there are some reports on genetic differences of hsp70 or hsp90 among silkworm genotypes such as comparison of nistari and jingsong (hosseini moghaddam et al., 2008), nistari and nb4d2 (velu et al., 2008), c. nichi, pure mysore and nb4d2 strains (joy and gopinathan, 1995) and pure mysore and nb4d2 (sosalegowda et al., 2010). velu et al. (2008) reported little differences in hsp70 expression between two resistant and sensitive strains on agarose gel (semiquantitative) after a mild heat shock (41 °c for one hour) but li et al.)2011(using qrt-pcr method showed that expression of hsp70 in jingsong (a thermosensitive commercial strains) was significantly more than nistari breed )a thermotolerant multivoltine breed in tropical region). it means that thermo-sensitive breed induced strongly hsp70 mrna after heat shock treatments. this result was consistent to our study which among four genotypes, the sensitive hybrids (103×104 and 107×110), as japanese maternal parents, had the highest gene expression. garbuz et al. (2002) reported that among drosophila species and strains originating from different climatic zones, some of species and strains exhibited positive correlation between hsp70 expression and thermotolerance and for others negative correlation. table 3 average larval mortality in the four silkworm hybrids for both thermal treatment and control groups hybrids lo 1* lo 2 107 ×110 (treatment) 0.70 ± 0.07c 1.84 ± 0.02a 107×110 (control) 0.13 ± 0.02d 0.93 ± 0.01d 110 ×107 (treatment) 0.50± 0.08cd 1.66 ± 0.01b 110×107 (control) 0.09d 0.57 ± 0.005e 103×104 (treatment) 1.66± 0.07a 2.02± 0.003a 103×104 (control) 0.34 ± 0.02d 1.23 ± 0.07c 104 ×103 (treatment) 1.19± 0.03b 1.91± 0.005a 104 ×103 (control) 0.09d 0.7 ± 0.04d a-e different superscripts within a column indicate significant differences (p < 0.0001) * average larval mortality is log-transformed values (lo1: first period of measuring mortality; lo2: second period of measuring mortality) 60 fig. 2 larval mortality percentage by treatment and control groups in the studied silkworm hybrids (lo1: first period of measuring mortality; lo2: second period of measuring mortality; average larval mortality is log-transformed values) in the current study hsp70 and hsp90 expression was up-regulated in the fat body of silkworm larvae after severe (45 °c) but non-lethal heat shock. . li et al. (2011) observed higher expression of hsp70 in fat body rather than testis and ovary in a thermosensitive strain of silkworm. velu et al. (2008) reported mid gut and fat body tissues had higher hsp70 expression than the cuticle and silk gland tissues. keshan et al. (2014) studied hsp90 expression in various tissues of silkworm and indicated that a higher hsp90 expression in all examined larval tissues after mild (39 °c) and mild to severe (42 °c) heat treatments. the highest expression was observed just immediately after heat exposure (time zero) in all silkworm hybrids. to our knowledge, this is the first study where the differences of hsp gene expression were evaluated for reciprocal crosses in the silkworm. in the silkworm, high thermotolerance in fifth instar larvae reflects its adaptation to high temperature (manjunatha et al., 2010). a large difference was also observed between the genotypes in terms of heat tolerance. the hybrids with japanese maternal parents were more sensitive than the chinese ones, which was in line with other researches (vasudha et al., 2006; firdose and reddy, 2009). hsieh et al. (1995) by comparing chinese and japanese maternal parents demonstrated that feng, a chinese strain, was the most tolerant strain followed by japanese races, kuo and j-09, while another chinese strain, c-54 was most susceptible. in different studies, different genotypes had different reactions to the heat stress. this diversity among silkworm varieties is related to their genetic background and related silkworm breeding plan. our results showed that this kind of differences could be detected in molecular levels. further investigations of these differences can help us to understand the mechanisms of protecting cells against environmental stresses and also identify variation of hsps genes expression among susceptible/ tolerant strains and hybrids of silkworm. conclusion the results of this study showed that both hsp70 and hsp90 have been up-regulated in the treated larvae. the hybrids with japanese female parents (107×110 and 103×104) were more thermosensitive than two others, moreover hsp90 and hsp70 were also significantly expressed in these two hybrids after heat shock. comparison of larval mortality among four hybrids revealed that the hybrid 103×104 had the highest mortality rate and 110×107 had the lowest mortality rate. high expression of hsps in thermo-sensitive hybrids showed importance of hsps in survivability. generally, it can be concluded that the expression patterns of high molecular weight hsp can 61 determine the various levels of thermotolerance in the different silkworm genotypes. acknowledgement we are grateful to the iran silk research center (guilan province) for cooperation in silkworm rearing. this work was supported by biotechnology committee of university of guilan under their block grant. references bettencourt br, hogan cc, nimali m, drohan bw. inducible and constitutive heat shock gene expression responds to modification of hsp70 copy number in drosophila melanogaster but does not compensate for loss of thermotolerance in hsp70 null flies. bmc biol. 6: 5-15, 2008. chavadi vb, sosalegowda ah, boregowda mh. impact of heat shock on heat shock proteins expression, biological and commercial traits of bombyx mori. insect sci. 13: 243-250, 2006. choi bg, hepat r, kim y. rna interference of a heat shock protein, hsp70, loses its protection role in indirect chilling injury to the beet armyworm, spodoptera exigua. comp. biochem. physiol: 168a: 90-95, 2014. cui yd, lu mx, du yz. cloning of the heat shock protein 70 gene from chilo suppressalis and the analysis of its expression characteristics under heat stress. acta. entamologica sin. 53: 841848, 2010. firdose am, reddy ys. racial features in the heat tolerance limits in the silkworm, bombyx mori. acta entom. sinica 52: 478-486, 2009. garbuz dg, molodtsov vb, velikodvorskaia vv, evgenev mb, zatsepina og. evolution of the response to heat shock in genus drosophila. 2002. russian genet. 8: 925-936, 2002. gray em. thermal acclimation in a complex life cycle: the effects of larval and adult thermal conditions on metabolic rate and heat resistance in culex pipiens (diptera: culicidae). j. insect physiol. 59: 1001-1007, 2013. hosseini moghaddam sh. principles of sericulture, university of guilan, iran, 2005. hosseini moghaddam sh, du x, li j, cao j, zhong b, chen yy. proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, bombyx mori, exposed to heat shock exposure. biotechnol. bioproc. eng. 13: 624-631, 2008. hsieh fk, yu sj, su sy, peng sj. studies on the thermotolerance of the silkworm, bombyx mori. chinese j. entom. 15: 91-101, 1995. huang lh, kang l. cloning and interspecific altered expression of heat shock protein genes in two leaf miner species in response to thermal stress. j. insect physiol. 16: 491-500, 2007. huang lh, wang cz, kang l. cloning and expression of five heat shock protein genes in relation to cold hardening and development in the leafminer, liriomyza satia. j. insect physiol. 55: 279-285, 2009. jensen p, overgaard j, loeschcke v, schou mf, malte h, kristensen tn. inbreeding effects on standard metabolic rate investigated at cold, benign and hot temperatures in drosophila melanogaster. j. insect physiol. 62: 11-20, 2014. jiang xf, zhai hf, wang l, luo lz, thomas ws, zhang l. cloning of the heat shock protein 90 and 70 genes from the beet armyworm, spodoptera exigua, and expression characteristis in relation to thermal stress and development. cell stress chaperone 17: 67-80, 2012. joy o, gopinathan ktp. heat shock response in mulberry silkworm races with different thermotolerances. j. biosci. 20: 499-513, 1995. kajiwara h, itou y, imamaki a, nakamura m, mita k, ishizaka m. proteomic analysis of silkworm fat body. j. insect biotechnol. sericol. 75: 4756, 2006. keshan b, paul s, bembem th, sanathoibi devi k. tissue-specific expression patterns of heat shock protein 90 transcripts in silkworm, bombyx mori. j. entomol. zool. stud. 2: 53-59, 2014. li j, hosseini moghaddam sh, du x, zhong b, chen yy. comparative analysis on the expression of inducible hsps in the silkworm, bombyx mori. mol. biol. rep. 34: 3915-3923, 2011. livak kj, schmittgen td. analysis of relative gene expression data using real-time quantitative pcr and the 2−δδct method. methods 25: 402408, 2001. mahroof r, zhu ky, subramanyam b. changes in expression of heat shock proteins in tribolium castaneum (coleoptera: tenebrionidae) in relation to developmental stage, exposure time, and temperature. ann. entomol. soc. am. 98: 100-107, 2005. manjunatha hb, rajesh rk, aparna hs. silkworm thermal biology: a review of heat shock response, heat shock proteins and heat acclimation in the domesticated silkworm, bombyx mori. j. insect sci. 10: 16, 2010. michaud mr, teets nm, peyton jt, blobner bm, denlinger dl. heat shock response to hypoxia and its attenuation during recovery in the flesh fly, sarcophaga crassipalpis. j. insect physiol. 57: 203-213, 2011. roberts rj, agius c, saliba c, bossier p, sung yy. heat shock proteins (chaperones) in fish and shellfish and their potential role in relation to fish health: a review. j. fish dis. 33: 789-801, 2010. rinehart jp, delinger dl. heat-shock protein 90 is down-regulated during pupal diapause in the flesh fly, sarcophaga crassipalpis, but remains responsive to thermal stress. insect mol. biol. 9: 641-645, 2000. rinehart jp, li a, yocum gd, robich rm, hayward sal, denlinger dl. up-regulation of heat shock proteins is essential for cold survival during insect diapause. proc. natl. acad. sci. usa 104: 11130-11137, 2007. shen y, gong yj, gu j, huang lh, feng ql. physiological effect of mild thermal stress and its induction of gene expression in the common 62 cutworm, spodoptera litura. j. insect physiol. 61: 34-41, 2014. sonoda s, ashfaq m, tsumuki h. cloning and nucleotide sequencing of three heat protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, plutella xylostella (l.) and their expression in relation to developmental stage and temperature. arch. insect biochem. physiol. 62: 80-90, 2006. sosalegowda ah, kundapur rr, boregowda mh. molecular characterization of heat shock proteins 90 (hsp83) and 70 in tropical strains of bombyx mori. proteom. 10: 2734-2745, 2010. vasudha bc, aparna hs, manjunatha hb. impact of heat shock on heat shock proteins expression, biological and commercial traits of bombyx mori. insect sci. 13: 243-250, 2006. velu d, ponnuvel km, qadri smh. expression of the heat shock protein genes in response to thermal stress in the silkworm bombyx mori. int. j. industrial entomol. 16: 21-27, 2008. wegele h, muller l, buchner j. hsp70 and hsp90 a relay team for protein folding. rev. physiol. biochem. pharmacol. 151: 1-44, 2004. yang lh, jiang ho, liu yh, dou w, wang jj. molecular characterization of three heat shock protein70 genes and their expression profiles under thermal stress in the citrus red mite. j. mol. biol. rep. 39: 3585-3596, 2012. zhang xy, zhang mz, zheng cj, liu j, hu hj. identification of two hsp90 genes from the marine crab, portunus trituberculatus and their specific expression profiles under different environmental conditions. comp. biochem. physiol. 150c: 465-473, 2009. zhao l, jones wa. expression of heat shock protein genes in insect stress responses. inv. surv. j. 9: 93-101, 2012. zheng ww, yang dt, wang jx, song qs, gilbert li, zhao xf. hsc70 binds to ultra-spiracle resulting in the up-regulation of 20hydroxyecdsone-responsive genes in helicoverpa armigera. mol. cell. endocrinol. 315: 282-291, 2010. isj 7: 211-220, 2010     211 isj 7: 211-220, 2010 issn 1824-307x review echinoderm immunity f ramírez-gómez1, je garcía-arrarás2 1department of biology, university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747, usa 2department of biology, university of puerto rico, p.o. box 23360, upr station, río piedras, san juan, pr 00931-3360, usa accepted september 27, 2010 abstract echinoderms are exclusively marine animals that, after the chordates, represent the second largest group of deuterostomes. their diverse species composition and singular ecological niches provide at the same time challenges and rewards when studying the broad range of responses that make up their immune mechanisms. two types of responses comprise the immune system of echinoderms: a cellular response and a humoral one. cell-based immunity is carried by the celomocytes, a morphologically heterogeneous population of free roaming cells that are capable of recognizing and neutralizing pathogens. celomocytes present diverse morphologies and functions, which include phagocytosis, encapsulation, clotting, cytotoxicity, wound healing among others. humoral immunity is mediated by a wide variety of secreted compounds that can be found in the celomic fluid and play important roles in defense against infection. compounds such as lectins, agglutinins, perforins, complement and some cytokines make up some of the humoral responses of echinoderms. recent advances in the field of molecular biology, genomics and transcriptomics have allowed for the discovery of new immune genes and their products. these discoveries have expanded our knowledge of echinoderm immunity and are setting up the stage for future experiments to better understand the evolution of the immune mechanisms of deuterostomes. key words: comparative immunology; echinoderm; immunity; celomocytes; genes introduction the phylum echinodermata is a very diverse group of marine animals that have sparked the interests of scientists for over a century. significant discoveries have been made using echinoderms in the areas of cell biology, developmental biology and immunology. five classes comprise the phylum: asteroidea (sea stars or starfish), crinoidea (crinoids or feather stars), ophiuroidea (brittle stars), echinoidea (sea urchins and sand dollars) and holothuroidea (sea cucumbers or holothurians). even though research has been done on all echinoderm classes, one group excels as the favorite of scientists: the echinoids. thus, sea urchins have become one of the classical animal models and have been particularly exploited in studies of fertilization and developmental biology. similarly, in the field of echinoderm immunology, sea urchins comprise the group that has been most ___________________________________________________________________________ corresponding author: francisco j ramirez-gomez university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747-2300, usa e-mail: framirez@umassd.edu extensively studied (smith et al., 2006). furthermore, the availability of the genome sequence for the purple sea urchin (strongylocentrotus purpuratus) has allowed for in depth studies into the genetic aspects of its immune response (hibino et al., 2006; rast et al., 2006). this trend has helped advance the field and at present, echinoderms are catching the attention of comparative immunologists. however, due to the inherent diversity of the echinoderm phylum, general assumptions cannot be easily established and what is true for one specific class may not apply to others. interest in echinoderm immunobiology also originates form the aquaculture field. although little known in the western hemisphere, holothurian and echinoid cultures are an important economic activity in asia. with an increasing demand for sea urchin roe and trepang (a generic name for sea cucumbers), commercial culture venues have increased in order to maintain the demands for these organisms. with increase in aquacultures one observes an increase in diseases, mainly infections, and therefore an increase interest in understanding how the organisms protect themselves from mailto:framirez@umassd.edu     212 pathogenic threat. the present review will attempt to summarize the latest published research work on the echinoderm immune system with a special emphasis on non-echinoid groups. the review focuses on the different immune components and mechanisms present in the phyla and highlights how rich, diverse and complex this group of animals can be. general aspects of echinoderm immunity in terms of their immune systems, echinoderms display the same basic types of responses that most multicellular (including vertebrates) animals do. they can recognize self from non-self and, if a foreign material (e.g., microorganism/pathogen) enters the body cavity, they can readily neutralize it and dispose of it (yui and bayne, 1983; dybas and fankboner, 1986; jans et al., 1996; glinski and jarosz, 2000). additionally, echinoderms possess very good wound-healing capabilities, a key feature that also plays an important role in one of the best known characteristics of the group: regeneration of lost body parts. these defense mechanisms are mediated by cellular and humoral responses, with several homologous and analogous components found in other invertebrates and vertebrates alike. in fact, it is their key position in the evolutionary tree, being invertebrate deuterostomes (thus sharing a common evolutionary branch with vertebrates) that makes the study of their immune system a very interesting and exciting field. therefore, this advantageous phylogenetic position allows for comparisons between immune mechanisms that have been well studied in vertebrates with those of their echinoderm counterparts. thus, echinoderms can provide important information on the evolution of the immune response. as in many other systems, echinoderm immune responses can be divided between cellular and humoral responses. cellular responses are mediated by the celomocytes, which are free roaming cells that occupy the celomic cavity but can also infiltrate tissues and organs. these cells circulate in the celomic fluid and exert the vast majority of immune functions. on the other hand, humoral responses are defined by the broad variety of molecules present in the celomic fluid. these molecules are capable of recognizing and neutralizing foreign material, promoting cell migration and agglutination and also playing roles in wound healing ( ryoyama, 1973; kanungo, 1982; canicatti et al., 1992; smith and davidson, 1992). cellular components celomocytes are a very abundant and diverse cell types that are present in all echinoderms. these cells are heterogeneous in morphology, size, relative abundance and function, which make a single standard classification for all echinoderms a difficult task. extensive research has been done during the past century on the morphological aspects of celomocytes. comprehensive reports on the celomocyte types of different echinoderms classes have also been published (kindred, 1924; boolootian and giese, 1958, 1959; boolootian, 1962; endean, 1966). these studies clearly show the wide variety of cell morphologies present in the echinoderm celomic fluid. however, the absence of a standard reference among groups and particularly, differences in terminology and even specimen preparation, contribute to the existing heterogeneity. nonetheless, some types of celomocytes can be found in all classes, while others have been considered to be specific to certain classes. these cell types are summarized in table 1 along with the particular functions that have been ascribed to certain cells. the distribution of these cell types is highly variable among species and also even at the individual level. for example, in some sea star species the vast majority (> 90 %) of celomocyte types are amebocytes, while other cell types seem to be exclusive of certain groups (e.g., holothurian crystal cells). table 2 summarizes the general distribution of celomocytes in three echinoderm classes (echinoids, holothuroids and asteroids) and how they differ depending on the group and the species. this cell distribution is also very dynamic, changing in accordance to the physiological or immune state of the animal. for example, in the sea star asterias rubens specific sub-populations of amoebocytes increase in number after injection of gram-positive bacteria while other sub-groups remain unchanged (coteur et al., 2002). our studies with the sea cucumber holothuria glaberrima have shown that the total number of celomocytes remains unchanged after challenges with diverse pathogen associated molecular patterns (pamps). however, the distribution of particular sub-types changes after immuno-stimulation, e.g., lymphocytes numbers diminish, while phagocytes increase (ramirezgomez et al., 2010). from all the celomocyte types, probably the one that is present in all the echinoderm classes is the phagocyte/amebocyte type. this cell ranges in size from 3 to 20 μm and its main characteristic is its ability to phagocytize other cells or foreign particles (endean, 1966). other roles have been attributed to phagocytes, most of them immune related, demonstrating that this cell type is the main effector of the echinoderm immune system. in fact, the discovery of these cells in the sea star, back in the late 1800’s by russian zoologist ilya metchnikoff gave rise to the field of cellular immunity, for which he was awarded the nobel prize in 1908 (metchnikoff, 1891). amebocyte roles include: graft rejection, chemotaxis, reactive oxygen species production, encapsulation, cytotoxicity, immune gene expression, agglutination and clotting reactions (gross et al., 1999, 2000; beck et al., 2001; coteur et al., 2001; lin et al., 2001; hillier and vacquier, 2003; clow et al., 2004; matranga et al., 2005; sun et al., 2008). several authors subcategorize phagocytes according to their size and morphology, but since these classifications are not the same for all echinoderms, some sub-types may overlap or on the other hand can be rendered as a different cell type altogether. lymphocytes are another cell type that might be present in all echinoderms (endean, 1966), but it is most frequently found in holothurians and some sea stars     213 table 1 summary of celomocyte types reported for echinoderm classes. e: echinoidea, h: holothuroidea, a: asteroidea, c: crinoidea, o: ophiuroidea. cell type present in class role reference discoidal cell e, h polygonal cell e small phagocyte e, h amebocytes /phagocytes e, h, a, c, o phagocytosis, clotting, encapsulation, chemotaxis, opsonisation, graft rejection (coteur et al., 2002; de faria and da silva, 2008; eliseikina and magarlamov, 2002; endean, 1966; matranga et al. 2005; ramirez-gomez et al., 2010; smith et al., 2006) colored spherule e, h, c antibacterial activity (de faria and da silva, 2008; endean, 1966; smith et al., 2006) colorless spherule e, h, a, c, o antibacterial, inflammation, wound healing, ecm remodeling (coteur et al., 2002; de faria and da silva, 2008; eliseikina and magarlamov, 2002; endean, 1966; garcia-arraras et al., 2006; ramirez-gomez et al., 2010; smith et al., 2006) lymphocyte e, h, a progenitor cells (coteur et al., 2002; eliseikina and magarlamov, 2002; endean, 1966; ramirez-gomez et al., 2010; xing et al., 2008) vibratile e, h, a, o celomic fluid movement, clotting (de faria and da silva, 2008; eliseikina and magarlamov, 2002; endean, 1966; matranga et al., 2005; pinsino et al., 2008; ramirez-gomez et al., 2010; smith et al., 2006; xing et al., 2008) crystal cells h osmoregulation (eliseikina and magarlamov, 2002; endean, 1966; ramirezgomez et al., 2010; xing et al., 2008) hemocytes h, a, o oxygen transport (eliseikina and magarlamov, 2002; endean, 1966; pinsino et al., 2008) (smith, 1981). these are small cells (4-6 μm), with a large nucleus and a thin layer of cytoplasm whose only common characteristic with their vertebrate namesakes is their morphology. lymphocytes are regarded as progenitor cells and may be the precursors of other celomocyte types (xing et al., 2008; ramirez-gomez et al., 2010). they can show phagocytic capabilities but this may reflect an intermediate state of maturity before becoming phagocytes (ramirez-gomez et al., 2010). spherule cells (spherulocytes) are present mostly in echinoids and holothuroids (endean, 1966; eliseikina and magarlamov, 2002; smith et al., 2006; de faria and da silva, 2008; xing et al., 2008; ramirez-gomez et al., 2010) and in at least one sea star species (penn, 1979). they are characterized by the presence of vesicles in their cytoplasm, some containing pigment (red, yellow, green, brown) other being colorless. spherulocytes range in sizes from 8 to 20 μm and their distribution varies substantially between species. they have been associated with antibacterial activity (johnson, 1969; service and wardlaw, 1984; haug et al., 2002), inflammatory responses (pagliara and canicatti, 1993), extracellular matrix remodeling (garcia-arraras et al., 2006), and wound healing (san miguel-ruiz and garcia-arraras, 2007). another cell type present in echinoids and holothuroids are the vibratile cells. these are cells whose size ranges from 6 to 20 μm and are highly motile due to the presence of a flagellum. their distribution varies accordingly to the species and their function is still not completely determined. they have been associated with clotting reactions (bertheussen and seijelid, 1978) and are also thought to be involved in the movement of the celomic fluid (xing et al., 2008). crystal cells seem to be exclusive of holothurians, these cells display a very regular geometric morphology (rhomboidal or hexagonal) and present a crystal inclusion within their cytoplasm (endean, 1966). their role is still not well defined, but it is likely that they play osmoregulatory roles (xing et al., 2008).     214 table 2 summary of celomocyte distribution in the celomic fluid in three echinoderm classes and six different species. l.var: lytechinus variegatus; s. purp: strongylocentrotus purpuratus; e. luc: echinometra lucunter; h. glab: holothuria glaberrima; a. jap: apostichopus japonicus; a. rub: asterias rubens. cell type echinoidea holothuroidea asteroidea l. var (borges et al., 2005) s. purp (smith et al., 2006) e. luc (de faria and da silva, 2008) h. glab (ramirezgomez et al., 2010) a. jap (xing et al., 2008) a. rub (coteur et al., 2002) lymphocytes n.f. .n.f n.f. 60 % 59 % n.f. phagocytes/ amebocytes > 60 % 40-80 % 77 % 30 % 17 % 80-95 % colored spherules < 40 % 7-40 % 1 % n.f. n.f. n.f. colorless spherules + 3-25 % 3 % 5 % 23 % n.f. vibratile cells + 11-20 % 19 % < 1 % n.a. n.f. n.f., not found; n.a., not accounted. n.f., not found. + spherules and vibratile cells were accounted together. interestingly, echinoderm cellular immunology has escaped the classical characterization of their components by phenotyping (identification of cellspecific epitopes expressed on cell membranes) as vertebrate lymphocytes do. the lack of definite surface markers for celomocytes has helped maintain the confusion in distinguishing between specific cell types and sub-types, limiting it to just morphological observations. however, this trend is slowly changing, as demonstrated by studies with sea urchin celomocytes, in which a sub-population of phagocytes was defined by nk cell surface markers and characterized by their cytotoxic properties (lin et al., 2001). furthermore, monoclonal antibodies were generated against these cells showing a successful identification of a specific cytotoxic phagocyte sub-type (lin et al., 2007). our research group has also identified subgroups of sea cucumber celomocytes using monoclonal antibodies, each sub-population showing distinct characteristics and different responses to immunostimulation (ramirez-gomez et al., 2010). similarly, a recent study in the sea cucumber apostichopus japonicus, has also led to the development of a monoclonal antibody that specifically recognizes spherulocytes. initial characterization of the antigen being recognized by this antibody resulted in the identification of a 136 kda protein according to western blotting (li et al., 2010). however, what is still missing is the full characterization of the antigens these antibodies are recognizing (protein sequences and cloning). future experiments where cell markers are used to describe celomocyte populations and compare these populations among the different echinoderm classes should be the basis for a clear and universal classification of echinoderm celomocytes. celomocyte origin the origin of celomocytes is still a matter of debate. two theories have been proposed to address this issue: one involving specific organs or tissues as the source of celomocytes while the other points at the celomocytes themselves as selfreplicating cells (bossche and jangoux, 1976; matranga et al., 2005). potential cytopoietic organs include the axial organ, tiedemann bodies, polian vesicles, connective tissue and the celomic epithelium (endean, 1966). the latter has received particular attention in studies involving the sea star a. rubens, showing the epithelial origin of sea star celomocytes (bossche and jangoux, 1976; holm et al., 2008). even though no direct evidence have been proposed for a self-replicating population of celomocytes, the idea of a circulating stem cell has not been ruled out and if anything has become more attractive in view of recent findings of stem cells in other metazoans (handberg-thorsager et al., 2008; watanabe et al., 2009; funayama, 2010). it must be stated that the evidence to ascertain the origin of celomocytes is far from definitive, and would not be acceptable by modern scientific standards. thus, it is necessary for scientists to use modern methodologies to verify the celomocyte origins proposed by past investigators. humoral components echinoderms present a wide rich variety of secreted immune molecules. they have been the subjects of extensive research, even to the point of potential medical applications (kelly, 2005). as mentioned before, the humoral components present in celomic fluid of echinoderms are capable of     215 recognizing foreign matter, neutralizing or destroying pathogens, inducing or enhancing cellular responses (opsonization) and helping during wound healing. a well-known group of recognition molecules are the lectins, which recognize carbohydrate moieties on the surface of host cells (self) and of bacteria and fungi (non-self). several lectins have been identified from the celomic fluid of echinoderms, where they play important roles in opsonization, lytic cytotoxicity, clot formation and wound repair (gross et al., 1999). different lectins with specific recognition abilities have been found in asteroids (kamiya et al., 1992), and holothuroids (matsui et al., 1994; gowda et al., 2008a, b). echinoidin, a c-type lectin (calcium-dependent) identified in a sea urchin also possess an rgd sequence, suggesting an additional role in cell-tocell adhesion (giga et al., 1987; ozeki et al., 1991). moreover, the c-type lectin cel-iii from the sea cucumber cucumaria miniata, which possess a strong hemolytic activity have been transgenically expressed in mosquitoes and shown to successfully impair malaria parasite development (yoshida et al., 2007). other humoral factors include hemolysins, that interact with plasma membranes and form holes in the membrane of cells causing lysis of target cells (canicatti, 1990, 1991). hemolysins have been identified in sea stars (leonard et al., 1990), sea urchins (ryoyama, 1973; stabili et al., 1992) and sea cucumbers (canicatti and parrinello, 1985). agglutinins are another type of humoral factor that play roles in cell aggregation, encapsulation and clotting and have also been studied in wound repair. they have been found in echinoids (ryoyama, 1973; canicatti et al., 1992), the sea star asteria pectinifera (kamiya et al., 1992) and in the sea cucumber holothuria polii (canicatti and parrinello, 1985). in vertebrates a well-known group of effector molecules are the cytokines, which play a wide variety of roles in the immune response. in echinoderms, homologues of cytokines have also been identified. the first glance at an echinoderm cytokine came from the sea star a. forbesi, in which a humoral factor named the sea star factor was isolated and found to possess cytokine-like properties (prendergast and suzuki, 1970; prendergast and liu, 1976; kerlin et al., 1994). furthermore, interleukin-like molecules were identified in the sea star, e.g., a protein with il-1 activity and an il-6 like molecule ( beck et al., 1989, 1993; beck and habicht, 1991a, 1991b, 1996). however, none of these findings resulted in a definite identification of the cytokine factor or the cloning of the corresponding gene(s). the issue appears to be complicated since no il-1 homologues were found in the sea urchin genome. however, other members of the cytokine network (mostly pro-inflammatory) have indeed been found, e.g., tnf and il-17 (hibino et al., 2006). another important humoral factor present in echinoderms is the complement protein family. most of the components of the alternative and lectin pathways have been identified in the sea urchins, being the purple sea urchin the first invertebrate in which a complement system was identified (smith et al., 1996; smith, 2001). initial evidence gathered from sea urchins, sea cucumbers and sea stars hinted at the presence of a complement system in echinoderms (kaplan and bertheussen, 1977; parrinello et al., 1979; bertheussen 1981a, 1981b, 1982, 1983; bertheussen and seljelid, 1982), but they were mostly from complement-derived or dependent activity and no definite identification of a complement protein was achieved. smith and colleagues (1996, 1998) successfully identified the first echinoderm (and invertebrate) homologue of the c3 component and later another complement protein was found (factor b) (smith et al., 1998). a recent publication reported the finding of a c3 complement homologue in the sea star a. rubens, whose expression is also induced by lps stimulation (mogilenko et al., 2010). additional components of the system have been identified from the sea urchin genome, suggesting that echinoderms possess a complement pathway mostly directed towards opsonization, since the components of canonical terminal pathway could not be found (hibino et al., 2006). molecular studies and the genomic era the vast majority of molecular studies have been done in echinoids, particularly the purple sea urchin s. purpuratus. a broad number of immune genes have been identified from the sea urchin since the early 1990’s and pinnacled with the publication of the s. purpuratus genome (sodergren et al., 2006). an in depth analysis of the immune repertoire contained within the sea urchin genome can be found in the publications of hibino et al. (2006) and rast et al. (2006). however, other species of echinoderms have also been the subject of molecular studies in order to better understand their immune responses. these studies altogether benefit greatly the advancement of the field, providing further insights into the genetic and molecular aspects of echinoderm immunity. echinoderm molecular immunogenetics has evolved in parallel with the technologies available for its study. starting with gene-by-gene approaches, in which single genes were analyzed at a time and their immune roles determined. an example of this is the case of the profilin gene, an actin binding and cytoskeletal modification protein, expressed in celomocytes and up-regulated after injury and lps injections (smith et al., 1992, 994, 1995). then, when sequencing technologies became accessible, high-throughput sequencing projects were launched, mostly screening cdna libraries. in the late 1990’s a survey of a cdna library from lps-activated celomocytes provided the first glimpses of the immune repertoire of an echinoderm (smith et al., 1996). several interesting findings were made in this study on sea urchins, beginning with the discovery of an echinoderm complement and a collection of putative immune effector genes that set the basis for future comparative studies between echinoderm species. our research group has been dwelling into the molecular immune aspects of holothurians since the     216 year 2000, when a homologue of the acute phase response protein serum amyloid a (saa) was identified for the first time in an invertebrate (santiago et al., 2000). its expression was found mostly in intestinal tissues during the regeneration process but also after immune stimulation with lps. saa mrna was found to be overexpressed following an immune challenge not only in the intestine (santiago-cardona et al., 2003) but also in celomocytes (ramirez-gomez et al., 2008). additionally, a series of immune-related genes were identified in the holothurian from intestinal cdna libraries. this identification was mostly done by sequence comparison with other immune genes present in other organisms and whose immune role was clearly defined. the expression of these holothurian immune genes was corroborated in celomocytes to determine if they were part of the gene repertoire of these cells. in addition their expression was analized after an lps challenge (ramirez-gomez et al., 2008). among these genes we found a c-type lectin, ferritin, cathepsin, toposome and an alpha 2 macroglobulin domain (a2m)-containing protein. one sequence of particular interest was a homologue of the dd104 protein from the sea urchin, which is up-regulated in celomocytes after injury and infection (rast et al., 2000) with the holothurian dd104 following a similar pattern but with higher expression levels in celomocytes after lps injection. recently, analysis of the expression of immune-related genes has also been done in embryos and larvae of the sea cucumber, a. japonicus. nine genes were studied: six of them (heat shock proteins -70, -90 and -gp96; thymosin-beta, ferritin and dd104) showed no changes upon lps challenge while the remaining three (mannan-binding c-type lectin, lysozyme and serine proteinase inhibitor) were found to be upregulated upon challenge (yang et al., 2010). the advent of array technologies that allow for studies on the expression of multiple genes at the same time, have opened the door for the identification of potential novel genes. many of these genes were missed in previous approaches probably due to their lack of homology to known genes. this approach was first carried out with the sea urchin, comparing immune-stimulated and immunoquiescent animals. an unexpected diversity of genes was found to be differentially expressed and, more interestingly, a set of novel genes, the 185/333 family of proteins, were then identified (nair et al., 2005). this family of genes represents a highly variable set of proteins that are involved in the immune response of the sea urchin (reviewed in ghosh et al., 2010). we have also used immune activation and microarray technologies to compare lps-injected sea cucumbers with seawater-injected controls and thus, identify immune-responsive genes in the holothurians. we have found 50 unique sequences differentially expressed after lps stimulation. the vast majority of these sequences showed no known homologies in the databases (ramirez-gomez et al., 2009). ongoing efforts are being done to further characterize these unknown genes. by complete sequencing of the mrnas we expect to find similarities and/or conserved domains that will provide either proper identification, or the characterization of novel holothurian lpsresponsive genes. an interesting case derived from our microarray study, is the echinoderm mayor yolk protein (myp) and its closely related protein, toposome. in our microarray, the holothurian myp gene was one of the top genes that showed differentially expression following lps injection. echinoderm myp was initially identified as an unconventional iron-binding vitellogenic protein making up to 50 % of the protein content of the sea urchin celomic fluid (brooks and wessel, 2002). it is synthesized in the digestive tract and also binds zinc ions (unuma et al., 2007, 2009). a possible immune role for this protein had been suggested due to its affinity for iron, making it an excellent bacteriostatic agent. our results from the holothurian microarray, have shown that myp mrna is up-regulated after lps stimulation in the digestive tract but its expression remains unchanged in celomocytes (ramirez-gomez et al., 2009). nonetheless, anti-myp labeling is found in phagocytic lymphocytes (ramirez-gomez et al., 2010). the toposome protein (which is closely related to myp), functions as an adhesion protein in the sea urchin embryo (cervello and matranga, 1989; scaturro et al., 1998; noll et al., 2007), but is also related to stress and injury respones (cervello et al., 1994; matranga et al., 2005; pinsino et al., 2007). we have found toposome mrna to be expressed in h. glaberrima celomocytes at relatively high levels that remain unchanged after lps stimulation (ramirez-gomez et al., 2008) as well as in intestinal tissues, in which its levels remained unchanged also (ramirez-gomez et al., 2009). these results show that both myp and toposome are indeed associated with the immune response but also suggest additional roles that might not be part of the traditional functions associated with celomocytes but might be associated with the immune functions of the digestive tract. now that we have entered the genomic era, further advances are expected as genome sequencing technologies become faster and more economically accessible. the s. purpuratus genome represents a cornerstone in echinoderm research that can be used to compare findings from other echinoderm species. however, as presented here, the great diversity of the animals within the echinoderm phylum suggest that having the genome of only one member of only one echinoderm class will not be enough to understand the echinoderm immune system. take for example one of the most diverse set of genes found in the sea urchin genome: the nlr gene family (nucleotide-binding domain, leucine-rich repeat containing proteins). these genes encode cytoplasmic pattern recognition proteins, which in humans are represented by about 20 genes (inohara and nunez, 2003). however, in the sea urchin 203 nlr predicted genes can be found. similar to the vertebrate counterparts, the major site of expression of the sea urchin nlrs is the gut (hibino et al., 2006). nonetheless, we were not able to identify sequences for this gene family in any of our holothurian intestinal cdna libraries nor in our intestinal microarray studies. this may reflect key     217 differences in gene repertoires between these two species related to their phylogenetic divergence. this variety of gene repertoires may also be attributed to differences in habitat and developmental history, and to differences in the microbe flora that challenges the organisms. these differences will eventually shape the type of immune responses that organisms react to. therefore, we still need more information on immune-related genes present in other species from as many different groups as possible in order to have a better understanding of the molecular events that are involved with the echinoderm immune response. concluding remarks echinoderm immunity is a challenging yet promising field to study. the large diversity of echinoderm species, with different internal organs (most of them with little physiological information as to their functions) and different lifestyles make it difficult to identify those tissues or cells that might be playing an immune role. moreover, different species might be responding to different immune challenges not usually associated with other animal groups (think about the fact that echinoderms occupy large number of niches in the benthic zone). an additional complication is the difficulty in establishing the immune status of the animals used in experimentation. for example, in studies by smith and colleagues, sea urchins were kept in aquaria in what appeared to be an immunoquiescent status. in this scenario it is difficult to compare the lps response of these animals to that of animals that have been directly collected from the wild. nonetheless, overcoming these difficulties can provide exciting and rewarding goals. among these, are the identification of novel immune associated genes and proteins and the characterization of new immune signaling pathways. moreover, the key phylogenetic position of echinoderms in the tree of life assures that whatever we learn about echinoderm immunity will help us understand the evolution of metazoan immune systems. references beck g, ellis t, zhang h, lin w, beauregard k, habicht gs, truong n. nitric oxide production by coelomocytes of asterias forbesi. dev. comp. immunol. 25: 1-10, 2001. beck g, habicht gs. primitive cytokines: harbingers of vertebrate defense. immunol. today 12: 180183, 1991a. beck g, habicht gs. purification and biochemical characterization of an invertebrate interleukin 1. mol. immunol. 28: 577-584, 1991b. beck g, habicht gs. characterization of an il-6-like molecule from an echinoderm (asterias forbesi). cytokine 8: 507-512, 1996. beck g, o'brien rf, habicht gs. invertebrate cytokines: the phylogenetic emergence of interleukin-1. bioessays 11: 62-67, 1989. beck g, o'brien rf, habicht gs, stillman dl, cooper el, raftos da. invertebrate cytokines. iii: invertebrate interleukin-1-like molecules stimulate phagocytosis by tunicate and echinoderm cells. cell. immunol. 146: 284-299, 1993. bertheussen k. endocytosis by echinoid phagocytes in vitro. ii. mechanisms of endocytosis. dev. comp. immunol. 5: 557-564, 1981a. bertheussen k. endocytosis by echinoid phagocytosis in vitro. i. recognition of foreign matter. dev. comp. immunol. 5: 241-250, 1981b. bertheussen k. receptors for complement on echinoid phagocytes. ii. purified human complement mediates echinoid phagocytosis. dev. comp. immunol. 6: 635-642, 1982. bertheussen k. complement-like activity in sea urchin coelomic fluid. dev. comp. immunol. 7: 21-31, 1983. bertheussen k, seijelid r. echinoid phagocytes in vitro. exp. cell res. 111: 401-12, 1978 bertheussen k, seljelid r. receptors for complement on echinoid phagocytes. i. the opsonic effect of vertebrae sera on echinoid phagocytosis. dev. comp. immunol. 6: 423431, 1982. boolootian ra. the perivisceral elements of echinoderm body fluids. amer. zool. 2: 275284, 1962. boolootian ra, giese ac. coelomic corpuscles of echinoderms. biol. bull. 115: 53-63, 1958. boolootian ra, giese ac. clotting of echinoderm coelomic fluid. j. exp. zool. 140: 207-229, 1959. borges jc, jensch-junior be, garrido pa, mangiaterra mb, silva jr. phagocytic amoebocyte sub populations in the perivisceral coelom of the sea urchin lytechinus variegatus (lamarck, 1816). j. exp. zool. a comp. exp. biol. 303: 241-248, 2005. bossche jp, jangoux m. epithelial origin of starfish coelomocytes. nature 261: 227-228, 1976. brooks jm, wessel gm. the major yolk protein in sea urchins is a transferrin-like, iron binding protein. dev. biol. 245: 1-12, 2002. canicatti c. hemolysins: pore-forming proteins in invertebrates. experientia 46: 239-244, 1990. canicatti c. binding properties of paracentrotus lividus (echinoidea) hemolysins. comp. biochem. physiol. 98a: 463-468, 1991. canicatti c, pagliara p, stabili l. sea urchin coelomic fluid agglutinin mediates coelomocyte adhesion. eur. j. cell biol. 58: 291-295, 1992. canicatti c, parrinello n. hemaglutinin and hemolysin level in coelomic fluid from holothuria polii (echinodermata) following sheep erythrocyte injection. biol. bull. 168: 175182, 1985. cervello m, arizza v, lattuca g, parrinello n, matranga v. detection of vitellogenin in a subpopulation of sea urchin coelomocytes. eur. j. cell biol. 64: 314-319, 1994. cervello m, matranga v. evidence of a precursorproduct relationship between vitellogenin and toposome, a glycoprotein complex mediating cell adhesion. cell. differ. dev. 26: 67-76, 1989. clow la, raftos da, gross ps, smith lc. the sea urchin complement homologue, spc3,     218 functions as an opsonin. j. exp. biol. 207: 2147-255, 2004. coteur g, danis b, fowler sw, teyssie jl, dubois p, warnau m. effects of pcbs on reactive oxygen species (ros) production by the immune cells of paracentrotus lividus (echinodermata). mar. pollut. bull. 42: 667-672, 2001. coteur g, debecker g, warnau m, jangoux m, dubois p. differentiation of immune cells challenged by bacteria in the common european starfish, asterias rubens (echinodermata). eur. j. cell biol. 81: 413-418, 2002. de faria mt, da silva jr. innate immune response in the sea urchin echinometra lucunter (echinodermata). j. invertebr. pathol. 98: 5862, 2008. dybas l, fankboner pv. holothurian survival strategies: mechanisms for the maintenance of a bacteriostatic environment in the coelomic cavity of the sea cucumber, parastichopus californicus. dev. comp. immunol. 10: 311-330, 1986. eliseikina mg, magarlamov ty. coelomocyte morphology in the holothurians apostichopus japonicus (aspidochirota: stichopodidae) and cucumaria japonica (dendrochirota: cucumariidae). russian j. mar. biol. 28: 197202, 2002. endean r. the coelomocytes and coelomic fluids. in: boolootian ra (ed), physiology of echinodermata, intersciences, new york, 1966. funayama n. the stem cell system in demosponges: insights into the origin of somatic stem cells. dev. growth differ. 52: 114, 2010. garcia-arraras je, schenk c, rodrigues-ramirez r, torres ii, valentin g, candelaria ag. spherulocytes in the echinoderm holothuria glaberrima and their involvement in intestinal regeneration. dev. dyn. 235: 3259-3267, 2006. ghosh j, buckley km, nair sv, raftos da, miller c, majeske aj, et al. sp185/333: a novel family of genes and proteins involved in the purple sea urchin immune response. dev. comp. immunol. 34: 235-245, 2010. giga y, ikai a, takahashi k. the complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin anthocidaris crassispina. homologies with mammalian and insect lectins. j. biol. chem. 262: 6197-6203, 1987. glinski z, jarosz j. immune phenomena in echinoderms. arch. immunol. ther. exp. (warsz.) 48: 189-193, 2000. gowda nm, goswami u, khan mi. purification and characterization of a t-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (holothuria scabra). fish shellfish immunol. 24: 450-458, 2008a. gowda nm, goswami u, khan mi. t-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (holothuria scabra): possible involvement in differential recognition of bacteria. j. invertebr. pathol. 99: 141-145, 2008b. gross ps, al-sharif wz, clow la, smith lc. echinoderm immunity and the evolution of the complement system. dev. comp. immunol. 23: 429-442, 1999. gross ps, clow la, smith lc. spc3, the complement homologue from the purple sea urchin, strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes. immunogenetics 51: 1034-1044, 2000. handberg-thorsager m, fernandez e, salo e. stem cells and regeneration in planarians. front. biosci. 13: 6374-6394, 2008. haug t, kjuul ak, styrvold ob, sandsdalen e, olsen om, stensvag k. antibacterial activity in strongylocentrotus droebachiensis (echinoidea), cucumaria frondosa (holothuroidea), and asterias rubens (asteroidea). j. invertebr. pathol. 81: 94-102, 2002. hibino t, loza-coll m, messier c, majeske aj, cohen ah, terwilliger dp, et al. the immune gene repertoire encoded in the purple sea urchin genome. dev. biol. 300: 349-365, 2006. hillier bj, vacquier vd. amassin, an olfactomedin protein, mediates the massive intercellular adhesion of sea urchin coelomocytes. j. cell. biol. 160: 597-604, 2003. holm k, dupont s, skold h, stenius a, thorndyke m, hernroth b. induced cell proliferation in putative haematopoietic tissues of the sea star, asterias rubens (l.). j. exp. biol. 211: 25512558, 2008. inohara n, nunez g. nods: intracellular proteins involved in inflammation and apoptosis. nat. rev. immunol. 3: 371-382, 2003. jans d, dubois p, jangoux m. defensive mechanisms of holothuroids (echinodermata): formation, role and fate of intracoelomic brown bodies in the sea cucumber holothuria tubulosa. cell tissue res. 283: 99-106, 1996. johnson pt. the coelomic elements of sea urchins (strongylocentrotus). 3. in vitro reaction to bacteria. j. invertebr. pathol. 13: 42-62, 1969. kamiya h, muramoto k, goto, r, sakai m. lectins in the hemolymph of a starfish, asterina pectinifera: purification and characterization. dev. comp. immunol. 16: 243-250, 1992. kanungo k. in vitro studies on the effect of cell-free coelomic fluid calcium, and/or magnesium on clumping of coelomocytes of the sea star asteria forbesi. biol. bull. 163: 438-452, 1982. kaplan g, bertheussen k. the morphology of echinoid phagocytes and mouse peritoneal macrophages during phagocytosis in vitro. scand. j. immunol. 6: 1289-1296, 1977. kelly ms. echinoderms: their culture and bioactive compounds. prog. mol. subcell. biol. 39: 139165, 2005. kerlin rl, cebra jj, weinstein pd, prendergast ra. sea star factor blocks development of tdependent antibody secreting clones by preventing lymphokine secretion. cell. immunol. 156: 62-76, 1994. kindred je. the cellular elements in the perivisceral fluid of echinoderms. biol. bull. 46: 228-251, 1924.     219 leonard la, strandberg jd, winkelstein ja. complement-like activity in the sea star, asterias forbesi. dev. comp. immunol. 14: 1930, 1990. li q, li y, li h, wang y, xu d. production, characterization and application of monoclonal antibody to spherulocytes: a subpopulation of coelomocytes of apostichopus japonicus. fish shellfish immunol. 29: 832-838, 2010. lin w, grant s, beck g. generation of monoclonal antibodies to coelomocytes of the purple sea urchin arbacia punctulata: characterization and phenotyping. dev. comp. immunol. 31: 465475, 2007. lin w, zhang h, beck g. phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, arbacia punctulata. j. exp. zool. 290: 741-750, 2001. matranga v, pinsino a, celi m, natoli a, bonaventura r, schroder hc, et al. monitoring chemical and physical stress using sea urchin immune cells. prog. mol. subcell. biol. 39: 85110, 2005. matsui t, ozeki y, suzuki m, hino a, titani k. purification and characterization of two ca(2+)dependent lectins from coelomic plasma of sea cucumber, stichopus japonicus. j. biochem. 116: 1127-1133, 1994. metchnikoff i. lectures on the comparative pathology of inflammation delivered at the pasteur institute in 1891, dover, new york, 1891. mogilenko da, kudriavtsev iv, orlov sv, kharazova ad, polevshchikov av. [expression of the starfish complement component c3 gene homologue under the influence of bacterial lipopolysaccharide]. mol. biol. (mosk.) 44: 7484, 2010. nair sv, del valle h, gross ps, terwilliger dp, smith lc. macroarray analysis of coelomocyte gene expression in response to lps in the sea urchin. identification of unexpected immune diversity in an invertebrate. physiol. genomics 22: 33-47, 2005. noll h, alcedo j, daube m, frei e, schiltz e, hunt j, et al. the toposome, essential for sea urchin cell adhesion and development, is a modified iron-less calcium-binding transferrin. dev. biol. 310: 54-70, 2007. ozeki y, matsui t, titani k. cell adhesive activity of two animal lectins through different recognition mechanisms. febs lett. 289: 145-147, 1991. pagliara p, canicatti c. isolation of cytolytic granules from sea urchin amoebocytes. eur. j. cell biol. 60: 179-184, 1993. parrinello n, rindone d, canicatti c. naturally occurring hemolysins in the coelomic fluid of holothuria polii delle chiaie (echinodermata). dev. comp. immunol. 3: 45-54, 1979. penn pe. wound healing in the tropical intertidal asteroid, napanthia belcheri (perrier). amer. zool. 19: 1006, 1979. pinsino a, thorndyke mc, matranga v. coelomocytes and post-traumatic response in the common sea star asterias rubens. cell stress chaperones 12: 331-341, 2007. pinsino a, thorndyke mc, matranga v. coelomocytes and post-traumatic response in the common sea star asterias rubens. cell stress chaperones 12: 331-341, 2008. prendergast ra, liu sh. isolation and characterization of sea star factor. scand. j. immunol. 5: 873-880, 1976. prendergast ra, suzuki m. invertebrate protein simulating mediators of delayed hypersensitivity. nature 227: 277-279, 1970. ramirez-gomez, f, aponte-rivera f, mendezcastaner l, garcia-arraras je. changes in holothurian coelomocyte populations following immune stimulation with different molecular patterns. fish shellfish immunol. 29: 175-185, 2010. ramirez-gomez f, ortiz-pineda pa, riveracardona g, garcia-arraras je. lps-induced genes in intestinal tissue of the sea cucumber holothuria glaberrima. plos one 4: e6178, 2009. ramirez-gomez f, ortiz-pineda pa, rojascartagena c, suarez-castillo ec, garciaarraras je. immune-related genes associated with intestinal tissue in the sea cucumber holothuria glaberrima. immunogenetics 60: 5771, 2008. rast jp, pancer z, davidson eh. new approaches towards an understanding of deuterostome immunity. curr. top. microbiol. immunol. 248: 3-16, 2000. rast jp, smith lc, loza-coll m, hibino t, litman gw. genomic insights into the immune system of the sea urchin. science 314: 952-956, 2006. ryoyama k. studies on the biological properties of coelomic fluid of sea urchin. i. naturally occurring hemolysin in sea urchin. biochim. biophys. acta 320: 157-165, 1973. san miguel-ruiz, j. e. and garcia-arraras, j. e. common cellular events occur during wound healing and organ regeneration in the sea cucumber holothuria glaberrima. bmc dev. biol. 7: 115, 2007. santiago p, roig-lopez jl, santiago c, garciaarraras je. serum amyloid a protein in an echinoderm: its primary structure and expression during intestinal regeneration in the sea cucumber holothuria glaberrima. j. exp. zool. 288: 335-344, 2000. santiago-cardona pg, berrios ca, ramirez f, garcia-arraras je. lipopolysaccharides induce intestinal serum amyloid a expression in the sea cucumber holothuria glaberrima. dev. comp. immunol. 27: 105-110, 2003. scaturro g, zito f, matranga v. the oligomeric integrity of toposome is essential for its morphogenetic function. cell biol. int. 22: 321326, 1998. service m, wardlaw ac. echinochrome-a as a bactericidal substance in the coelomic fluid of echinus esculentus (l). comp. biochem. physiol. 79b: 161-165, 1984. smith lc. the complement system in sea urchins. in: beck g, m. sugumaran m, cooper c (eds), phylogenetic perspectives on the vertebrate immune systems: adv. exp. med. biol, pp 363    220 372, kluwer academic/plenum publishing co., new york, 2001. smith lc, britten rj, davidson eh. spcoel1: a sea urchin profilin gene expressed specifically in coelomocytes in response to injury. mol. biol. cell 3: 403-414, 1992. smith lc, britten rj, davidson eh. lipopolysaccharide activates the sea urchin immune system. dev. comp. immunol. 19: 217224, 1995. smith lc, chang l, britten rj, davidson eh. sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. j. immunol. 156: 593-602, 1996. smith lc, davidson eh. the echinoid immune system and the phylogenetic occurrence of immune mechanisms in deuterostomes. immunol. today 13: 356-362, 1992. smith lc, harrington mg, britten rj, davidson eh. the sea urchin profilin gene is specifically expressed in mesenchyme cells during gastrulation. dev. biol. 164: 463-474, 1994. smith lc, rast jp, brockton v, terwilliger dp, nair sv, buckley k, et al. the sea urchin immune system. inv. surv. j. 3: 25-39, 2006. smith lc, shih cs, dachenhausen sg. coelomocytes express spbf, a homologue of factor b, the second component in the sea urchin complement system. j. immunol. 161: 6784-6793, 1998. smith vj. invertebrate blood cells. in: ratcliffe na, riowley af (eds), the echinoderms, academic press, new york, pp 513-562, 1981. sodergren e, weinstock gm, davidson eh, cameron ra, gibbs ra, angerer rc, et al. the genome of the sea urchin strongylocentrotus purpuratus. science 314: 941-952, 2006. stabili l, pagliara p, metrangolo m, canicatti c. comparative aspects of echinoidea cytolysins: the cytolytic activity of spherechinus granularis (echinoidea) coelomic fluid. comp. biochem. physiol. 101a: 553-556, 1992. sun y, jin l, wang t, xue j, liu g, li x, et al. polysaccharides from astragalus membranaceus promote phagocytosis and superoxide anion (o2-) production by coelomocytes from sea cucumber apostichopus japonicus in vitro. comp. biochem. physiol. 147c: 293-298, 2008. unuma t, ikeda k, yamano k, moriyama a, ohta h. zinc-binding property of the major yolk protein in the sea urchin implications of its role as a zinc transporter for gametogenesis. febs j. 274: 4985-4998, 2007. unuma t, konishi k, kiyomoto m, matranga v, yamano k, ohta h, et al. the major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae. mol. reprod. dev. 76: 142-150, 2009. watanabe h, hoang vt, mattner r, holstein tw. immortality and the base of multicellular life: lessons from cnidarian stem cells. semin. cell dev. biol. 20: 1114-1125, 2009. xing k, yang hs, chen my. morphological and ultrastructural characterization of the coelomocytes in apostichopus japonicus. aquat. biol. 2: 85-92, 2008. yang a, zhou z, dong y, jian b, wang x, chen z, et al. expression of immune-related genes in embryos and larvae of sea cucumber apostichopus japonicus. fish shellfish immunol. 29: 839-845, 2010. yoshida s, shimada y, kondoh d, kouzuma y, ghosh ak, jacobs-lorena m., et al. hemolytic c-type lectin cel-iii from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development. plos pathog 3: e192, 2007. yui ma, bayne cj. echinoderm immunology: bacterial clearance by the sea urchin strongylocentrotur purpuratus. biol. bull. 165: 473-486, 1983.   research report isj 4: 119-126, 2007 issn 1824-307x research report a morphological and functional characterization of bombyx mori larval midgut cells in culture g cermenati, p corti, s caccia, b giordana, m casartelli dipartimento di biologia, università degli studi di milano, 20133 milano, italy accepted november 12, 2007 abstract recent studies have shown that bombyx mori larval midgut can transport proteins unaltered following the transcellular pathway by transcytosis. the numerous steps involved in this complex process are still unknown in the insect midgut, and a promising tool to elucidate this aspect is the availability of single midgut cells in culture suitable for transport experiments. mature midgut cells in culture were obtained from stem cells isolated from b. mori larvae cultured in grace’s medium supplemented with 20-hydroxyecdysone (20-he) and α-arylphorin. after three weeks, up to 60 % of the cultured cells were differentiated into columnar and goblet cells, the two predominant cell types in the midgut epithelium. these cells presented in vitro the same shape, morphology and polarity recorded in vivo, even if their dimensions were slightly reduced. columnar cells displayed a well developed cytoskeletal arrangement, with actin filaments highly organized within the thick brush border and distributed in faint filaments in the cell cytoplasm. microtubules formed a substantial net just beneath the brush border and ran longitudinally from the apical to the basal pole of the cell. cultured cells homogenates displayed aminopeptidase n and alkaline phosphatase activity, proving that these two enzymes, involved in vivo in the intermediate and final digestion, are expressed also in vitro. the columnar cells differentiated in culture were able to internalize two model proteins with quite different transport rates. key words: bombyx mori larval midgut; stem cells; columnar cells in culture; cytoskeletal scaffolding; digestive enzymes; protein uptake introduction the lepidopteran midgut is formed by a folded epithelial cells monolayer, separated from underlying muscles and tracheae by a thin basal membrane and composed by three main cell types, goblet, columnar, stem cells (cioffi, 1979; baldwin and hakim, 1991), and by fewer endocrine cells. goblet cells have a peculiar shape, with a basally located nucleus and a cavity lined by an apical plasma membrane forming numerous microvilli, where a v-h+-atpase pump (wieczorek et al., 1989) and a k+/2h+ antiport (azuma et al., 1995) are expressed. the combined activity of these two transporters generates the high electrical voltage, the active secretion of k+ and the extreme luminal ___________________________________________________________________________ corresponding author morena casartelli dipartimento di biologia università degli studi di milano via celoria 26, 20133 milano, italy e-mail: morena.casartelli@unimi.it alkalinisation typical of the lepidopteran midgut epitehelium. columnar cells have an almost cylindrical shape with a central nucleus, an apical thick brush border and deep infoldings of the basal plasma membrane (cioffi, 1979; baldwin and hakim, 1991). this cell type is involved in nutrient digestion (terra and ferreira, 2005) and absorption (giordana et al., 1982, 1998). the small stem cells, roughly conicalor spindle-shaped with a large nucleus, are located at the base of the epithelium (turbeck, 1974; baldwin and hakim, 1991): they are the only cell type that undergoes mitosis (loeb and hakim, 1996) and their proliferation initiates in proximity of the moults (baldwin and hakim, 1991). in the last decade primary cultures of mature cells from several lepidopteran species were obtained successfully from the isolated midgut stem cells maintained in a culture medium integrated with 20hydroxyecdisone and α-arylphorin, a subunit isolated from a perivisceral fat body extract of manduca sexta pupae (sadrud-din et al., 1994, 119 1996; loeb and hakim, 1996; blackburn et al., 2004). differentiation to goblet and columnar cells requires the presence of factors released in the medium by the actively developing cell culture (sadrud-din et al., 1996). more recently, some of the peptides that, acting like the mammalian growth factors, induce cell differentiation have been identified (loeb et al., 1999; goto et al., 2001; loeb and jaffe, 2002). we have recently shown that bombyx mori larval midgut isolated and perfused in vitro can transport two selected proteins unaltered from the lumen to the haemolymph side of the epithelium by transcytosis (casartelli et al., 2005, 2007). the availability of single mature columnar cells in culture may represent the best tool to study in detail the multiple steps involved in this complex pathway. we have therefore standardised a primary culture of larval midgut cells from b. mori and examined some morphological features of the isolated cells grown and differentiated in culture. we also investigated if they expressed some of the digestive enzymes detected in vivo and if they could internalize two different proteins, albumin labelled with fluorescein isothiocyanate (fitc-albumin) and green fluorescent protein (gfp). materials and methods experimental animals bombyx mori eggs and the artificial diet (cappellozza et al., 2005) were provided by crainstitute for sericulture (padova, italy). larvae were reared under controlled conditions (25 ± 1 °c, 65-70 % rh, 12l:12d photoperiod). histological analysis of iv instar midgut epithelium during pre-moult, moult and v instar feeding period silkworms at the above indicated stages of development were anaesthetized with co2. the midgut was explanted, deprived of the peritrophic membrane and malpighian tubules and fixed in pycric acid, formaldehyde and glutaraldehyde (pafg) at room temperature for 2 h and then at 4 °c over-night according to ermak and eakin (1976). the samples were then washed in 0.1 m cacodylate buffer (ph 7.4), postfixed in 1 % osmium tetroxide in the same buffer for 2 h, washed in distilled water and left for 2 h in 2 % uranyl acetate. after dehydratation in a graded ethanol series, samples were embedded in epon resin and the polymerisation was performed at 60 °c for 48 h. semithin sections were cut with a reichert ultracut e microtome and observed at the light microscope (axiovert 200m equipped with axiocam hrm, zeiss, germany). preparation of midgut cells cultures larvae of b. mori at the end on the iv instar, just before the iv moult, were anaesthetized with co2 and surface-sterilized by consecutive immersions, lasting approximately 2 min each, in the following solutions: 10 % (v/v) detergent (pharma soap medical); 2 % (v/v) p-hydroxybenzoic acid methyl ester (sigma), prepared from a stock solution of 15 % p-hydroxybenzoic acid methyl ester (w/v) in 95 % ethanol; 0.1 % (v/v) sodium hypochlorite. silkworms were cut between the second and the third pair of thoracic legs and behind the third pair of abdominal appendages to exclude the foregut and the hindgut, and the peritrophic membrane along with the enclosed intestinal contents was removed. the central part of the larva was transferred to a petri dish containing a sterile physiological solution composed of 47 mm kcl, 20.5 mm mgcl2, 20 mm mgso4, 5.3 mm k2hpo4, 5.6 mm kh2po4, 1 mm cacl2, 75 mm sucrose at ph 7, 0.2 % (v/v) gentamicin (50 mg/ml, sigma), 0.01 % (v/v) antibiotic-antimycotic solution 1x (sigma). to this solution was added 0.003 ‰ (v/v) sodium hypochlorite. the ventral cuticle was cut longitudinally and the midgut, deprived of muscles and silk glands, was isolated. dissected midguts from 8-10 animals were cut along the longitudinal axis and rinsed twice (10 min for each rinse) in the above mentioned sterile physiological solution added with 0.003 ‰ (v/v) sodium hypochlorite, then again twice (for 10 min each) in the sterile physiological solution. midguts were pooled into a strainer (100 μm mesh size), placed in a petri dish containing few ml of the latter solution and left under mild agitation for 1 h. in these conditions, the loosely attached stem cells migrated away from the tissue. the tissue within the sieve was discarded and the free cells in the filtrate were collected and pelletted by gentle centrifugation at 400xg for 5 min. cells were then resuspended in growth medium, composed by a mixture of 67.4 % grace’s insect medium (gibco), 11.2 % 100 mm koh, 6.7 % fetal bovine serum (gibco), 0.5 % vitamins mix (composed by, in mg/100ml: 300 riboflavin, 150 pyridoxine hydrochloride, 150 thiamine hydrochloride, 150 folic acid, 600 nicotinic acid, 600 pantothenic acid, 12 biotin, 1.2 vitamin bb12), 0.018 ‰ antibiotic-antimycotic solution 1x (sigma), 0.1 % gentamicine (50 mg/ml, sigma). cultured cells were supplemented with 6x10 m 20hydroxyecdysone (sigma) and 100 ng/ml αarylphorin (purified according to blackburn et al., 2004 in insect bio-control laboratory, usda, beltsville, md, usa), kindly donated by prof rs hakim, howard university, washington, dc, usa. all the solutions used were routinely sterilized by filtration (nalgene, 0.2 μm pore size) prior to use. three ml of the cell suspension in growth medium were distributed in the wells (35 mm in diameter) of six well plates. cultures were incubated at 25 °c. one ml of medium from each well was routinely replaced with 1 ml of fresh medium once a week. -8 viability of cells in culture, recognition and count of stem cells, differentiating cells and mature cells along six weeks cell viability was checked with the trypan blue test in the initial stem cell culture and every seven days: viable cells excluded the dye, whereas dead cells became blue. an aliquot of the cell culture was removed, the cells were centrifuged for 5 min at 400xg and then resuspended in a known amount of the physiological solution (see above). an aliquot of the suspension was mixed with 0.4 % (w/v) trypan blue (sigma) (2:1). after 2 min, viable and dead cells were counted under the inverted microscope using a haemocytometer slide (burker). viable cells 120 were then classified in four different categories (stem, differentiating, columnar and goblet cells) on the basis of their morphological features and counted every seven days for six weeks. differences between the four categories along this experimental period were tested by student’s t test. immunodetection of microtubules in cultured columnar cells three weeks old cultured cells were pelletted at 400xg for 5 min and resuspended in the suitable volume of physiological solution (see above). cells were then fixed and permeabilized for 5 min with ice-cold methanol. after 3 rinses in pbs, cells were incubated for 15 min in pbs containing 1 % bovine serum albumin (bsa) and for 1 h with the anti-αtubulin mouse igg (sigma), diluted 1:500 in pbs with 1 % bsa. the cells were then washed 3 times in pbs plus 1 % bsa and incubated for 1 h with alexa fluor 488-conjugated anti-mouse igg antibody raised in donkey (molecular probes) diluted 1:1000 in pbs plus 1 % bsa. after 3 rinses in pbs, the samples were mounted in dabco (sigma)-mowiol (calbiochem), covered with a cover-slip and examined with a clsm tcs sp2 aobs (leica microsystems heidelberg gmbh, germany) equipped with an argon ion laser (458, 476, 488, 496 or 514 nm excitation), two hene lasers (543, 594 and 633 nm excitation) and tunable emission wavelength collection. a 63x leica oil immersion plan apo (na1,4) objective and a 2x zoom were used for all the experiments. detection of actin filaments in cultured columnar cells three weeks old cultured cells were pelletted at 400xg for 5 min and resuspended in the appropriate volume of physiological solution (see above). cells were then incubated for 10 min in 4 % paraformaldehyde in pbs, washed 3 times in pbs and permeabilized for 4 min with 0.1 % triton-x100 in pbs. the cells were washed 3 times in pbs and then incubated for 20 min with 4.3 μg/ml tritcphalloidin. after 3 rinses in pbs, the samples where mounted in dabco (sigma)-mowiol (calbiochem), covered with a cover-slip and examined with a confocal microscope (see above). enzymes assay three weeks old midgut cells in culture were pelletted by gentle centrifugation at 400xg for 5 min and resuspended in a small amount of physiological solution (see above). after three washes, the pellet was resuspended in a buffer solution (100 mm mannitol, 10 mm hepes-tris at ph 7.2) and lysated in the eppendorf vial with a motor for pellet pestle (sigma). protein concentration in the lysate was determined according to bradford (1976) with bsa as standard. all enzymatic assays were conducted under conditions in which products formation depended linearly on enzyme concentration. aminopeptidase n and alkaline phosphatase activities were determined at 25 °c by measuring the release of p-nitroaniline from l-leucine-pnitroanilide in 40 mm tris-hcl at ph 7.5 or of pnitrophenol from p-nitrophenylphosphate in 1 m trishcl at ph 8, respectively. enzymes activities were determined in triplicate or quadruplicate in a pharmacia biotech ultrospec 3000 spectrophotometer. proteins internalization in cultured columnar cells three weeks old cultured cells were pelletted by gentle centrifugation at 400xg for 5 min and resuspended in a small volume of physiological solution (see above). cells were incubated at 25 °c for 2 h in the presence of 1.4 μm fitc-albumin (sigma) or 1.5 μm gfp (vector etc). at the end of the incubation, the cells were washed three times with the physiological solution, fixed for 10 min with 4 % paraformaldehyde in pbs and then rinsed three times in pbs. cells were mounted in dabco (sigma)-mowiol (calbiochem) and examined with a confocal microscope (see above). results stem cells differentiation in culture and morphology of midgut cells in vivo and in vitro to isolate the largest possible number of stem cells from b. mori midgut, we performed an histological analysis of the tissue in three different instances of the larval development. in fig. 1 is shown the midgut epithelium dissected from larvae immediately before the iv moult (a), during the iv moult (b) or in the middle of the v instar during the feeding period (c). in the period just before the last larval-larval moult, the stem cells are located in numerous nidi at the base of the epithelium (fig. 1a). during the moult (fig. 1b), the stem cells proliferate and then differentiate, each of the newly developed cell inserting between the mature cells of the iv instar epithelium. in the v instar, during the feeding period (fig. 1c), very few single stem cells are visible, far less numerous than in the pre-moult period. therefore, the largest number of stem cells can be isolated from b. mori larval midguts in the period just preceding the iv moult. to monitor cell development in culture with time, we followed the growth, differentiation and percentage distribution of each cell type for 42 days (figs 2, 3), by identifying the different cell types every 7 days on the basis of their morphological features. stem cells were round, with a diameter of 4-8 μm (fig. 2a) and some of them could be observed in mitosis (fig. 2b). cells in an early stage of differentiation were round, with long tenuous membrane projections and numerous granules in the cytoplasm (fig. 2c): as suggested by sadruddin et al. (1996), these spherical cells with uniformly distributed microvilli appears to correspond to the initial phase of differentiation of columnar cells, that in a more advanced phase were triangular in shape (fig. 2d), differing from adult ones for their small dimension (10-25 μm). young columnar (fig. 2e) and goblet (fig. 2g) cells had the same shape of the respective mature cells (figs 2f, h) but their dimensions were smaller (between 25-30 μm). mature columnar cells were characterized by a well developed apical membrane with numerous microvilli, a centrally placed nucleus and a cylindrical or cubical shape (fig. 2f), while mature goblet cells were flask-like and presented the typical wide cavity, the apical valve and a basally located 121 fig. 1 semithin sections of the midgut epithelium of bombyx mori larvae dissected immediately before the iv moult (a), during the moult (b) and in the v instar feeding period (c). stem cells nidi (a), proliferating cells (b) and a single stem cell (c) are indicated by arrows. bars = 10 μm. nucleus (fig. 2h). both these cells showed most of the apparent morphological features seen in vivo but they were never as tall as those of the original epithelium (60 to 80 μm, fig. 1). as shown in fig. 3, in the filtrate collected after a mild agitation of the pre-moult midgut tissue, the stem cells represented the 87.4 ± 3.6 % (3 determinations) of the total viable cells present in the medium. after six days in culture, the reduction of stem cells was accompanied by an increase of differentiating, columnar and goblet cells. at the end of the following 7 days, the percentage of stem cells were further decreased, while differentiating cells were the most represented cell type. from the third week on, less than 10 % were stem cells and the remaining cells were represented by percentage values not statistically different (ranging between 27.6 ± 3.4 % (3 determinations) and 41.4 ± 5.3 % (3 determinations)) of columnar, goblet and differentiating cells. all along the experimental period here considered, viable cells were 79.2 ± 5.1 % (21 determinations) of the total cells present in the culture. the columnar cells used for the experiments reported in this paper came from three weeks-old cultures, although the cells maintained the same functional properties till day 42 (data not shown). organisation of the cytoskeleton in cultured columnar cells we examined the distribution of microtubules and actin filaments in columnar cells. as shown in figure 4 b, a large number of microtubules ran parallel to the apical cell surface, forming a dense network just below the apical microvilli. deeper down the cell, numerous bundles of microtubules are oriented longitudinally along the basal-apical axis of the cell. phalloidin stained conspicuously actin filaments within the brush border microvilli of columnar cells both in the initial phase of differentiation (fig. 5a) and in the mature phase (fig. 5b). in the latter figure a number of filaments running from the basolateral membrane deep into the cytoplasm were also detectable. enzymes activity columnar cells in vivo are responsible for the production of the different classes of enzymes involved in the digestion of ingested nutrients (terra and ferreira, 2005). we investigated if the activity of two enzymes currently used as marker enzymes of the apical membrane of midgut columnar cells, i.e. leucine-aminopeptidase n (apn) and alkaline phosphatase (alp), could be detected in the lysate of three weeks old cell cultures. although after 20 days in culture columnar cells represented only the 28.9 ± 2.7 % (3 determinations) of all the cells in culture (fig. 3), an activity of both enzymes could be measured: apn and alp specific activities (mu/mg of protein) were 890 ± 88 (5 determinations) and 131 ± 12 (4 determinations) respectively. proteins absorption in cultured columnar cells we have shown that fitc-albumin is transported across the lepidopteran larval midgut in vitro by transcytosis (casartelli et al., 2005), entering the cell through the apical membrane and being released in the haemocoel across the basolateral membrane. at variance, the green fluorescent protein gfp fed in vivo to the hemipteran lygus hesperus reached without degradation the haemocoel following apparently a paracellular pathway across the junctional complex (habibi et al., 2002). we investigated if mature columnar cells in culture were able to internalize these two proteins, by incubating the cells for 2 h in the presence of 1.4 μm fitc-albumin or 1.5 μm gfp. the fluorescent proteins taken up by columnar cells after 2 h of incubation were detected by confocal laser microscopy: while columnar cells internalized abundantly fitc-albumin (fig. 6a), gfp could not be detected inside the cells (fig. 6b). intracellular fitc-albumin was never diffused uniformly into the cytoplasm but was always localized in vesicular structures clearly visible as spots. 122 fig. 2 morphology of cultured midgut cells. brightfield images (acquired with confocal laser scanning microscope) of fixed cells: stem cell (a); stem cell in mitosis (b); differentiating cells (c, d); columnar (e) and goblet (g) cells in an early phase of differentiation; and fully differentiated (f, h). bars = 5 μm (a, b); 10 μm (c-h). discussion detailed information on the mechanisms involved in peptide and protein absorption by the insect gut can have a considerable impact on the development of new delivery strategies for orally administered insecticidal proteins targeting haemocoelic receptors. proteins are absorbed by the insect midgut in vivo (fishman et al., 1984; modespacher et al., 1986; zlotkin et al., 1992; benyakir and shochat, 1996; powell et al., 1998; habibi et al., 2002; kurahashi et al., 2005), and recent studies performed in vitro in b. mori larval midgut showed that two selected proteins crossed unaltered the intestinal barrier by transcytosis (casartelli et al., 2005, 2007). we considered that midgut columnar cells in primary culture could be a powerful tool to identify the different steps involved in this composite transcellular pathway. sadrud-din et al. (1994, 1996) obtained primary cultures of midgut cells from stem cells isolated from m. sexta larval epithelium, identifying the conditions in vitro in which the stem cells could survive, multiply and differentiate. stem cells from the insect midgut can be easily removed from the tissue because they are not linked to the other cells by junctions and their proliferation in vitro can be induced by ecdysone or 20-hydroxyecdysone (smagghe et al., 2005) and by fat body extracts or its derivates (hakim et al., 2007), while differentiation is stimulated by various factors produced by the mature and differentiating cells in the culture (loeb et al., 2004). following the same method, we isolated the stem cells from the midguts of pre-moult iv instar b. mori larvae and obtained their proliferation and differentiation in culture. we analysed the evolution of the culture by counting the total number of cells, testing their viability and determining the different cell types among the living cells every seven days for 6 weeks (fig. 3). the initial culture was almost exclusively composed of stem cells (87.4 ± 3.6 %, 3 determinations) but six days later all the cell types shown in figure 2 were already present. the progressive drop in stem cells observed in the following 21 days was compensated by the parallel increase in differentiating cells and in young and mature columnar and goblet cells. from the third week on, these three cell types represented about 30 % each of total living cells. the morphology of the different kind of cells shown in figure 2 is in large agreement with that reported by sadrud-din et al. (1996). it is worth noting that, following a drastic decrease after 27 days, stem cells fig. 3 percentage of the various viable cell types in culture in the different days since stem cells isolation. each bar represents the mean ± se of three different determinations. 123 fig. 4 brightfield (a) and confocal laser scanning micrographs (maximum projection) (b) of a typical cultured columnar cell in which is visible microtubules organisation. immunolocalisation of microtubules was performed with an anti-α-tubulin primary antibody visualised with an alexa fluor 488conjugated secondary antibody. bars =10 μm. number increases progressively in the subsequent two weeks, suggesting an enhanced production by the cell culture of specific proliferation factors, once reached a steady state. although our present report of the cell culture properties is referred only to six weeks (fig. 3), the culture maintains almost unaltered the characteristics shown here for up to three months (data not shown). the integrity of the cytoskeleton is fundamental for the maintenance of cell shape and polarity and for the correct localization of membrane proteins in the apical and basal domains of the cell surface. efficient targeting of vesicles loaded with the specific proteins, from the golgi apparatus to the correct membrane domain, requires an intact microtubule organization (reviewed by yeaman et al., 1999). it has been shown in enterocytes and other polarized epithelia that disruption of microtubule architecture by colchicine or nocodazole leads to a preferential alteration of the delivery to the apical rather than to the basolatelal membrane. in rat intestinal epithelium, the microtubules organizing center(s), identified with anti γ-tubulin antibodies, is/are located as a band close to the sub-apical space near the terminal web, from which the fast growing positive ends of microtubules grow towards the basolateral membrane, forming bundles of apical-basal filaments along the cell axis, while the negative ends are apically located (waschke and drenckhahn, 2000). in the subapical-space microtubules still run in parallel and only few are oriented obliquely or perpendicularly, but they never cross the terminal web (waschke and drenckhahn, 2000). microtubules distribution from the basal to the apical pole in b. mori midgut cells largely follows this scheme but, at variance with mammalian cells, in the insect they form a well developed felt just under the brush border (fig. 4b). this particular structure could be related to the lack in insect columnar intestinal cells of a terminal web organized as that of mammals (hull and staehelin, 1979; bonfanti et al, 1992; gibson and perrimon, 2003). as a matter of fact, in caco-2 cells monolayer, an intestinal model epithelium, in which a complete terminal web presumably does never fully differentiate, microtubules in the apical cytoplasm have a network-like arrangement (waschke and drenckhahn, 2000). the disposition of actin cytoskeleton in the mature cell shown in fig. 5b follows that classically described for polarized columnar cells (yeaman et al., 1999): it is highly organized within the apical microvilli and some filaments running from the basolateral membrane deep into the cytoplasm were also visible. well structured actin filaments were also observable in columnar cells in an earlier phase of differentiation (fig. 5a). as reported in the results, some of the differentiating cells were round shaped with microvilli uniformly distributed all over the surface (fig. 2c). sadrud-din et al. (1996) suggested that these cells corresponded to the initial phase of differentiation of columnar cells. the cell reported in fig. 5a, a representative of columnar cells in a more advanced stage, suggests that the differentiation of the basolateral membrane, at least from a morphological point of view, is a delayed process. the midgut epithelium performs in vivo the intermediate and final digestion of nutrients (terra and ferreira, 2005). two enzymes involved in this process highly abundant in the midgut epithelium are aminopeptidase n (apn) and alkaline phosphatase (alp). different isoforms of apn were characterized in the insect midgut (terra and ferreira, 2005), where they play a critical role in the final digestion of polypeptides by catalyzing the hydrolysis of amino acid residues at the n-terminal end. alkaline phosphatases, also present in the midgut epithelium as different isoforms (ferreira and fig. 5 brightfield and confocal laser scanning micrographs (maximum projection) of typical cultured columnar cells, in the initial phase of differentiation (a) and in the mature phase (b), in which is visible microfilaments organisation. actin filaments were visualised with tritc-phalloidin. bars = 10 μm. 124 fig. 6 brightfield and confocal laser scanning micrographs of a single optical section in a middle cell focal plane (where the nucleus was clearly evident) of cultured midgut columnar cells incubated for 2 h in the presence of 1.4 μm fitc-albumin (a) or 1.5 μm gfp (b). in the figure typical columnar cells are reported. bars = 10 μm. terra, 2005), are responsible for the removal of the phosphate groups from various phosphorylated substrates, an important step to allow the subsequent absorption of the molecule. both enzymes are expressed by columnar cells in culture, since their activity could be measured in the cell lysate. finally, we tested the ability to take up proteins from the medium by mature columnar cells. fitcalbumin, a protein actively absorbed by transcytosis by b. mori midgut epithelium in vitro (casartelli et al., 2005), was already detected inside the cell after 20 min (not shown) and was uniformly distributed as numerous spots after 2 h (fig. 6a). conversely, gfp, a protein that was supposed to reach the haemocel in the hemipteran lygus hesperus following the paracellular pathway (habibi et al., 2002), was not found inside columnar cells after 2 h (fig. 6b), intracellular traces of the protein becoming apparent only after 16 h (not shown). it is therefore feasible that the appearance of this protein in the haemocoel in vivo could be due to its passive leakage through the paracellular route rather than by an active process like transcytosis. in conclusion, b. mori columnar cells, differentiated from stem cells in vitro, maintained in culture the typical morphological features of the epithelium in vivo and were able to perform at least some of the normal absorptive and digestive functions. of particular interest for us was the ability of the cells to act selectively as regards the uptake of two model proteins. the very rapid internalization by the cell of fitc-albumin designates this protein as an excellent tool to assess the specific mechanisms involved in endocytosis and to finely characterize the multiple sequence of intracellular events involved in transcytosis. acknowledgements this work was supported by the italian ministry of education university and research (cofin 2004, project no. 2004077251; cofin 2006, project no. 20060794417). we are indebted to prof. rs hakim for his support and advise on the preparation of midgut cell cultures. we also thank dr r marotta e dr c di benedetto for their suggestions in the preparation of the histological samples. references azuma m, harvey wr, wieczorek h. stoichiometry of k+/h+ antiport helps to explain extracellular ph 11 in a model epithelium. febs lett. 361: 153-156, 1995. baldwin km, hakim rs. growth and differentiation of the larval midgut epithelium during molting in the moth manduca sexta. tissue cell 23: 411422, 1991. bonfanti p, colombo a, heintzelman mb, mooseker ms, camatini m. the molecular architecture of an insect midgut brush border cytoskeleton. eur. j. cell biol. 57: 298-307, 1992. ben-yakir d, shochat c. the fate of immunoglobulin g fed to larvae of ostrinia nubilalis. entomol. exp. appl. 81: 1-5, 1996. blackburn mb, loeb mj, clark e, jaffe h. stimulation of midgut stem cell proliferation by manduca sexta α-arylphorin. arch. insect biochem. physiol. 55: 26-32, 2004. bradford mm. a rapid sensitive method for the quantification of protein utilizing the principle of dye-protein binding. anal. biochem. 72: 248254, 1976. cappellozza l, cappellozza s, saviane a, sbrenna g. artificial diet rearing system for silkworm bombyx mori (lepidoptera bombycidae): effect of vitamin c deprivation on larval growth and cocoon production. appl. entomol. zool. 40: 405-412, 2005. casartelli m, corti p, leonardi mg, fiandra l, burlini n, pennacchio f, et al. absorption of albumin by the midgut of a lepidopteran larva. j. insect physiol. 51: 933-940, 2005. casartelli m, corti p, cermenati g, grimaldi a, fiandra l, santo n, et al. absorption of horseradish peroxidase in bombyx mori larval midgut. j. insect physiol. 53: 517-525, 2007. cioffi m. the morphology and fine structure of the larval midgut of a moth (manduca sexta) in relation to ion transport. tissue cell 11: 467469, 1979. ermak th, eakin rm. fine structure of the cerebral and pygidial ocelli in chone ecaudata (polychaeta: sabellidae). j. ultrastruct. res. 54: 243–260, 1976. fishman l, primor n, zlotkin e. the selective permeability of fleshfly midgut to an orally toxic cobra venom cardiotoxin. j. exp. biol. 108: 441-451, 1984. gibson mc, perrimon n. apicobasal polarization: epithelial form and function. curr. opinion cell biol. 15: 747-752, 2003. 125 goto s, takeda m, loeb mj, hakim rs. immunohistochemical detection of a putative insect cytochine, midgut differentiation factor 1 (mdf-1) in midgut columnar cells of heliothis virescens. invert. reprod. devel. 40: 117-124, 2001. giordana b, sacchi vf, hanozet gm. intestinal amino acid absorption in lepidopteran larvae. biochim. biophys. acta 692: 81-88, 1982. giordana b, leonardi mg, casartelli m, consonni p, parenti p. k+-neutral amino acid symport of bombyx mori larval midgut: a system operative in extreme conditions. am. j. physiol. 274: r1361-r1371, 1998. habibi j, brandt sl, coudron ta, wagner rm, wright mk, backus ea, huesing je. uptake, flow and digestion od casein and green fluorescent protein in the digestive system of lygus hesperus knight. arch. insect biochem. physiol. 50: 62-74, 2002. hakim rs, blackburn mb, corti p, gelman db, goodman c, elsen k, et al. growth and mitogenic effects of arylphorin in vivo and in vitro. arch. insect biochem. physiol.64, 63-73, 2007. hull be, staehelin la. the terminal web. a reevaluation of its structure and function. j. cell biol. 81: 67-82, 1979. kurahashi h, atiwetin p, nagaoka s, miyata s, kitajima s, sugimura y. absorption of mulberry root urease to the hemolymph of the silkworm bombyx mori. j. insect physiol. 51: 1055-1061, 2005. loeb mj, hakim rs. insect midgut epithelium in vitro: an insect stem cell system. j. insect physiol. 42: 1103-1111, 1996. loeb mj, jaffe h, gelman db, hakim rs. two polypeptide factors that promote differentiation of insect midgut stem cells in vitro. arch. insect biochem. physiol. 40: 129-140, 1999. loeb mj, jaffe h. peptides that elicit midgut stem cell differentiation isolated from chymotryptic digests of hemolymph from lymantria dispar pupae. arch. insect biochem. physiol. 50: 8596, 2002. loeb mj, coronel n, natsukawa d, takeda m. implications for the functions of the four known midgut differentiation factors: an immunohistologic study of heliothis virescens midgut. arch. insect biochem. physiol. 56: 720, 2004. modespacher up, rudin w, jenni l, hecker h. transport of peroxidase through the midgut epithelium of glossina m. morsitans (diptera, glossinidae). tissue cell. 18: 429-236, 1986. powell ks, spence j, bharathi m, gatehouse jh, gatehouse amr. immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper, nilaparvata lugens (stal). j. insect physiol. 44: 529-539, 1998. sadrud-din sy, hakim rs, loeb mj. proliferation and differentiation of midgut cells from manduca sexta, in vitro. invert. reprod. dev. 26: 197-204, 1994. sadrud-din sy, loeb mj, hakim rs. in vitro differentiation of isolated stem cells from the midgut of manduca sexta larvae. j. exp. biol. 199: 319-325, 1996. smagghe g, vanhassel w, moeremans c, de wilde d, goto s, loeb mj, et al. stimulation of midgut stem cell proliferation and differentiation by insect hormones and peptides. ann. n.y. acad. sci. 1040: 472-475, 2005. terra wr, ferreira c. biochemistry of digestion. in: gilbert li, iatrou k, gill ss (eds), comprehensive molecular insect science, oxford, elsevier, 4: 171-224, 2005. turbeck bo. a study of the concentrally laminated concretions, ‘spherites’, in the regenerative cells of the midgut of lepidopterous larvae. tissue cell 6: 627-640, 1974. waschke j, drenckhahn d. uniform apicobasal polarity of microtubules and apical location of γtubulin in polarized intestinal epithelium in situ. eur. j. cell biol. 79: 317-326, 2000. wieczorek h, weerth s, schindlbeck m, klein u. a vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut. j. biol. chem. 264: 11143-11148, 1989. yeaman c, grindstaff kk, nelson wj. new perspectives on mechanisms involved in generating epithelial cell polarity. physiol. rev. 79: 73-98, 1999. zlotkin e, fishman l, shapiro jp. oral toxicity to fleshflies of a neurotoxic polypeptide. arch. insect biochem. physiol. 21: 41-52, 1992. 126 acknowledgements isj106r.pdf isj 2: 132-141, 2005 issn 1824-307x review hyperglycemic stress response in crustacea s lorenzon brain center, department of biology, university of trieste, italy accepted september 27, 2005 abstract blood glucose level in crustaceans is controlled by the crustacean hyperglycemic hormone (chh), released from the eyestalk neuroendocrine centres both under physiological and environmental stress conditions. hyperglycemia is a typical response of many aquatic animals to pollutants and stress and, in crustaceans, increased circulating chh and hyperglycemia are reported to result from exposure to several environmental stressors. biogenic amines and enkephalin have been found to mediate the release of several neurohormones from crustacean neuroendocrine tissue and a model of the controlling network is proposed. key words: crustacea; glucose; crustacean hyperglycemic hormone (chh); stress response; neuroendocrine control introduction hyperglycemia is a typical response of many aquatic animals to harmful physical and chemical environmental changes. in crustaceans increased circulating crustacean hyperglycemic hormone (chh) titres and hyperglycemia are reported to occur following exposure to several environmental stressors (durand et al., 2000; lorenzon et al., 1997; 2002; santos et al., 2001) in intact but not in eyestalkless animals (fig.1), suggesting a chh mediated response (fingerman et al., 1981; reddy and bhagyalakshimi, 1994; reddy et al., 1996; lorenzon et al., 2000, 2004a). toxicity induced by a pollutant is the result of interaction of the compound or one of its metabolites, with the biochemical events involved in the homeostatic control of a physiological process (brouwer et al., 1990). physiological processes are mostly coordinated by hormones. anthropogenic chemicals can alter the hormonal (endocrine) systems of wildlife and the corresponding author: simonetta lorenzon brain center, department of biology, university of trieste, via giorgieri 7, i-34127 trieste, italy e-mail: lorenzon@units.it effects of organic and inorganic contaminants on functions regulated by hormones in crustaceans are being investigated with increased frequency because several of these phenomena could be used as biomarkers of environmental contamination. heavy metals and organic compounds have been found to negatively affect hormonally-regulated functions, such as reproduction, molting, blood glucose level and pigmentary effectors in crustaceans (fingerman et al., 1998; depledge and billinghurst, 1999). therefore, biosentinel parameters and “early warning” of toxicity can be identified by looking for alterations in endocrine patterns (fingerman et al., 1996). neurosecretory structures in the eyestalk are the most important components of the neuroendocrine system of the stalk-eyed crustaceans. the hemolymph glucose concentration is mainly controlled by the chh synthesized within the x-organ (xo) and released from the sinus gland (sg) complex in the eyestalk (abramowitz et al., 1944; fingerman, 1987). biogenic amines and enkephalin (l/m-enk) control the release of neurohormones from the crustacean neuroendocrine tissue. serotonin (5-ht) is involved in regulating important aspects of behaviour and a variety of systemic physiological functions. 5-ht has long been known (bauchau and mengeot, 1966) to have a potent hyperglycemic effect in several crustacean species (lorenzon et al., 1999, 2004b; lee et al., 132 2000; komali et al., 2005;), while dopamine (da) and enkephalin showed conflicting results in different species (sarojini et al., 1995; lorenzon et al., 1999, 2004b; zou et al., 2003; komali et al., 2005). the crustacean hyperglycemic hormone (chh) multiple forms of the chh represent one member of an eyestalk neuropeptide family (bocking et al, 2001), that includes the moult inhibiting hormone (mih) and the gonad inhibiting hormone (gih): the chh/mih/gih family. these neuropeptides, synthesized in the xo, a cluster of neuron perikarya located in the medulla terminalis of the eyestalk, are transported to and stored in the axon terminals forming a neurohemal organ named sg and released by exocytosis into the hemolymph (fig. 2). the main function of chh is the regulation of hemolymph sugar level: chhs are also involved in other functions such as reproduction (de kleijn et al., 1998; de kleijn and van herp, 1998), molting (chung et al., 1999; webster et al., 2000), lipid metabolism (santos et al., 1997), stress response (lorenzon et al., 1997; 2002;chang et al., 1999; durand et al., 2000; santos et al., 2001) and hydromineral regulation (spanings-pierrot et al., 2000; serrano et al., 2003). on the basis of the primary structure, the chh/mih/gih family can be divided into two subfamilies (de kleijn et al., 1995; lacombe et al., 1999): the chh sub-family characterized by the chh precursor-related peptide (cprp) and the mih/gih sub-family without cprp. the prepropeptide chh consists of a signal peptide, cprp and a peptide with 72–74 amino acids. usually, the mature peptide has an amidated carboxyl terminus (de kleijn and van herp, 1998; lacombe et al., 1999), which is important in conferring hyperglycemic activity in penaeus japonicus as evidenced by bioassay of recombinant peptide (katayama et al., 2003). in several crustacean species, different isoforms of chh exist. in the american lobster homarus americanus, chh-a (8.583 da) and chh-b (8.638 da) have been found, with different actions during the female biannual reproductive cycle (de kleijn et al., 1995). role of biogenic amines and enkephalin in blood glucose regulation neurotransmitters such as 5-ht, da and l/m-enk play a fundamental role in hormone modulation (fingerman et al., 1994) and at the same time their level and functions can be altered by pollutants (amiard-triquet et al., 1986, reddy et al., 1997). 5-ht is well known as a neurotransmitter in crustaceans on several grounds, and its levels have been measured in the nervous system and hemolymph of various crustacean species (elofsson et al., 1982; laxmyr, 1984; kulkarni and fingerman 1992), thus suggesting a possible role as a neurohormone (rodriguez-soza et al., 1997). in crustaceans 5ht is linked with discrete circuits that control movements of the foregut, escape behaviour, locomotion and posture as well as with higher-order behaviours such as aggression (sosa et al., 2004). in addition 5-ht levels are sensitive to environmental stress. 5-ht has long been known to have a potent hyperglycemic effect in several crustacean species (bauchau and mengeot, 1966; keller and beyer 1968; lüschen et al., 1993; kuo et al., 1995; santos et al., 2001). in our laboratory (lorenzon et al., 1999, 2004b) we have demonstrated that 5-ht elevates blood glucose in palaemon elegans, astacus leptodactylus and squilla mantis. however no such effects were found in eyestalkless individuals of these species, suggesting the involvement of the eyestalk hormone chh in the hyperglycemic response. in all the species injection of the antagonist, ketanserin and cph (cyproheptadine, 5-ht1 receptor inhibitor) were able to inhibit the hyperglycemic effect of 5-ht. 5-ht1 like receptors seemed to be more likely involved in mediating 5-ht action, as cph was a more effective antagonist than ketanserin (5-ht2 receptor inhibitor and also putative da antagonist). these data agree with those by lee et al. (2000) in procambarus clarkii suggesting that 5-ht induced hyperglycemia is mediated by 5-ht1 and 5-ht2 like receptors. using elisa very recently we have demonstrated in p. elegans that injection of 5-ht induced a rapid and massive release of chh from the eyestalk into the hemolymph followed by hyperglycemia. on the contrary da did not significantly affect chh release and hyperglycemia (lorenzon et al. 2005). da and enkephalins showed conflicting results in different species (table 1). injection of da induced marked decrease in blood glucose levels in p. elegans and s. mantis (lorenzon et al., 1999, 2004b). on the other hand injection of the da receptor blocker inhibits the effects on blood glucose, apparently allowing the release of chh. these findings are in contrast with those by lüschen et al., (1993) for carcinus maenas, kuo et al. (1995) for penaeus monodon and komali et al. (2005) for macrobrachium malcolmsonii where da induced hyperglycemia in intact animals. as for enkephalins, l/m-enk elicited hypoglycemic response in intact s. mantis but not in eyestalkless individuals (lorenzon et al. 2004a). these results confirm those of jaros (1990), lüschen et al. (1991), rothe et al. (1991) and sarojini et al. (1995) who reported that l/m-enk induced hypoglycemia in uca pugilator, c. maenas and p. clarkii respectively. on the other hand l-enk induced hyperglycemic response in intact but not in eyestalkless a. leptodactylus (lorenzon et al. 2004). these observations are consistent with our previous findings in p. elegans (lorenzon et al., 1999) and also with recent reports on oziotelphusa senex senex (reddy and basha, 2001), on the mud crab scylla serrata (reddy and kishori, 2001) and in the two prawns, penaeus indicus and metapenaeus monocerus (kishori et al., 2001). in s. mantis injection of the opioid antagonist naloxone reversed the inhibitory effect on blood glucose of l-enk while in a. leptodactylus an additive effect on hyperglycemia was recorded (lorenzon et al., 2004b). all these results corroborate the commonly held view that 5-ht, is a potent hyperglycemic effector and exerts its effect through chh release from the 133 fig.1 stress response in crustacea. medulla terminalis xo-sinus gland complex (mtxosg), mediated by modulation of electrical activity of xo cells (saenz et al., 1997). a detailed reconstruction of the underlying neural circuitry suffers from lack of precise identification of neurosecretory cell types, contrasting results of electrophysiological evidence and discrepancies due to interspecific differences (glowik et al., 1997; saenz et al., 1997). finally 5-ht appears to provide a major control mechanism for glucose mobilization whereas da and l/m-enk act as modulators whose plasticity in use or actions varied among even closely related species. stress response stress induced by changes in environmental parameters, emersion, handling and transport during commercial processes requires homeostatic regulation that brings about behavioural and physiological alterations in aquatic animals. hemolymph glucose concentration can change significantly with altered physiological and environmental conditions. exposure to air during commercial transport and hypoxia are reported to induce hyperglycemia in many crustacean species like the spiny lobster, jasus edwardsii (morris and oliver 1999; speed et al., 2001), the crab, eriocheir sinensis (zou et al., 1996), the spider crab, maia squinado (durand et al., 2000) and the norway lobster, nephrops norvegicus (spicer et al., 1990). moreover hyperglycemia is reported in the giant prawn, macrobrachium rosenbergii as a response to cold shock (kuo and yang, 1999). blood glucose level increased in p. elegans and other crustacean species after injection of lipopolysaccharide (lps) and the hyperglycemic effect, is likely mediated by the chh since it does not occur in eyestalkless animals. it is dose-related and dependent on the different gram negative bacterial lps (lorenzon et al., 1997, 2002). heavy metals like cd, hg, and cu cause hyperglycemia in the freshwater prawn, macrobrachium kistenensis, the crab, barytelphusa canicularis (nagabhushanam and kulkarni, 1981, machele et al., 1989) and s. serrata (reddy and bhagyalakshmi, 1994). moreover, cdcl2 induces hyperglycemia in intact crayfish p. clarkii, but not in the absence of the eyestalks, suggesting a chh mediated response (reddy et al., 1996). our studies (lorenzon et al., 2000) on the effect of heavy metals on blood glucose levels in p. elegans showed that the intermediate sublethal concentrations of hg, cd and pb produced significant hyperglycemic responses while the highest concentrations elicited no hyperglycemia in 24 h. in contrast, animals exposed to cu and zn showed hyperglycemia even at high concentrations. this difference in response could be explained on the basis of the physiological roles these two essential 134 fig. 2 general organization of neuroendocrine tissues in the eyestalk of crustaceans. elements play in crustaceans, and consequent tolerance adaptations, as opposed to the toxic, xenobiotic heavy metals cd, hg and pb. on the other hand both groups of heavy metals failed to elicit a hyperglycemic responses in eyestalk ablated animals suggesting the involvement of mtxo-sg hormones, most likely chh. however, in spite of the richness of information regarding variations in blood glucose levels following stress, much less is known about the stress-induced variation in chh levels in the sinus gland and in the hemolymph. in the crayfish orconectes limosus subjected to hypoxia, blood chh titers raise within 15 min (keller and orth, 1990). in cancer pagurus emersion induced an increase in the hemolymph chh after 4 h (webster, 1996). using elisa chang et al. (1998) observed variation in the blood chh in homarus americanus following exposure to various environmental stresses. emersion was found to be a potent stimulator of blood chh while temperature and salinity variations were less effective. moreover an increase in water temperature increased blood chh in c. pagurus and p. clarckii (wilcockson et al. 2002; zou et al., 2003). in c. maenas it has been shown that the concentration of the chh in the hemolymph increases dramatically during molting from 1-5 fmol 100µl-1 in the intermolt up to 150-200 fmol 100µl-1 during ecdysis (chung et al., 1999). variation in the hemolymph chh titer were also observed in n. norvegicus infected by the parasitic dinoflagellate hematodinium sp. (stentiford et al., 2001). using elisa and bioassay tests we have recently demonstrated the relationship between an environmental stressor and the release of chh from the eyestalk into the hemolymph and the hyperglycemic response in the shrimp, p. elegans (lorenzon et al., 2004a). moreover with this work we validated the use of a cross reactive antibody, antinenchh, to assess chh level in the eyestalk and hemolymph of p. elegans. with the help of standard immunocytochemistry the antibody had previously been tested for recognition of chh in the eyestalks of different species belonging to systematic groups increasingly remote in the phylogenetic tree: the decapods a. leptodactylus, n. norvegicus, p. elegans, munida rugosa and the stomatopod s. mantis (giulianini et al., 2002). finally we have quantified the variations in the hemolymph chh after a challenge with different stressors. in p. elegans exposure to copper induced a dose-related rapid and massive release of chh from the eyestalk into the hemolymph at the higher, lethal concentration while a gradual and reduced discharge was observed at the lower concentration (fig. 3). the relationship between exposure to a toxicant and release of the chh was confirmed by variation in blood glucose with a dose related hyperglycemia that peaked 2 h after exposure to copper (fig. 4). animals exposed to sublethal concentrations of hg showed similar quantitative and time course relations between toxicant, release of chh from the eyestalk, increment of hormone level in the hemolymph and subsequent hyperglycemia as already described for copper contamination. interestingly, however, the highest, lethal concentration induced the release of chh from the eyestalk into the hemolymph but was not followed by a significant variation in blood glucose (figs 5, 6). this situation could be related to the high toxicity of hg which may interfere with the finer mechanisms that regulate hyperglycemic response. it is neither due to synaptic blockage of the superimposed neuronal release network (lorenzon et al., 1999) nor limited release of circulating chh as high levels of chh are discharged from the sg into the hemolymph. it is not due to inhibition of peripheral receptors on glycogenolytic target organs: indeed native sg homogenate injected into eyestalkless shrimps exposed to lethal concentration of hg for 3 h is still able to cause hyperglycemia (lorenzon et al., 2000). high concentrations of hg, instead, may change the functionality of the prepro-chh processed during secretory steps and due to its ability to bind cysteines six of which represent a highly conserved feature of the peptide structure (lacombe et al., 1999) hg might alter the active configuration of the peptide, as seen in other systems (rodgers et al., 2001), but not its immunoreactivity. moreover hg is known to impair osmoregulatory mechanisms in the crab, eriocheir sinensis (péqueux et al., 1996); and inhibit acetylcholinesterase activity in p. clarkii (devi and fingerman, 1995). the altered response in p. elegans exposed to high concentrations of hg may also be related to physiological modifications induced by hg at a different systemic level (lorenzon et al., 2004a). cu contamination induced variations of 5-ht of the eyestalk and hemolymph of p. elegans (lorenzon et al., 2005). the release of 5-ht from the eyestalk appears to be very rapid and dose dependent. in the hemolymph 5-ht peak occurs after 30 min and again the concentration of circulating 5ht is dose dependent. after 1 h the level of 5-ht slowly decreases to the basal level (fig. 7). 135 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 a ch h ( p m o l s g e -1 ) time (h) cu ++ 0.1 mg l -1 cu ++ 5 mg l -1 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 b ch h ( p m o l m l -1 ) time (h) cu ++ 0.1 mgl -1 cu ++ 5 mgl -1 fig. 3 time course of chh in the eyestalk homogenates (a) and in the hemolymph (b) of p. elegans after exposure to different concentrations of cu++ and in relation to untreated controls. values are expressed as means ± sd (n=4 repeated measures). 0,5 1 2 3 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 3,0 3,2 3,4 in cr e m e n t o f g ly ce m ia time (h) cu ++ 0.1 mg l -1 cu ++ 5 mg l -1 control fig. 4 time course of glycemia in the hemolymph of p. elegans after exposure to different concentrations of cu++ and in relation to untreated controls. values of increment given as: [(experimental value)/ (value displayed by the same animal at 0 h)]–1, are expressed means ± sd (n=10 repeated measures). the release of 5-ht from the eyestalk into the hemolymph after cu exposure precedes in its time course the release of chh, confirming its role as neurotransmitter acting on chh neuroendocrine cells. the rapid and massive release of 5-ht from the eyestalk of individual species following exposure to cu might have induced release of the chh resulting in hyperglycemia in intact but not in eyestalkless animals. lastly contamination with different doses of lps, a bacterial thermostable endotoxin from e. coli, confirms the dose-related and convergent chain of events that leads to hyperglycemia. this suggests that blood glucose elevation is a general-purpose response to stressors and is likely to perform a protective role (lorenzon et al., 2004a). conclusion in spite of the vastness of information on hyperglcemic stress response in crustacea, there still exist many questions. in the scheme presented in figure 8 a possible model of the controlling network is proposed. stressors have been demonstrated to release the chh and 5-ht from the eyestalk leading to an increase in their hemolymph concentrations. 5-ht exerts a positive influence inducing the release of chh from the sg into the blood. the chh then acts upon the target organs to release more than normal level glucose resulting in hyperglycemia. the da 136 0,5 1 2 3 control 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 a ch h ( pm ol s g -1 ) time (h) hg ++ 0.1 mg l -1 hg ++ 0.5 mg l -1 hg ++ 5 mg l -1 control 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 btime (h) ch h ( p m o l m l -1 ) hg ++ 0.1 mg l -1 hg ++ 0.5 mg l -1 hg ++ 5 mg l -1 control fig. 5 time course of chh in the eyestalk homogenates (a) and in the hemolymph (b) of p. elegans after exposure to three different concentrations of hg++ and in relation to untreated control. values are expressed means ± sd (n=4 repeated measures). 0,5 1 2 3 5 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 in cr e m e n t o f g ly ce m ia time (h) hg ++ 0.1 mg l -1 hg ++ 0.5 mg l -1 hg ++ 5 mg l -1 control fig. 6 time course of glycemia in the hemolymph of p. elegans after exposure to three different concentrations of hg++ and in relation to untreated controls. values of increment given as: [(experimental value)/ (value displayed by the same animal at 0 h)]–1, are expressed means ± sd (n=10 repeated measures). receptor blocker, spiperone, inhibited the hypoglycemic action of da and was found not to affect the ability of l/m-enk to produce hypoglycemia. on the other hand, naloxone blocked the action of both l/m-enk and da, thereby allowing the release of chh (sarojini et al., 1995, lorenzon et al., 1999). apparently da and l-enk produced hypoglycemia by inhibiting chh release. these results suggest that in the chain of neurons terminating at the neuroendocrine cells that secrete chh, dopaminergic neurons precede enkephalinergic neurons. we also suggest a role for the hemocytes in the hyperglycemic stress response as stressors affect both the total (thc) and the differential haemocyte count and that exocytosis of chh granules from the eyestalk neuroendocrine cells can be elicited either by an early release from hemocytes of cytokines and/or other circulating messengers like 5-ht. moreover lps treated eyestalkless animals undergo less haemocytopenia than intact individuals. this suggests that previous chh release and hyperglycemia can cause a decrease in thc, which eventually exerts a protective function (lorenzon et al., 2002). in summary it may be said that indicators of stress responses are useful in assessing the shortterm well-being or long-term health status of an animal (fossi et al., 1997; paterson and spanoghe, 1997) and, such indicators have received considerable attention in commercially important species of decapod crustaceans (paterson 137 0 0,5 1 2 3 control 0 5 10 15 20 25 30 35 40 a hemolymph cu ++ 5 mg l -1 cu ++ 0.1 mg l -1 time(h) 5 -h t ( n g m l -1 ) 0 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 b eyestalk 5 -h t ( n g m l -1 ) time (h) cu ++ 5 mg l -1 cu ++ 0.1 mg l -1 fig. 7 time course (0.5-3 h) of 5-ht in the hemolymph (a) and in the eyestalk (b) of p. elegans after exposure to different concentrations of cu++ and in relation to untreated controls. values are expressed means ± sd (n=4 repeated measures). fig. 8 possible model of hyperglycemic stress response controlling network. in the scheme: continuous arrow=demonstrated effect, dotted arrow= hypothesized effect, red arrow= stimulation, blue arrow= inhibition, green arrow=release. 138 and spanoghe, 1997; chang et al., 1999). a number of researchers have suggested different methods for quantifying the stress responses in crustaceans; which include the measurement of different hemocyte types in the hemolymph (jussila et al., 1997 lorenzon et al., 1999, 2001), and the physiological, biochemical (paterson and spanoghe, 1997; stentiford et al.. 1999), and molecular changes in the tissues and the hemolymph (fossi et al., 1997). thus variations in the hemolymph glucose concentration in the hemolymph and of the chh level in relation to stressors could be used as a tool to monitor a variety of stress responses. acknowledgements the author is grateful to prof. ea ferrero and dr. pg giulianini for useful discussion and comments on the manuscript. the constructive comments of anonymous referees are kindly acknowledged. this work was supported by grants n° 4c186 and 6d4 from the italian mipaf to eaf, and by grant from murst “giovani ricercatori” project to sl. this work was also part of the phd research project of sl. references abramowitz a, hisaw fl, papandrea dn. the occurrence of a diabetogenic factor in the eyestalk of crustaceans. biol. bull. 86: 1-5, 1944. amiard-triquet c, amiard jc, ferrand r, andersen ac, dubuois mp. disturbance of a met-enkephaline-like hormone in the hepatopancreas of crab contaminated by metals. ecotox. envir. saf. 11: 198-209, 1986. bauchau ag, mengeot jc. sérotonine et glycémie chez les crustacés. experientia 22: 238-239, 1966. bocking d, dirkensen h, keller r. the crustacean neuropeptides of the chh/mih/gih family: structures and biological activities. in: korand w (ed), the crustacean nervous system, springer, berlin, germany, pp 84-97, 2001. brouwer a, murk aj, koeman jh. biochemical and physiological approaches in ecotoxicology. funct. ecol. 4: 275-281, 1990. chang es, keller r, chang sa. quantification of crustacean hyperglycemic hormone by elisa in hemolymph of the lobster, homarus americanus, following various stress. gen. comp. endocrinol. 111: 359-366, 1998. chang es, ghang sa, beltz bs, kravitz ea. crustacean hyperglycemic hormone in the lobster nervous system: localization and release from cells in the subesophageal ganglion and thoracic second roots. j. comp. neurol. 414: 50-56, 1999. chung js, dircksen h, webster sga. remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab carcinus maenas. proc. natl. accad. sci. usa 96: 13103-13107, 1999. depledge mh, billinghurst z. ecological significance of endocrine disruption in marine invertebrate. mar. poll. bull. 39: 32-38, 1999. de kleijn dpv, van herp f. involvement of the hyperglycemic neurohormone family in the control of reproduction in decapod crustaceans. invert. reprod. dev. 33: 263–72, 1998. de kleijn dpv, de leeuw eph, van den berg mc, martens gjm, van herp f. cloning and expression of two mrnas encoding structurally different crustacean hyperglycemic hormone precursors in the lobster homarus americanus. biochem. biophys. acta 1260: 62–66, 1995. de kleijn dpv, janssen kp, waddy sl, hegeman r, lai wy, martens gj, et al. expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female american lobster homarus americanus. j. endocrinol. 156: 291–298, 1998. devi m, fingerman m. inhibition of acetylcholinesterase activity in the central nervous system of the red swamp crayfish procambarus clarkii, by mercury, cadmium and lead. bull. environ. contam. toxicol. 55: 746-750, 1995. durand f, devillers n, lallier fh, regnault m. nitrogen excretion and change in blood components during emersion of the subtidal spider crab maia squinado (l.). comp. biochem. physiol. 127a: 259-271, 2000. elofsson r, laxmyr l, rosengren e, hanson c. identification and quantitative measurements of biogenic amines and dopa in the central neurons and hemolymph of the crayfish pacifastacus leniusculus (crustacea). comp. biochem. physiol. 71: 201-205, 1982. fingerman m, hanumante mm, deshpande ud, nagabhushanam r. increase in the total reducing substances in the hemolymph of the freshwater crab, barytelphusa guerini, produced by a pesticide (ddt) and an indolealkylamide (serotonin). experientia 37: 178-189, 1981. fingerman m. the endocrine mechanisms of crustaceans. j. crust. biol. 7: 1-24, 1987. fingerman m, nagabhushanam r, sarojini r, reddy ps. biogenic amines in crustaceans: identification, localisation and roles. j. crust. biol. 14: 413-437, 1994. fingerman m, devi m, reddy ps, katayayani r. impact of heavy metal exposure on the nervous system and endocrine-mediate process in crustaceans. zool. stud. 35: 1-8, 1996. fingerman m, jackson nc, nagabhushanam r. hormonally-regulated functions in crustaceans as biomarkers of environmental pollution. comp. biochem. physiol. 120c: 343-350, 1998. fossi mc, savelli c, casini s, franchi e, mattei n, corsi, i. multi-response biomarker approach in the crab carcinus aestuarii experimentally exposed to benzo(a)pyrene, polychlorobiphenyls and methyl-mercury. biomarkers 2: 311-319, 1997. giulianini pg, pandolfelli n, lorenzon s, ferrero ea, edomi p. an antibody to recombinant crustacean hyperglycaemic hormone of nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan crustacea. cell tissue res. 307: 243254, 2002. glowik rm, golowasch j, keller r, marder e. d-glucosesensitive neurosecretory cells of the crab cancer borealis and negative feedback regulation of the blood glucose level. j. exp. biol. 200: 1421-1431, 1997. jaros pp. enkephalins, biologically active neuropeptides in invertebrates, with special reference to crustaceans. in: wiese k, krenz wd, tautz j, reichert h, jussila j, jago j, tsuetnenko e, dunstan b, evans, lh (eds), total and differential haemocyte counts in western rock lobsters (panulirus cygnus george) under post-harvest stress. mar. freshw. res. 48: 863-867, 1997. katayama h, nagata k, ohira t, yumoto f, tanokura m, nagasawa h. the solution structure of molt-inhibiting hormone from the kuruma prawn marsupenaeus japonicus. j. biol. chem. 278: 9620-9623, 2003. keller r, beyer j. zur hyperglykämischen wirkung von serotonin und augenstielextrakt beim flusskrebs orconectes limosus. z. vgl. physiol. 59: 78-85, 1968. keller r, orth, h. hyperglycemic neuropeptides in crustaceans. in epple a, scanes c, stetson m. (eds) progress in comparative endocrinology, wiley liss, new york, pp. 265-271, 1990 kishori b, premasheela b, ramamurthi r, reddy s. evidence for hyperglycemic effect of methionineenkephalin in the prawns penaeus indicus and 139 metapenaeus monocerus. gen. comp. endocrinol. 123: 90-99, 2001. komali m, kalarani v, venkatrayulu ch, reddy dcs. hyperglycemic effects of 5-hydroxytryptamine and dopamine in the freshwater prawn macrobrachium malcolmsonii. j. exp. zool. 303: 448-455, 2005. kulkarni gk, fingerman m. qualitative analysis by reverse phase high performance liquid chromatography of 5hydroxytryptamine in the central nervous system of the swamp crayfish procambarus clarkii. biol. bull. 182: 341347, 1992. kuo c, hsu c, lin c. hyperglycemic effects of dopamine in the tiger shrimp, penaeus monodon. aquaculture 135: 161-172, 1995. kuo cm, yang yh. hyperglycemic responses to cold shock in the freshwater giant prawn, macrobrachium rosenbergii. j. comp. physiol. 169b: 49-54, 1999. lacombe c, greve p, martin g. overview on the subgrouping of the crustacean hyperglycemic hormone family. neuropeptides 33: 71-80, 1999. laxmyr l. biogenic amines and dopa in the central nervous system of decapod crustaceans. comp. biochem. physiol. 77c: 139-143, 1984. lee c, yau s, liau c, huang w. serotoninergic regulation of blood glucose levels in the crayfish, procambarus clarkii: site of action and receptor characterization. j. exp. zool. 286: 596-605, 2000. lee c, yang p, zou h. serotoninergic regulation of crustacean hyperglycemic hormone secretion in the crayfish, procambarus clarkii. physiol. biochem. zool. 74: 376-382, 2001. lorenzon s, giulianini pg, ferrero ea. lipopolysaccharideinduced hyperglycemia is mediated by chh release in crustaceans. gen. comp. endocrinol. 108: 395-405, 1997. lorenzon s, pasqual p, ferrero ea. biogenic amines control blood glucose level in the shrimp palaemon elegans. in f.b. schram (ed), the biodiversity crisis and crustacea crustacean issues 12, balkema rotterdam, pp 4714801999. lorenzon s, francese m, ferrero ea. heavy metal toxicity and differential effects on the hyperglycemic stress response in the shrimp palaemon elegans. arch. environ. contam. toxicol. 39: 167-176, 2000. lorenzon s, francese m, smith vj, ferrero ea, heavy metal affect the circulating haemocyte number in the shrimp palaemon elegans. fish shellfish immunol. 11: 459-472, 2001. lorenzon s, pasqual p, ferrero ea. different bacterial lipolysaccharides as toxicants and stressors in the shrimp palaemon elegans. fish shellfish immunol. 13: 27-45, 2002. lorenzon s, edomi p, giulianini pg, mettulio r, ferrero ea. variation of crustacean hyperglycemic hormone (chh) level in the eyestalk and hemolymph of the shrimp palaemon elegans following stress. j. exp. biol. 207: 4205-4213, 2004a. lorenzon s, brezovec s, ferrero ea. species-specific effects on hemolymph glucose control by serotonin, dopamine and l-enkephalin and their inhibitors in squilla mantis and astacus leptodactylus (crustacea). j. exp. zool. 301: 727-736, 2004b. lorenzon s, edomi p, giulianini pg, mettulio r, ferrero ea. role of biogenic amines and chh in the crustacean hyperglycemic stress response. j. exp. biol., 2005 (in press). lüschen w, buck f, willig a, jaros pp. isolation, sequence analysis and physiological properties of enkephalins in the nervous tissue of the shore crab carcinus maenas (l.) proc. natl. accad. sci. usa 88: 8671-8675, 1991. lüschen w, willig a, jaros pp. the role of biogenic amines in the control of blood glucose level in the decapod crustacean, carcinus maenas. comp. biochem. physiol. 105: 291-296, 1993. machele pr, khan ak, sarojini r, nagabhushanam r. copper and cadmium induces changes in blood sugar level of crab, barytelphusa canicularis. uttar pradesh j. zool. 9: 113-115, 1989. morris s, olivier s. circulatory, respiratory and metabolic response to emersion and low temperature of jasus edwardsii: simulation studies of commercial shipping methods. comp. biochem. physiol. 122: 299-308, 1999. nagabhushanam r, kulkarni gk. freshwater palaemonid prawn, macrobrachium kistenensis (tiwari)-effect of heavy metal pollutants. proc. indian natl. sci. acad. b 47: 380-386, 1981. ollivaux, c., dircksen h, toullec j, soyez d. enkephalinergic control of the secretory activity of neurons producing stereoisomers of crustacean hyperglycemic hormone in the eyestalk of the crayfish orconectes limosus. j. comp. neurol. 444: 1-9, 2002. paterson bd, spanoghe pt. stress indicators in marine decapod crustaceans, with particular reference to the grading of western rock lobsters (panulirus cygnus) during commercial handling. mar. freshw. res. 48: 829–834, 1997. péqueux a, bianchini a, gilles r. mercury and osmoregulation in the euryhaline crab, eriocheris sinensis. comp. biochem. physiol. 111c: 149-155, 1996. reddy ps. a neurotransmitter role of methionine-enkephalin in causing hyperglycemia in the fresh water crab oziothelphusa senex senex. curr. sci. 76: 1126-1128, 1999. reddy ps, basha mr. on the mode of action of methionine-enkephalin, fk33-824 and naloxone in regulating the hemolymph glucose level in the freshwater field crab oziotelphusa senex senex. z. naturforsch. 56: 629-632, 2001. reddy ps, bhagyalakshmi a. change in oxidative metabolism in selected tissues of the crab scylla serrata in response to cadmium toxicity. ecotoxicol. environ. saf. 29: 255-264, 1994. reddy ps, katayayani rv, fingerman m. cadmium and naphthalene induced hyperglycemia in the fiddler crab uca pugilator: differential modes of action on the neuroendocrine system. bull. environ. contam. toxicol. 56: 425-431, 1996. reddy ps, kishori b. methionine-enkephalin induces hyperglycemia through eyestalk hormone in the estuarine crab scylla serrata. biol. bull. 201: 17-25, 2001. reddy ps, tuberty sr, fingerman m. effects of cadmium and mercury on ovarian maturation in the red swamp crayfish, procambarus clarkii. ecotoxicol environ saf. 37: 62-65, 1997. rodgers js, hocker jr, hanas rj, nwosu ec, hanas js. mercuric ion inhibition of eukaryotic transcription factor binding to dna. biochem. pharmacol. 61: 1543-1550, 2001. rodriguez-soza l, picones a, rosete gc, islas s, aréchiga h. localization and release of 5-hydroxytryptamine in the crayfish eyestalk. j. exp. biol. 200: 3067-3077, 1997. rothe h, lüschen w, asken a, willig a, jaros pp. purified crustacean enkephalin inhibits release of hyperglycemic hormone in the crab carcinus maenas. comp. biochem. physiol. 99c: 57-62, 1991. saenz f, garcia u, aréchiga h. modulation of electrical activity by 5-hydroxytryptamine in crayfish neurosecretory cells. j. exp. biol. 200: 3079-3090, 1997. santos ea, keller r, rodriguez e, lopez l. effects of serotonin and fluoxetine on blood glucose regulation in two decapod species. braz. j. med. biol. res. 34: 7580, 2001. santos ea, nery le, keller r, goncalves aa. evidence for the involvement of the crustacean hyperglycemic 140 hormone in the regulation of the lipid metabolism. physiol. zool. 70: 415–420,1997. sarojini r, nagabhushanam r, fingerman m. dopaminergic and enkephalinergic involvement in the regulation of blood glucose in the red swamp crayfish procambarus clarkii. gen. comp. endocrinol. 97: 160-170, 1995. serrano l, blanvillain g, soyez d, charmantier g, grousset e, aujoulat f, et al., putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish astacus leptodactylus. j. exp. biol. 206: 979-988, 2003. sosa ma, spitzer n, edwards dh, baro dj. a crustacean serotonin receptor: cloning and distribution in the thoracic ganglia of crayfish and freshwater prawn. j. comp. neurol. 473: 526-537, 2004 soyez d, laverdure am, kallen j, van herp f. demonstration of a cell-specific isomerization of invertebrate neuropeptides. neuroscience 82: 935942,1998 spanings-pierrot c, soyez d, van herp f, gompel m, grousset e, charmantier g. involvement of crustacean hyperglycemic hormone in the control of gill ion transport in the crab pachygrapsus marmoratus. gen. comp. endocrinol. 119: 340-350, 2000. speed sr; baldwin j, wong rj wells rmg. metabolic characteristic of muscles in the spiny lobster, jasus edwardsii, and responses to emersion during simulated live transport. comp. biochem. physiol. 128b: 435-444, 2001. spicer ji, hill ad, taylor ac, strang rhc. effect of aerial exposure on concentrations of selected metabolites in the blood of the norwegian lobster nephrops norvegicus (crustacea: nephropidae). mar. biol. 105: 129-135, 1990. stentiford gd, neil dm, coombs gh. changes in the plasma free amino acid profile of the norway lobster, nephrops norvegicus, at different stages of infection by a parasitic dinoflagellates (genus hematodinium) dis. aquat. organ. 38: 151–157, 1999. stentiford gd, chang es, chang sa, neil dm. carbohydrate dynamics and the crustacean hyperglycemic hormone (chh): effects of parasitic infection in norway lobsters (nephrops norvegicus). gen. comp. endocrinol. 121: 13-22, 2001. webster sg, dircksen h, chung js. endocrine cells in the gut of the shore crab carcinus maenas immunoreactive to crustacean hyperglycemic hormone and its precursor-related peptide. cell tissue res. 300: 193205, 2000. webster sg. measurement of crustacean hyperglycaemic hormone levels in the edible crab cancer pagurus during emersion stress. j. exp. biol. 199: 1579-1585, 1996. wilcockson dc, chung js, webster sg. is crustacean hyperglycemic hormone precursor-related peptide a circulating neurohormone in crabs? cell. tissue res. 307:129-38, 2002. zou h, juan c, chen s, wang h, lee c. dopaminergic regulation of crustacean hyperglycemic hormone and glucose levels in the hemolymph of the crayfish procambarus clarkii. j. exp. zool. 298: 44-52, 2003. zou e, du n, lai w. the effects of severe hypoxia on lactate and glucose concentrations in the blood of the chinese freshwater crab eriocheir sinensis (crustacea: decapoda). comp. biochem. physiol 114a: 105-109, 1996. 141 visions and perspectives isj 9: 89-92, 2012 issn 1824-307x visions and perspectives contribution of invertebrate models to aging and longevity studies e ottaviani1, c franceschi 2, 3 1 department of biology, university of modena and reggio emilia, modena, italy 2 department of experimental pathology, university of bologna, bologna, italy 3 cig-interdepartmental center "l galvani", university of bologna, bologna, italy accepted may 28, 2012 abstract this paper summarizes pros and cons of the invertebrate models involved in aging and longevity. the worm caenorhabditis elegans and the fruit fly drosophila melanogaster are the two models that have given the major contributions on this topic. furthermore, we also discuss the possible contribution of recent theories on aging and inflammation to understand the complex phenotype of aged soma, from invertebrates to humans. key words: aging; longevity; invertebrates   introduction according to kirkwood (1985) animal species keep a balance between energy investments in maintenance, growth and repair on one hand and reproductive activity on the other hand, and this balance is related to aging. despite the great variability present among the animal kingdom, invertebrates and vertebrates use a common pool of highly conserved molecules combinatorially assembled under the constrain of selection for fitness (ottaviani et al., 1997, 2007; franceschi et al., 2000). we surmized that same or similar molecules found in invertebrates are present in vertebrates and in these higher forms of life their function remains basically similar. however, nature has apparently made new uses of these old molecules, while at the same time evolving towards more complex and centralized functions and organs (ottaviani et al., 1991). the molecular mechanisms able to affect aging and longevity are also involved in the capability of the organisms to cope with a variety of stressors (franceschi et al., 2000). furthermore, a prediction of these arguments is that the processes that extend lifespan and longevity in invertebrates will have a counterpart in vertebrates, including humans. however, one of the major differences between invertebrates and vertebrates is the appearance of the acquired immune response, characterized by high levels of specificity and memory. likely, the ___________________________________________________________________________ corresponding author: enzo ottaviani department of biology university of modena and reggio emilia via campi 231/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it immune system is, at a higher level of biological organization and complexity, the counterpart of the anti-stress response network identified in invertebrates as the major determinant of survival. in this context, invertebrates respond to stressors utilizing the same basis set of molecules found in vertebrates, but in the last, the stress response becomes more specialized and specific, involving an evolutionary well maintained network of responses (ottaviani and franceschi, 1996). the study of the mechanisms that underlie aging and longevity was conducted primarily in invertebrates without forgetting yeast, while human studies were considered secondary and only recently they have assumed considerable importance. with regards the human model, we have proposed the inflamm-aging theory, that represents the major characteristic of the aging process (franceschi et al., 2000). indeed, the ability to cope with a variety of stressors and the concomitant progressive increase in the proinflammatory status is considered a major cause related to a continuous, lifelong antigenic load and stress. in this paper the main invertebrate species used as models for the study of aging and longevity are reported. the invertebrate models used in aging and longevity studies the worm caenorhabditis elegans and the fruit fly drosophila melanogaster are the two models that have given the major contributions to the knowledge of the molecular mechanisms underpinning aging and longevity. it should be noted that in 1978, 451     89 references were listed on this topic for d. melanogaster (van heukelem, 1978). c. elegans is important for the study of aging as in this metazoan it was shown for the first time that single-gene mutations are able of extending maximum lifespan. in particular, the mutations of 4 genes age-1, daf-2, spe-26 and clk-1 induce a life extension of more than 40 %. the overexpression of tkr-1 in transgenic worms increases the longevity 40 100 % and confers increased resistance to heat and ultraviolet irradiation (see for review lithgow, 1996; murakami and johnson, 1998). the effects on c. elegans longevity and resistance to infection were also observed in worms fed with lactobacilli and bifidobacteria (ikeda et al., 2007; komura et al., 2010). with regards d. melanogaster, spencer et al. (2003) found a group of gene called “aging genes” (methuselah, indy, inr, chico, superoxide dismutase) that extend the fly lifespan by up to 85 %. further experiments using axenic cultures and antibiotic treatment showed that the presence of bacteria during the first week of adult life of flies enhanced longevity by 30 35 %. conversely, the presence of bacteria in the last stage of life caused a slight decrease (brummel et al., 2004). the addition of escherichia coli to the diet significantly prolonged the fly longevity in the two oregon r d. melanogaster strains, selected for different longevities: a short-life with an average adult lifespan of 10 days and a long-life standard r strain with an average adult lifespan of 50 days. furthermore, it has also been observed modifications on the structure and the histochemical reactivity of the fat body. the increased survival was associated with a great amount of glycogen accumulated in fat body cells from both strains. in aged control animals, fed with standard diet, lipid droplets were seen to be stored in fat body of shortlived, but not long-lived flies (franchini et al., 2012). for what the increased survival is concerned, it is important to mention that recently blagosklonny and hall (2009) suggest a new view, i.e., that "excessive growth is driving for aging", involving basic shared molecular pathways and processes such as the evolutionary conserved tor pathway. tor, the target of the antifungal drug rapamycin, has been described from yeast saccharomyces cerevisiae to higher eukaryotes, and its decreased activity has been found to slow aging in yeast, c. elegans and d. melanogaster (katewa and kapahi, 2011; mccormick et al., 2011). a different approach in the study of aging and longevity is represented by the medfly populations of ceratitis capitata in the wild (carey et al., 2008), where unlike the laboratory animals, it is not possible to control the experimental conditions. in this study, a new method for estimating age structure in insect populations was proposed, and it revealed that the major modifications were found in field populations, demonstrating that middle-aged individuals are common in the wild, and revealing the extraordinary lifespan of wild-caught insects. according to abel et al. (2009), bivalves are another excellent model to study aging since several parameters can be evaluated. for instance, the shell can provide information for determining the individual age and, at the same time, provides information on changes in environmental conditions that could influence the life of animals. it was also noted how different molluscan lifestyles regulate the balance between ros production and antioxidant defense, playing an important role in the determination the maximum lifespan. buick and ivany (2004) suggested that one of the processes in extending bivalve lifespan from high latitudes could be the seasonal limitations of light and food availability. however, other valuable model for the aging study are potentially available among marine invertebrates characterized by the ''negligible senescence", i.e., animals that do not show an increase in mortality rate or a decrease in fertility, physiological function or disease resistance with age (finch and austad, 2001). in this context, a list is reported by bodnar (2009). a new theoretical scenario within an evolutionary perspective, the theoretical field of aging has been dominated by few major theories, such as the mutation accumulation theory (medawar, 1952), the antagonistic pleiotropy theory (williams, 1957) and the disposable soma theory (kirkwood, 1977). in this paper we would like to discuss some of the implications of the more recent and above-mentioned conceptualization of blagosklonny and hall (2009). this quasiprogrammed hypothesis prompted us in extending the inflamm-aging theory to invertebrates. indeed, inflammation is an ancestral and highly conserved process which plays a fundamental physiological role for survival from invertebrates to homo sapiens. in a more general perspective, the age-related inflammatory process, which develops and increases with aging, owing to a lifelong persistent antigenic load as well as to other stimuli (accumulation of senescent cells), might be interpreted as a quasi-programmed extension to later life of the physiological tendency to activate inflammation and tissue repair crucial for survival in young age. accordingly, we not only predict a major role of the gut microbiota in life extension in the invertebrates (ottaviani et al., 2011), but we also suggest that inflamm-aging, and the underpinning molecular mechanisms and pathways, will also play a prominent role in invertebrate models of life extension. this extension to invertebrates of the inflamm-aging theory can be complemented by the recently advanced hypothesis of metaflammation (hummasti and hotamisligil, 2010). this conceptualization suggests that an excess intake of nutrients is the driving force triggering the inflammatory status characteristic of obesity and metabolic diseases, such as metabolic syndrome and diabetes in humans and mice. thus, at least two inflammatory process, inflamm-aging and metaflammation, have been identified, which likely affect aging, age-related diseases and longevity. we surmize that a theoretical merging of inflammaging and metaflammation could be useful to understand, at least in part, a variety of major topics in the aging field, such as the negative effects of excess nutrient and the positive effects of caloric restriction, the positive effects of inflammation for     90 table 1 major advantages and disadvantages of invertebrate model systems in comparison to humans _____________________________________________________________ advantages • the relative simplicity of their systems • the rapid generation time • the short life-span • the small size • standardization of the environmental conditions, including diet, temperature, among others • easy possibility to perform genetic studies disadvantages • inbreeding • artificial environment, including diet, temperature, among others • altered cycle of day and night • less complex immune system • scarce knowledge of the gut microbiota • scarce knowledge on mitochondrial dna • scarce knowledge of the pathology _____________________________________________________________ survival against infectious agents and the negative effects of inflamm-aging, among others. concluding remarks in our perspective the take home message of the this paper can be summarized in the table 1 where we list the major advantages and disadvantages of invertebrate model systems in comparison to humans, within a "balanced" view which appreciate what and how the different models, including humans can contribute for a better understanding of the highly complex phenotype of aged soma. references abele d, brey t, philipp e. bivalve models of aging and the determination of molluscan lifespans. exp. gerontol. 44: 307-315, 2009. blagosklonny mv, hall mn growth and aging: a common molecular mechanism. aging (albany ny) 1: 357-362, 2009. bodnar ag. marine invertebrates as models for aging research. exp. gerontol. 44: 477-484, 2009. brummel t, ching a, seroude l, simon af, benzer s. drosophila lifespan enhancement by exogenous bacteria. proc. natl. acad. sci. usa 101: 12974-12979, 2004. buick dp, ivany lc. 100 years in the dark: extreme longevity of eocene bivalves in antarctica. geology 32: 921-924, 2004. carey jr, papadopoulos nt, müller hg, katsoyannos bi, kouloussis na, wang jl, et al. age structure changes and extraordinary lifespan in wild medfly populations. aging cell 7: 426-437, 2008. finch ce, austad sn. history and prospects: symposium on organisms with slow aging. exp. gerontol. 36: 593-597, 2001. franceschi c, bonafe m., valensin s, olivieri f, de luca m, ottaviani e, et al. inflamm-aging. an evolutionary perspective on immunosenescence. ann. ny acad. sci. 908: 244-54, 2000. franchini a, mandrioli m, franceschi c, ottaviani e. morpho-functional changes of fat body in bacteria fed drosophila melanogaster strains. j. mol. histol. 43: 243-251, 2012. hummasti s, hotamisligil gs. endoplasmic reticulum stress and inflammation in obesity and diabetes. circ. res. 107: 579-591, 2010. ikeda t, yasui c, hoshino k, arikawa k, nishikawa y. influence of lactic acid bacteria on longevity of caenorhabditis elegans and host defense against salmonella enterica serovar enteritidis. appl. environ. microbiol. 73: 6404-6409, 2007. katewa sd, kapahi p. role of tor signaling in aging and related biological processes in drosophila melanogaster. exp. gerontol. 46: 382-390, 2011. kirkwood bl. comparative and evolutionary aspects of longevity. in: finch ce, schneider el (eds), handbook of the biology of ageing, 2nd ed, van nostrand reinhold, new york, pp 27-44, 1985. kirkwood tbl. evolution of ageing. nature 270: 301-304, 1977. komura t, yasui c, miyamoto h, nishikawa y. caenorhabditis elegans as an alternative model     91 host for legionella pneumophila, and protective effects of bifidobacterium infantis. appl. environ. microbiol. 76: 4105-4108, 2010. lithgow gj. invertebrate gerontology: the age mutations of caenorhabditis elegans. bioessays 18: 809-815, 1996. mccormick ma, tsai sy, kennedy bk. tor and ageing: a complex pathway for a complex process. philos. trans. r. soc. lond. b biol. sci. 366: 17-27, 2011. medawar pb. an unsolved problem of biology, hk lewis & co., london, 1952. murakami s, johnson te. life extension and stress resistance in caenorhabditis elegans modulated by the tkr-1 gene. curr. biol. 8: 1091-1094, 1998. ottaviani e, caselgrandi e, bondi m, cossarizza a, monti d, franceschi c. the "immuno-mobile brain": evolutionary evidence. adv. neuroimmunol. 1: 27-39, 1991. ottaviani e, franceschi c. the neuroimmunology of the stress response from invertebrates to man. prog. neurobiol. 48: 421-440, 1996. ottaviani e, franceschi f. the invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems. immunol. today 18: 169-174, 1997. ottaviani e, malagoli d, franceschi c. common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidences to in silico approaches. trends immunol. 28: 497-502, 2007. ottaviani e, ventura n, mandrioli m, candela m, franchini a, franceschi c. gut microbiota as a candidate for lifespan extension: an ecological/evolutionary perspective targeted on living organisms as metaorganisms. biogerontology 12: 599-609, 2011. spencer cc, howell ce, wright ar, promislow de. testing an 'aging gene' in long-lived drosophila strains: increased longevity depends on sex and genetic background. aging cell 2: 123-130, 2003. van heukelem wf. aging in lower animals, in: behnke ja, finch ce, moment b (eds), the biology of aging, plenum press, new york, pp 115-130, 1978. williams gc. pleiotropy, natural selection, and the evolution of scenescence. evolution 11: 398411, 1957.     92 microsoft word isj-2010-222     251 isj 7: 251-261, 2010 issn 1824-307x research report effects of bacillus thuringiensis var. kurstaki and medicinal plants on hyphantria cunea drury (lepidoptera: arctiidae) i zibaee1, ar bandani1, jj sendi2, r talaei-hassanloei1, b kouchaki2 1department of plant protection, agricultural and natural resources campus, university of tehran, karaj 31584, iran 2department of plant protection, college of agriculture, university of guilan, rasht, 41635-1314, iran accepted november 2, 2010 abstract the fall armyworm, hyphantria cunea drury (lepidoptera: arctiidae) is an insect native to north america that was recently introduced into iran resulting in severe damage to trees and agricultural production. an experiment was conducted to examine potential effects of medicinal plants, artemisia annua and lavandula stoechas and the insect pathogenic bacterium bacillus thuringiensis var. kurstaki on activities of digestive enzymes (α-amylase, αand β-glucosidase, lipase and proteases) and lactate dehydrogenase (ldh) in h. cunea by using two hosts, mulberry and sycamore. results showed that b. thuringiensis var. kurstaki and plant extracts when administered orally, affected the digestive enzyme profiles of h. cunea. combined effect of b. thuringiensis, a. annua and l. stoechas extracts on mulberry decreased the activities of digestive enzymes in a dose-related manner, except for β-glucosidase and lipase. when larvae were treated by different concentrations of the mentioned insecticides, ldh activity increased i.e. the higher activity was obtained by b. thurengiensis alone and b. thurengiensis and l. stoechas extracts together. the least activity was observed in the case of l. stoechas extracts alone on both hosts. physiological analysis would be particularly informative when using combination of biopesticides to enhance the efficiency of a safe management process. key words: hyphantria cunea; bacillus thuringiensis var. kurstaki; artemisia annua extract; lavandula stoechas extract; digestive enzymes; lactate dehydrogenase introduction the fall armyworm, hyphantria cunea drury (lepidoptera: arctiidae) is an insect native in north america that is presently distributed in many areas in the northern hemisphere (warren and tadic, 1970) and new zealand (kean and kumarasinghe, 2007). it has been introduced to different areas of europe and asia (li et al., 2001). since, 2002, h. cunea established itself in northern areas of iran, causing severe damage to trees. it is a multivoltine pest feeding on leaves of trees and hibernates as a pupa in soil around the damaged trees. research has been conducted to look for natural plant protection compounds such as botanical insecticides, antifeedants and microorganisms such as fungi and bacteria. bacillus thuringiensis is a gram-positive, soil dwelling bacterium which is commonly used as a ___________________________________________________________________________ corresponding author: idin zibaee department of plant protection, agricultural and natural resources campus, university of tehran, kardj, 31584, iran e-mail: izibaee@gmx.com pesticide. the genus artemisia is a member of a large plant family asteraceae (compositae) encompassing more than 300 different species of this diverse genus (shekari et al., 2008). the species a. annua, known as sweet worm wood, grows widely in europe and america and is grown in china, turkey, vietnam, afghanistan and australia (bhakuni et al., 2001; shekari et al., 2008). several isolated compounds from this species have shown antimalarial, antibacterial, antiinflamatory, plant growth regulatory and cytotoxicity (antitumor) activities (akhtar and isman, 2004). lavandula stoechas (french lavender), lamiaceae, occurs naturally in the mediterranean region and is a perennial shrub that grows to 30-100 cm tall. it was declared a toxic weed and has potential for use as a botanical insecticide. reduced efficacy of synthetic insecticides has been highlighted in the last two decades. the first alternative was b. thuringiensis berliner (bt), but the increasing number of reports on the resistance to bt led also to choose other biological insecticides such as those coming from plants (senthil nathana et al., 2006). several studies have investigated the     252 fig. 1 mortality probit of the bacillus thurengiensis, artemisia annua and lavandula stoechas in the presence of two hosts on the larvae of hyphantaria cunea. combination of bt with baculoviruses, however other combinations should be explored (senthil nathana et al., 2006). it is clear that botanical insecticides and microbes such as b. thuringiensis affect insect physiology in different ways including decrease of digestive enzyme activities. hence, in this paper research was conducted to examine potential effects of two botanical insecticides and bacterial toxin on activities of digestive enzymes and lactate dehydrogenase (ldh) in the h. cunea in the presence of two hosts, mulberry, an important tree in orchards, and sycamore, an important tree in urban areas, in order to find more suitable ways to decrease its population and damage.     253 materials and methods insects first instar larvae of hyphantria cunea were collected from the field and reared separately on mulberry and sycamore to reach 4th instar larvae in the laboratory at 27 ± 2 °c under a 14 h light:10 h dark photoperiod. these larvae were used to initiate the experiments. preparation of bacillus thurengiensis var. kurstaki and plant extracts a stock suspension of bacillus thuringiensis var kurstaki (109 spore/ml) was provided by giah company (iran) and a serial concentration prepared using distilled water. medicinal plant (artemisia annua and lavandula stoechas) leaves were collected, washed with distilled water and dried at room temperature in the shade. methanolic extraction was carried out according to the procedure described by shekari et al. (2008). briefly, 30 g of dried leaves were stirred with 300 ml of 85 % methanol in a flask, left for 48 h at 4 °c, then filtered through whatman no.4 filter paper. the solvent was removed by vacuum in a rotary evaporator and the dark green residue was dissolved in 10 ml acetone and used as a starting stock solution. further dilutions with either acetone or distilled water were used to prepare different concentrations. bioassay bioassays were performed with first instar larvae of h. cunea using 102, 104 and 106 spores/ml of bt on mulberry and 104, 106 and 108 spore/ml on sycamore. concentrations of 0.09, 0.22 and 0.42 % of a. annua on mulberry and concentrations of 0.13, 0.28 and 0.48 % 0n sycamore were used. concentrations of 0.02, 0.11 and 0.32 of l. stoechas on mulberry and concentrations of 0.13, 0.38 and 0.79 on sycamore were used. control leaves were treated with distilled water. for each treatment 30 larvae in three replicates were used in all the experiments and whole experiments were replicated twice. during the experiments, the larvae of each experimental condition were kept separately. the effective concentration (lc50) was calculated after 24 h using probit analysis (finney, 1971). fresh leaves were sprayed with different concentrations of the bt, a. annua, and l. stoechas and allowed to air dry. control leaves were treated with methanol alone. first instar larvae were starved for 4 h and then fed on leaves treated with the different concentrations of btk, a. annua, and l. stoechas. the uneaten leaves were removed every 24 h and the larvae were fed fresh treated leaves until larvae reached to fourth instar when the biochemical experiments were initiated. sample preparation for enzymatic assay enzyme samples from the midguts of fourth instar larvae were prepared based on zibaee and bandani (2009). briefly, larvae were randomly selected and their midguts were removed by dissection under a stereo microscope in ice-cold saline buffer (6 μmol/l nacl). the midgut was separated from the insect body, rinsed in-cold saline buffer, placed in a pre-cooled homogenizer and ground in 1 ml of universal buffer containing succinate (5 mm), glycine (2 mm) and 2morpholinoethanensulfonic acid (ph 7.2). the homogenates from both preparations were separately transferred to the 1.5 ml centrifuge tubes and centrifuged at 15000 rpm for 20 min at 4 °c. the supernatants were pooled and stored at -20 °c for subsequent analyses (zibaee and bandani, 2009). digestive enzyme assays α-amylase activity α-amylase activity was assayed by the dinitrosalicylic acid (dns) procedure (bernfeld, 1955), using 1% soluble starch (merck, darmstadt, germany) as substrate. twenty microliters of the enzyme were incubated for 30 min at 35 °c with 500 μl universal buffer and 40 μl soluble starch. the reaction was stopped by addition of 100 μl dns and heating in boiling water for 10 min. dns is a color reagent hence, the reducing groups released from starch by α-amylase action were measured by the reduction of dns. the boiling water stops the αamylase activity and catalyzes the reaction between dns and the reducing groups of starch. absorbance was then read at 540 nm. one unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35 °c. a blank sample without substrate with αamylase extract and a negative control containing no α-amylase extract with substrate were run simultaneously. all assays were performed in duplicate and each assay was repeated at least three times. αand β-glucosidase activity for solubilization of membrane hydrolyses (α, β-glocusidases) in triton x-100, membrane preparations were exposed to triton x-100 for 20 h at 40 ˚c, in a ratio of 10 mg of triton x-100/mg of protein, before being centrifuged at 15,000 rpm for 30 min. no sediment was visible after the centrifugation of this supernatant at 10,000 rpm for 60 min. the activity of the enzymes remains unchanged, at -20c, for periods of at least a month (ferreira and terra, 1983). the α, β-glucosidases activity was assayed by incubating 50 μl of enzyme solution with 75 μl of p-nitrophenyl-α-dglucopyranoside (pnαg) (5 mm), p-nitrophenyl-β-d glucopyranoside (pnβg) (5 mm) and 125 μl of 100 mm universal buffer (ph 5.0) at 37 °c for 10 min. the reaction were stopped by adding 2 ml of sodium carbonate (1 m) and read at 450 nm (ferreira and terra, 1983). lipase activity the enzyme assays were carried out as described by tsujita et al. (1989). thirty µl of midgut tissue extracts, 0.5 ml of universal buffer solution (1m) (ph 7.2), and 100 µl of p-nitrophenyl butyrate (50 mm), as substrate, were incorporated, mixed thoroughly and incubated at 37 °c. after 1 min, 100 µl distilled water was added to each tube (control and experimental samples) and absorbance was read at 405 nm. one unit of enzyme release 1.0     254 table 1 toxicity of bacillus thurengiensis, artemisia annua and lavandula stoechas extracts on the larvae hyphantaria cunea concentration1 bacillus thurengiensis artemisia annua lavandula stoechas mulberry sycamore mulberry sycamore mulberry sycamore ld10 95% confidence interval2 102 101-104 104 103-105 0.09 0.03-0.13 0.13 0.07-0.18 0.02 0.001-0.06 0.13 0.05-0.20 ld30 95 % confidence interval 104 103-106 106 105-107 0.22 0.16-0.28 0.28 0.22-0.35 0.11 0.03-0.18 0.38 0.27-0.51 ld50 95 % confidence interval 106 106-108 108 107-1010 0.42 0.33-0.63 0.48 0.38-0.71 0.32 0.20-0.49 0.79 0.20-0.57 l90 95 % confidence interval 109 108-1011 1012 1010-1014 1.93 1.06-8.14 1.76 1.05-5.48 4.12 1.69-5.46 4.77 2.28-8.17 slope±se 0.34-0.073 0.25-0.056 1.95±0.50 2.29±0.54 1.15±0.35 1.64±0.40 x2 (df) 4.70 4.44 2.46 2.37 2.83 0.82 p-value 0.48 1.14 0.72 0.70 0.16 0.64 1concentration of bacillus thurengiensis is spore/ml and plant extracts are percentage 2confidence limits have been calculated with 95 % confidence nanomole (10-9 mole) of p-nitrophenol per minute at ph 7.2 at 37 °c using p-nitrophenyl butyrate as substrate. the negative control tube was placed in a boiling water bath for 15 min to destroy the enzyme activity and then cooled prior to be added with the substrate. protease activity general protease activity of adult midguts was determined using azocasein as substrate (elpidina et al., 2001). the reaction mixture was 80 µl of 2 % azocasein solution in 40 mm universal buffer of specified ph and 30 µl enzyme. the reaction mixture was incubated at 37 °c for 60 min. proteolysis was stopped by addition of 300 µl of 10 % trichloroacetic acid (tca). appropriate blanks in which tca was added first to the substrate were prepared for each assay. precipitation was achieved by cooling at 4 °c for 120 min and the reaction mixture was centrifuged at 16,000 rpm for 10 min. an equal volume of 1 n naoh was added to the supernatant and the absorbance was recorded at 440 nm. lactate dehydrogenase (ldh) assay for evaluating lactate dehydrogenase (ldh), the king’s (1965) method was used. to standardize volumes, 0.2 ml nad+ solution was added to the test tubes and 0.2 ml of water was added to control test tubes, each containing 1 ml of the buffered substrate. 0.01 ml of the sample was also added to the test tubes. test tube samples were incubated for exactly 15 min at 37 ˚c and then arrested by adding 1 ml of color reagent (2,4-dinitrophenyl hydrazine) to each tube and the incubation continued for an additional 15 min. after the contents were cooled to room temperature, 10 ml of 0.4 n naoh was added to each tube to make the solutions strongly alkaline. at exactly 60 s after the addition of alkali to each tube, the intensity of color was measured at 440 nm. protein determination protein concentrations were measured according to the method of bradford (1976), using bovine serum albumin (bio-rad, münchen, germany) as a standard. statistical analysis the mortality and lethal concentration were obtained by using probit analysis (robertson et al., 2007) and polo-pc software (leora, 1987). in this case, significant differences among the concentrations were recorded when 95 % confidence intervals (ci) did not overlap. other data were compared by one-way analysis of variance (anova) followed by tukey's studentisized test when significant differences were found at p = 0.05 (sas, 1997). differences among samples were considered statistically significant (p < 0.05).     255 results dose-response relationships the results showed that plant extracts and bacterial toxins produced a dose response in the insect species on both host species (fig. 1, table 1). the ld50 values of b. thurengiensis were significantly different in sycamore and mulberry (table 1). in the case of a. annua extracts, no significant difference was observed between sycamore and mulberry, whereas extracts from l. stoechas produced effects similar to those observed for b. thurengiensis (table 1). effect of btk and plant extracts on digestive enzymes results showed that b. thuringiensis and plant extract affected the digestive enzymatic profiles of h. cunea at several concentrations by using oral ingestion treatment in the presence of two hosts (tables 2-6). when larvae fed on leaves treated by btk, activity of all digestive enzymes was decreased and showed a dose-related status (table 2). a. annua treatment decreased digestive enzyme activities in larvae feed on both mulberry and sycamore in a dos-related manner (table 3). treatment of leaves by l. stoechas demonstrated a slightly decrease on digestive enzymes except for protease and lipase. however, the effect of l. stoechas extracts on enzyme activities on sycamore was more with regard to mulberry (table 4). combined effect of b. thuringiensis and a. annua on mulberry showed that digestive enzyme activities decreased except for β-glucosidase and lipase (table 5). similar results were found in the case of b. thuringiensis and l. stoechas (table 6). effect of btk and plant extracts on lactate dehydrogenase activity table 7 shows effect of b. thuringiensis, a. annua and l. stoechas on ldh activity of h. cunea. results demonstrated that ldh activity increased by treating different concentrations of insecticides on larvae and the higher activity was obtained by b. thuringiensis alone and b. thuringiensis and l. stoechas together. the least activity was observed in the case of l. stoechas alone. similar results found when sycamore was used as host. discussion crude botanical components for various purposes were well known in traditional cultures for centuries (schmutter, 1990). their extracts and active ingredients are a good choice for different investigations including pest management tactics. here we observed that the treatment of b. thuringiensis, a. annua and l. stoechas separately as well as the combined effect of bacteria and plant extracts exerted a significant effect on h. cunea digestive enzyme and ldh when spread onto two plant hosts, namely mulberry and sycamore. several studies have shown that feeding is necessary for the stimulation of digestive enzyme activities (sibley, 1981; broadway and duffey, 1988). results demonstrated that sublethal doses of these biopesticides individually decreased digestive enzyme activities such as α-amylase, αand βglucosidase, lipase and protease and increased ldh activity. higher enzyme activities in the midgut of control insects are most probably due to consumption and utilization of large quantities of food (senthil-nathan et al., 2006). imbalance in enzyme-substrate complex and inhibition of peristaltic movement of the gut (hori, 1969) might have inhibited the enzyme activities in the treated insects (zibaee and bandani, 2009). it is clear that exposure of diet to botanical insecticides has significant effects on several enzyme activities found in the late instar larvae of h. cunea. botanical insecticides may interfere with the production of certain types of proteins (smirle et al., 1996; senthil-nathan et al., 2006). in the case of decreasing activity of digestive enzymes due to b. thuringiensis treatment, this bacterium causes damage to the epithelial cells of the midgut through crystalline parasporal bodies, which release the active toxin after digestion by serine proteases under the alkaline conditions in the intestinal fluid (senthil-nathan et al., 2006). therefore one would expect that such damage to the midgut would cause a decrease in digestive enzyme activities (eguchi et al., 1972; mathavan et al., 1989; smirle et al., 1996; senthil nathan et al., 2006). α-amylase is an endo-digestive enzyme that catalyzes the breakdown of 1:4-α-glucosidase bonds in polysaccharides and converts starches into maltose (disaccharide) and glycogen into glucose. in the many studies, pesticides including b. thuringiensis, synthetic chemicals and botanical components significantly decreased the activity of αamylase in the midgut of different insects (senthilnathan et al., 2006; shekari et al., 2008; zibaee et al., 2008; zibaee and bandani, 2009). saleem and shakoori (1987) showed that sublethal concentrations of pyrethroids decreased the α-amylase activity in larval gut of the beetle tribolium castaneum herbst (coleoptera: tenebrionidae). lee et al. (1994) showed that some igrs decreased the activity level of α-amylase and esterase in the treated larvae. ascher and ishaaya (2004) showed that the activity level of this enzyme increased 30 % in s. littoralis boisd (lepidoptera: noctuidae) treated with phentine acetate compared with control. senthilnathan et al. (2006) found that b. thuringiensis decreased the activity level of this enzyme; the activity was much lower when bacterial spores and botanical components were combined. zibaee et al. (2008) showed that along with elevation of spraying times, the activity level of α-amylase would sharply decrease in chilo suppressalis walker (lepidoptera: crambidae) larvae. shekari et al. (2008) demonstrated that α-amylase activity level decreased 24 h after treatment and sharply increased at 48 h after treatment with a. annua extract of the elm leaf beetle. similar results were found when adults of eurygaster integriceps puton (heteroptera: scutelleridae) fed on grain and water contained a. annua extract (zibaee and bandani, 2009). in this study we found that b. thuringiensis, a. annua and l. lavandula, individually, decreased activity level of α-amylase and the highest inhibitions were obtained when larvae had been fed on sycamore and combined exposures were made.     256 table 2 effect of bacillus thurengiensis on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of two different hosts treatment1 α-amylase α-glucosidase β-glucosidase protease lipase mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 2.44±0.20a 1.85±0.08a 2.72±0.34a 2.32±0.98a 2.91±1.35a 2.64±0.66a 3.54±0.00a 2.14±0.004a 4.00±0.008a 3.44±0.004a ld10 2.07±0.06ab 1.56±0.07ab 2.26±0.21a 1.62±0.40ab 3.74±0.36a 2.62±0.31a 3.08±0.001a 2.01±0.001ab 2.80±0.003b 2.8±0.004b ld30 1.69±0.06b 1.53±0.03b 1.60±0.32b 1.21±0.33b 2.84±0.12ab 1.77±0.60b 2.18±0.003b 1.13±0.00b 2.14±0.003c 2.02±0.002c ld50 1.15±0.14c 1.30±0.06b 1.58±0.44b 0.63±0.30c 2.45±0.30b 1.21±0.50b 0.71±0.001c 0.77±0.001c 1.49±0.003c 1.55±0.002c 1concentration of is b. thurengiensis spore/ml. ld10, ld30 and ld50 are 10 2, 104 and 106 on mulberry and 104, 106 and 108 on sycamore. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test. table 3 effect of artemisia annua extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of two different hosts treatment1 α-amylase α-glucosidase β-glucosidase protease lipase mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 1.87±0.09a 1.75±0.28a 2.05±0.54a 1.90±0.4a 3.88±1.03a 2.67±0.28a 3.80±0.00a 2.36±0.00a 3.43±0.00a 3.34±0.00a ld10 1.44±0.05b 1.69±0.05a 1.39±0.10b 1.55±0.27b 2.48±0.07c 2.50±0.39b 3.16±0.00ab 1.62±0.00ab 2.79±0.00b 2.61±0.00ab ld30 1.13±0.00c 1.19±0.04ab 1.24±0.28b ±0.950.09c 1.39±0.14c 1.55±0.21c 1.88±0.00b 0.80±0.00b 2.00±0.00c 2.01±0.00b ld50 0.84±0.06c 0.99±0.03b 0.52±0.19c 0.14±0.08d 0.28±0.49d 1.25±0.31d 0.76±0.00c 0.50±0.00c 1.47±0.00d 0.56±0.00c 1concentrations of plant extract are 0.09, 0.22 and 0.42 on mulberry and 0.13, 0.28 and 0.48 on sycamore as ld10, ld30 and ld50. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test.     257 table 4 effect of lavandula stoechas extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of two different hosts treatment1 α-amylase α-glucosidase β-glucosidase protease lipase mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 2.08±0.01a 1.97±0.03a 2.20±0.20a 1.49±0.91a 2.89±0.33a 2.61±0.21a 3.64±0.00a 3.51±0.00a 3.25±0.00a 2.77±0.02a ld10 2.05±0.03a 1.61±0.02b 1.77±0.65b 1.62±0.23 2.42±0.68a 2.46±0.48a 3.65±0.00a 3.43±0.00a 3.18±0.00a 2.49±0.00a ld30 1.89±0.03b 1.38±0.08b 2.37±0.74a 1.53±0.30a 2.71±0.12a 1.79±0.70a 3.42±0.00a 3.20±0.00a 2.91±0.00a 2.42±0.00a ld50 1.71±0.02b 1.11±0.05c 2.15±0.75a 1.54±0.34a 2.45±0.23a 1.41±0.23b 3.39±0.00a 3.25±0.00a 2.69±0.00a 2.38±0.00a 1concentrations of plant extract are 0.02, 0.11 and 0.32 on mulberry and 0.13, 0.38 and 0.79 on sycamore as ld10, ld30 and ld50. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test. table 5 combined effect of bacillus thurengiensis and artemisia annua extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae on mulberry as the host treatment1 α-amylase α-glucosidase β-glucosidase protease lipase control 2.01±0.01a 1.99±0.021 3.32±0.80a 3.93±0.00a 3.16±0.00a ld10 2.00±0.018a 1.52±0.016b 2.89±0.34a 3.93±0.00a 3.14±0.00a ld30 1.72±0.03ab 1.45±0.08b 2.96±0.87a 2.82±0.00ab 3.14±0.00a ld50 0.86±0.06c 0.69±0.32b 2.61±0.87a 0.91±0.00b 3.07±0.00a 1each ld value shows b. thurengiensis + plant extract concentration as ld10: 10 2+ 0.09, ld30: 10 4+0.22, ld50: 10 6+0.42. 2.means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test.     258 table 6 combined effect of bacillus thurengiensis and lavandula stoechas extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of mulberry leaves as the host treatment1 α-amylase α-glucosidase β-glucosidase protease lipase control 1.93±0.03a 1.92±0.10a 2.99±0.79a 3.35±0.00a 3.30±0.00a ld10 1.75±0.11ab 1.77±0.30a 2.76±1.01a 1.05±0.00b 2.50±0.00b ld30 1.24±±0.19b 1.34±0.14ab 3.12±0.75a 0.33±0.00b 1.87±0.00b ld50 0.38±0.13c 1.20±1.08b 2.52±1.00b 0.14±0.00b 1.03±0.00c 1each ld value shows b. thurengiensis + plant extract concentration as ld10: 10 2+ 0.02, ld30: 10 4+0.11, ld50: 10 6+0.32. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test. table 7 effect of bacillus thurengiensis, artemisia annua and lavandula stoechas extract on lactate dehydrogenase (µmol/min/mg protein) of hyphantaria cunea larvae on mulberry and sycamore as the host treatment1 bacillus thurengiensis artemisia annua lavandula stoechas b.t. + artemisia annua b.t. + lavandula stoechas mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 0.17±0.05d 0.21±0.10d 0.19±0.08c 0.18±0.03c 0.23±0.07c 0.19±0.07b 0.20±0.04c 0.25±0.09d 0.23±0.031d 0.17±0.06c ld10 0.26±0.08c 0.35±0.08c 0.21±0.09c 0.22±0.01c 0.26±0.06b 0.21±0.03b 0.27±0.06b 0.38±0.03c 0.40±0.08c 0.34±0.02b ld30 0.50±0.10b 0.49±0.08b 0.35±0.07b 0.45±0.05b 0.27±0.07b 0.29±0.06ab 0.53±0.1bc 0.67±0.09b 0.61±0.19b 0.39±0.04b ld50 0.71±0.14a 0.95±0.02a 0.57±0.07a 0.81±0.04a 0.54±0.10a 0.48±0.07a 0.67±0.17a 0.97±0.12a 0.96±0.21a 0.86±0.09a 1each ld value shows b. thurengiensis (b.t.) and plant extract alone and b.t. + plant extract concentration together in the presence of mulberry leaves as the host. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test.     259 the glycosidases catalyze the hydrolysis of terminal, non-reducing 1, 4-linked α-d-glucose residues with release of α-d-glucose. study of glucosidase in herbivorous insects is important not only for understanding digestion biochemistry but also for developing insect pest management strategies. plants produce a wide variety of allelochemicals which act as defensive compounds. these include alkaloids, cyanogenic and triterpenoid glycosides, phenols, flavenoids and nonprotein amino acids (hsiao, 1985). among these allomones, glycosides seem to play an important role in host plant resistance to insects. for example, tomatine, an alkaloid glycoside and rutin (quercetin 3-rutinoside) are involved in the resistance of tomato to the tomato fruitworm, heliothis zea fabricius (lepidoptera: noctuidae), by acting as feeding deterrents (pratviel-sosa et al., 1986). dimboa is another glycoside which is present in young corn tissues. the toxic action of these glycosides is due to their corresponding aglycones liberated by the action of β-glucosidase. in the current study, treatment of h. cunea larvae with sublethal concentrations of different biopesticides showed a reduction in the activity level of αand βglucosidases. the increase of plant extract concentrations on sycamore corresponded to a reduced enzymatic activity in larvae. this may be due to a drop in the consumption rates and leveling off or decline in food conversion efficiencies (zibaee and bandani, 2009). these decreasing activities were reported in other insects. hemmingi and lindroth (1999, 2000) studying effect of phenolic components in gypsy moth (lepidoptera, lymantriidae) and forest tent caterpillar (lepidoptera, lasiocampidae) demonstrated that glucosidase activities declined for both insect species when reared on diets with phenolic glycosides in addition to decreasing growth and increasing developmental time. zibaee and bandani (2009) found that a. annua extract significantly decreased activity of αand βglucosidases on e. integriceps adults so that the lowest activity was obtained at 25 % concentration of plant extract treatment of adults. lipases (triacylglycerol acylhydrolase; ec 3.1.1.3), which catalyses the hydrolysis of fatty acid ester bonds, are widely distributed among animals, plants and microorganisms (zibaee et al., 2009). it was found that b. thuringiensis and plant extracts decreased the activity level of lipase but in the case of l. stoechas no significant differences were observed. senthil nathan et al. (2006) showed that treating cnaphalocrocis medinalis (guenee) (lepidoptera: pyralidae), the rice leaffolder, by btk, nske, and vnle (azadirachtin and neem components) sharply decreased the activity level of lipase in the midgut. proteases hydrolyze proteins to amino acids classified as endopeptidases (ec 3.4.21-24) and exopeptidases (ec 3.2.4.11-19) based on their catalytic mechanism (pascual-ruiz et al., 2009). it was observed that biopesticides significantly decreased activity of protease in the midgut of h. cunea larvae especially on sycamore but no significant differences were obtained in the case of l. stoechas on both hosts. because proteases are necessary for activation of b. thuringiensis protoxin to active toxin, this biopesticide could be a logical choice for control of caterpillars due to high ph value and suitable activity of proteases in the their midgut. zibaee and bandani (2009) found that a. annua extract significantly decreased the activity of protease on e. integriceps adults so that the lowest activity were obtained when 25 % concentration of plant extract was used on adults. physiological conditions of h. cunea affects the activity of the tested enzymes and reflects the absorption, digestion, and transport of nutrients in the midgut. b. thurengiensis damages the epithelial cells of the midgut through crystalline parasporal bodies, so decreasing levels of digestive enzymes. this results in reduced phosphorous liberation for energy metabolism, decreased rate of metabolism and decreased rate of metabolite transport, maybe due to the direct effects on enzyme regulation and synthesis. ldh is an important glycolytic enzyme being present virtually in all tissues (kaplan and pesce, 1996). it is also involved in carbohydrate metabolism and has been used as an indicative criterion of exposure to chemical stress (wu and lam, 1997; diamantino et al., 2001) and as an index of anaerobic metabolism (chamberlin and king, 1998). activity level of ldh in culex after treatment with ddt, malathion and cyfluthrin decreased 58.88 %, 33.33 % and 66.66 %, respectively (arshad et al., 2002). senthil-nathan and kalviani (2005) showed that feeding of spodoptera litura on ricinus communis treated with azadirachtin and nucleopolyhedrovirus decreases the amount of this enzyme in midgut that demonstrates low nutritional efficiency of the larvae. similar results were also observed on effectiveness of melia azedarach on rice leaffolder (senthilnathan, 2006). results in our current study showed that reduction of digestive enzyme activities due to using different bio-pesticides on larvae fed on sycamore were higher than those of mulberry. in addition, induction of ldh activity on larvae fed on sycamore was higher than that of mulberry. there may be different reasons for these differences. first of all, sycamore trees have been planted extensively around the city but mulberry orchards exist in the limited areas. hence, sycamore trees are more available to larvae than mulberry. secondly, almost all mulberry trees in the area have been modified genetically for silkworm rearing and have less plant secondary metabolites than sycamore. this matter is being observed more obviously on activity of digestive enzymes in control. but, treatment of sycamore leaves by biopesticides has a synergistic relationship with secondary metabolites existing in the plant tissue and caused more reduction in the digestive enzyme activities. as a conclusion, a. annua and l. stoechas extracts had significant effects on the larvae of h. cunea so that they act synergistically with b. thurengiensis var. kurstaki toxin, causing reduction of digestive enzyme activity and elevation of ldh activity. h. cunea is a widely distributed pest which causes severe damages to trees in orchards. hence, widely spraying by common synthetic     260 insecticide has high environmental risks specially on human therefore, studies on biopesticides and their combinations are necessary specially for physiological effect to decrease the population density of the pest. physiological analysis would be particularly informative to get insight into which combinations of biopesticides enhance the efficiency of a safe management process. acknowledgment this study was supported by a university of tehran grant. we thank m allahyari for assistance. references abudulai m, shepard bm, mitchell pl. parasitism and predation on eggs of leptoglossus phyllopus (l.) (hemiptera: coreidae) in cowpea: impact of endosulfan sprays. j. agric. urban entomol. 18: 105-115, 2001. akhtar y, isman mb. comparative growth inhibitory and antifeedant effects of plant extract and pure allelochemicals on some phytophagous insect species. j. appl. entomol. 128: 32-38, 2004. arshad m, ahmad i, naqvi snh, kahkashan a. level of lactate dehydrogenase in resistant and susceptible strains of culicine mosquitoes of the karachi region after treatment with ddt, malathion and cyfluthrin. turkish j. zool. 26: 97-100, 2002. ascher krs, ishaaya i. antifeeding and protease and amylase inhibiting activity of phentin acetate in spodoptera littoralis larvae, pestic. biochem. physiol. 75: 326-336, 2004. bernfeld p. amylases, α and β. methods enzymol. 1: 149-158, 1955. bradford m. a rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254, 1976. broadway rm, duffey ss. the effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. j. insect physiol. 34: 1111-1117, 1988. chamberlin me, king me. changes in midgut active ion transport and metabolism during the fifth instar of the tobacco hornworm (manduca sexta). j. exp. zool. 280: 135-141, 1998. diamantino tc, amadeu e, soaresa mvm, guilherminoc l. lactate dehydrogenase activity as an effect criterion in toxicity tests with daphnia magna, straus. chemosphere 45: 553-560, 2001. eguchi m, sawaki m, suzuki y. multiple forms of midgut alkaline phosphatase in the silkworm: new band formation and the relationship between the midgut and digestive fluid. insect biochem. 2: 297-304, 1972. elpidina en, vinokurov ks, gromenko va, rudenskaya ya, dunaevsky ye, zhuzhikov, dp. compartmentalization of proteinases and amylases in nauphoeta cinerea midgut. arch. insect biochem. physiol. 48: 206-216, 2001. ferreira c, terra wr. physical and kinetic properties of a plasma-membrane-bound pdglucosidase (cellobiase) from midgut cells of an insect (rhynchosciara americana larva). biochem. j. 213: 43-51, 1983. finney dj. probit analysis, 3rd ed, cambridge university press. london, uk, 1971. gill ss, cowles ea, pietrantonio pv. the mode of action of bacillus thuringiensis endotoxins. ann. rev. entomol. 37, 615–635, 1992. hemmingi jdc, lindroth rl. effects of phenolic glycosides and protein on gypsy moth (lepidoptera: lymantriidae) and forest tent caterpillar (lepidoptera: lasiocampidae) performance and detoxication activities. environ. entomol. 29: 1108-1115, 2000. hemmingi jdc, lindroth rl. effects of light and nutrient availability on aspen: growth, phytochemistry and insect performance. j. chem. ecol. 26: 1687-1714, 1999. hsiao th. feeding behavior. in: comprehensive insect physiology biochemistry and pharmacology, vol. 9, pp 471-512, 1985. kean jm, kumarasinghe lb. predicting the seasonal physiology of fall webworm (hyphantria cunea) in new zealand. new zealand plant protec. 60: 279-285, 2007. king j. the dehydrogenases or oxidoreductases. lactate dehydrogenase, in: van nostrand d (ed), practical clinical enzymology, london, pp 83-93, 1965. lee hr, kim jw, lee i. studies on the toxicity of insect growth regulators against the fall armyworm (hyphantaria cunea drury) and the rice stem borer (chilo suppressalis walker) comparisonsa in enzyme activities. korean j. appl. entomol. 33: 88-95, 1994. leora software, polo-pc. 1987. a user guide to probit or logit analysis. leora software, berkeley, california, 1987. li yp, goto m, ito s, sato y, sasaki k, goto n. physiology of diapause and cold hardiness in the overwintering pupae of the fall webworm hyphantria cunea (lepidoptera: arctiidae) in japan. j. insect physiol. 47: 1181-1187, 2001. madigan m, martinko j. brock biology of microorganisms (11th ed). prentice hall publication, 2005. mathavan s, sudha pm, pechimuthu sm. effect of bacillus thuringiensis israelensis on the midgut cells of bombyx mori larvae: a histopathological and histochemical study. j. invertebr. pathol. 53: 217–227, 1989. pascual-ruiz s, carrillo l, alvarez-alfagemeyy f, ruiz m, castan p, ortego f. the effects of different prey regimes on the proteolytic digestion of nymphs of the spined soldier bug, podisus maculiventris (hemiptera: pentatomidae). bull. entomol. res. 99: 487-491, 2009. pratviel-sosa f, clemont s, percheron f, chararas c. studies on glycodidases and glucanases in thaumetopoea pitycocampa larvae. part 1. purificaiton and some properties of the αglucosidase. comp. biochem. physiol. 84: 7781, 1986. robertson jl, preisler hk, russell rm. poloplus: probit and logit analysis user’s guide. leora software, petaluna, ca, usa, 2007. saleem ma, shakoori ar. point effects of dimilin and ambush on enzyme activies of tribolium castaneum larvae. pestic. biochem. physiol. 29: 127-137, 1987.     261 sas institute. sas/stat user’s guide for personal computers. sas institute, cary, nc, 1997. schmutterer h. properties and potential of natural pesticides from the neem tree, azadirachta indica. ann. rev. entomol. 35: 271-297, 1990. senthil nathan s, kalaivani k. efficacy of nucleopolyhedrovirus (npv) and azadirachtin on spodoptera litura fabricius (lepidoptera: noctuidae). biol. control. 34: 93-98, 2005. senthil nathana s, chunga pg, murugan k. combined effect of biopesticides on the digestive enzymatic profiles of cnaphalocrocis medinalis (guene´e) (the rice leaffolder) (insecta: lepidoptera: pyralidae). ecol. environ. safe 64: 382-389, 2006. senthil-nathan ss. effects of melia azedarach on nutritional physiology and enzyme activities of the rice leaffolder cnaphalcrocis medinalis (guenee) (lepidoptera: pyralidae). pestic. biochem. physiol. 84: 98-108, 2006. shekari m, jalali sendi j, etenbari k, zibaee a, shadparvar a. effects of artemisia annua l. (asteracea) on nutritional physiology and enzyme activities of elm leaf beetle, xanthogaleruca luteola mull. (coleoptera: chrysomellidae). pestic. biochem. physiol. 91: 66-74, 2008. sibley rm. strategies of digestion and defaecation. in: townsend cr, calew p (eds), physiological ecology and evolutionary approach to resource use, blackwell publishers, pp 109-139, oxford, 1981. smirle mj, lowery dt, zurowski cl. influence of neem oil on detoxication enzyme activity in the obliquebanded leafroller, choristoneura rosaceana). pestic. biochem. physiol. 56: 220230, 1996. warren lo, tadic m. the fall webworm, hyphantria cunea (drury). arkansas. agri. experiments-station. bull. 759, 1-106, 1970. weinzierl r, henn t. alternatives in insect management: biological and biorational approaches. north central regional extension publication 401. cooperative extension service, university of illinois at urbanachampaign, 1991. wu rss, lam pks. glucose-6-phosphate dehydrogenase and lactate dehydrogenase in the green-lipped mussel (perna viridis). possible biomarker for hypoxia in the marine environment. water. res. 31: 138-142, 1997. zibaee a, bandani ar. effects of artemisia annua l. (asteracea) on the digestive enzymatic profiles and the cellular immune reactions of the sunn pest, eurygaster integriceps (heteroptera: scutellaridae), against beauveria bassiana. bull. entomol. res. 100: 185-196, 2010. zibaee a, bandani ar, ramzi s. lipase and invertase activities in midgut and salivary glands of chilo suppressalis (walker) (lepidoptera, pyralidae), rice striped stem borer. inv. surv. j. 5: 180-189, 2009. zibaee a, jalali sendi j, etebari k, alinia f, ghadamyari m. the effect of diazinon on some biochemical characteristics of chilo suppressalis walker (lepidoptera: pyralidae), rice striped stem borer. munis. entomol. zool. 3: 255-264, 2008. isj 5: 20-xx, 2008 isj 5: 20-29, 2008 issn 1824-307x research report a study on biochemical differences among five different groups of rice striped stem borer chilo suppressalis walker (lepidoptera: pyralidae) a zibaee1, jj sendi1, f alinia2, k etebari3 1department of plant protection, faculty of agriculture, the university of guilan, rasht 41635-1314, iran 2rice research institute of iran (rrii), rasht 41635-1658, iran 3department of sericulture, faculty of natural resources, the university of guilan, somehe sara, iran accepted february 21, 2008 abstract identification of biodiversity in different rice striped stem borer (chilo supprressalis) populations is very important to adopt suitable integrated pest management procedures. larvae were collected from five different regions in north of iran including gourabzarmikh (go), sheikhmahaleh (sh), rasht (ra), amol (am) and babol (ba). activity levels of five enzymes including alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alpha-amylase were evaluated in 4th instar larvae. in addition, five non-enzymatic compounds such as glucose, cholesterol, total protein, uric acid and urea were also measured. amount of measured compounds showed significant differences in all groups except for alanine aminotransferase and aspartate aminotransferase. hierarchical agglomerative clustering under upgma model demonstrated that ba population had the most genetic distance and was separated from other groups. in the second group, go population had the most genetic distance from others and two groups of ra and sh had the least genetic distances. key words: rice stripped stem borer; hierarchical agglomerative clustering; biochemical characteristics introduction the rice striped stem borer (chilo supprressalis) is a cosmopolitan and destructive pest in rice fields of the world (khanjani, 2006). this pest was introduced to iran in 1973 and has been widely distributed in all rice fields of iran. its distribution is random and aggregative with 2-3 generations per year (saeb and gramy, 2000). in north of iran, this pest has been distributed in all areas and its density is more than economic injury level (dezfoulian and moustofipoor, 1972). in 1995, it has been reported from other provinces of iran such as isfahan, shiraz, eilam and khozestan. severe damages have been reported from these areas (moghaddas and saiiad-nasiri, 1995). the chemical control especially organophosphorous compounds has been a common practice for more ___________________________________________________________________________ corresponding author: jalal jalali sendi department of plant protection faculty of agriculture the university of guilan rasht 41635-1314, iran e-mail: jjalali2001@yahoo.com than three decades (khosroshahi et al., 1979). however, other methods such as cultural practices and biological control with trichogramma spp., have been incorporated. in recent years, control of c. suppressalis has been concentrated on using resistance varieties and pheromone traps. saeb and mohammad-salehi (1998) studied 78 different varieties and showed that binam variety with 15 % white head was the most resistant one. saeb (1999) studying on different germplasts of rice showed that khazar variety was resistant to first generation of rice striped stem borer and susceptible to second generation. saeb (2002a) suggested that using pheromone traps including z-13, octadecenal, z-11, hexadecenal and z-9, hexadecenal was a usefull practice for rice striped stem borer control in north of iran. using different markers to determine the intraspecific biodiversity and better understanding of genetic polymorphisms has always been within the range of researchers' interests (chatterjee and data, 1992; eguchi, 1995; etebari and matindoost, 2004b; etebari et al., 2005). "biochemical marker" is a term used for some biochemical compounds, 20 which are able to demonstrate the differences between two species or different biotypes of the same species (stoikova et al., 1998). bartelett (1989) used these biochemical markers for identification and presence of biotypes in different species of heliothis. loxdale and brookes (1990) used the same markers to identify the biotypes of blackberry grain aphid (sitobiom avenae) in southeast england. there are many biochemical markers in insects which explicit differences among various individuals in the same population. the measurement of αamylase and invertase could divide the silkworm populations in two classes, one group with two generations and high silk production and the other with several generations and low production (chatterjee and data, 1992). chatterjee et al. (1993) reported that there is a significant correlation between some biochemical parameters of hemolymph and midgut fluid of the silkworm larvae of which the most important compounds are: amylase, invertase and alkalin phosphatase. the amount of these compounds in larval hemolymph depends on different factors such as the type of food, environmental conditions, genetics and etc. enzymes with respect to their genetic structure are less changeable than other biochemical compounds in larval hemolymph. generally for this aspect, different qualitative enzymatic analyses or isoenzymes are being utilized (etebari et al., 2005). in this study, larvae were collected from five different regions in north of iran including gourabzarmikh, sheikhmahaleh, rasht, amol and babol. activity levels of five enzymes including alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alpha-amylase and non-enzymatic coumponds such as total protein, glucose, cholesterol, urea and uric acid were evaluated in 4th instar larvae of rice striped stem borer. materials and methods insects the larvae of chilo suppressalis were collected from five different sites of rice fields including amol (am), babol (ba) in mazandaran province and sheikhmahaleh (sh), gouramzarmikh (go) and experimental blocks of rice research institute of iran, rasht (ra) in guilan province (300 larvae from each location) and reared on dorfac variety of rice, at 25 ± 1 ºc temperature, 70 ± 5 % rh and 16l:8d. thirthy larvae of fourth instar from each site were randomly selected and used in biochemical experiments with 3 replications. sample preparation and biochemical analysis whole larval body was homogenized in fluid nitrogen and samples of each region were diluted with phosphate buffer in weight to volume proportion and centrifuged for 10 min in 10,000 rpm. the supernatant was transferred to new tubes and was preserved at -20 ºc until the onset of the experiments. protein was measured based on biuret's method by utilizing a total protein assay kit (biochem co, iran). in this method, proteins makes a complex purplish blue with an alkaline copper solution, which its absorption value at 540 nm has a direct relation to the amount of whole body protein. to measure total cholesterol, richmonds's (1971) method was performed. the principles of this method are based on hydrolysis of cholesterol esters by cholesterol oxidase, cholesterol esterase and peroxidase. glucose was analyzed as a method described by siegert (1987). alanine aminotrasferase (alt) and aspartate aminotransferase (ast) were measured using thomas' (1998) procedure. method of mihara et al. (1988) was used to analyze alkaline phosphatase (alp) and p-nitrophenylphosphate is used as a substrate and light absorption was evaluated at 400 nm. uric acid contents were determined using uricase as described by valovage and brooks (1979); this enzyme produces a purplish color which has a direct correlation (at 500 nm) with uric acid concentration. urea was measured with urease gdh kit (biochem. co, iran). in this method, ammonia ion is produced by urease enzyme and second reaction was catalyzed by glutamate dehydrogenase. finally, reducing absorption rate was calculated at 340 nm. for evaluating lactate dehydrogenase (ldh), king’s method (1965) was used. based on this method, the catalytic potential of the enzyme in conversion of lactate to pyrouvat and simultaneously the reduction of nad+ to nadh is considered. alpha-amylase was measured using kondo et al (1988) method. in this method, cnpg3 substrate is used in which 2chloro-4-nitrophenol has been bound to maltoriose. cnpg3 is hydrolysed by alpha-amylase and its concentration is determined at 405 nm. statistical and clustering methodology all data were analyzed using sas software and tukey’s studentized range (hsd) test in a complete randomized design (sas, 1997). hierarchical agglomerative clustering was done using ntsys software, employing the method of average linkage between groups (romesburg, 1984) under upgma (unweighted pair-group method sing arithmetic average). the clustering was based on the squared euclidean distance. the average linkage between two groups are considered as the average of distance among all pairs of cases with one number from each group. hierarchical clustering analysis was carried out by considering all ten biochemical parameters. results the quantitative differences of analyzed compounds activity levels and the amount of biochemical compounds in 4th instar larvae from five groups of rice stripped stem borer have been represented in figs 1 and 2. in all larval groups, significant differences among enzyme activity levels and amount of non-enzymatic compounds were observed, except for ast and alt. activity of ast in different groups was fluctuating between 1,420 to 21 fig. 1 changes of nonenzymatic macromolecules in five populations of rice stem borer. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). 2,850 iu/l (table 1). the minimum value of this enzyme was measured in larvae of am and maximum value was in larvae of ra. the activity level of alt was less than ast and the minimum and maximum value of it was observed in ra and sh, respectively. however, these enzymes were not significantly different among various groups of larvae (fig. 1). the amount of alp had a significant difference in all populations (table 1). the highest amount of this enzyme (1,006 iu/l) was measured in am population and the lowest amount (261 iu/l) was evaluated in go population. activity levels of alphaamylase and ldh also showed significant differences in various populations of rice stripped stem borer. the maximum value of alpha-amylase and ldh were 40 iu/l, 1,040 iu/l in go and 21iu/l, 249 iu/l in sh, respectively. the measurement of five non-enzymatic compounds including total protein, cholesterol, uric 22 fig. 2 changes of enzymatic macromolecules in five populations of rice stem borer. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). acid, urea and glucose in the larvae of various regions demonstrated significant differences (fig. 2). the highest value of protein (2.6 g/dl) was measured in am and the lowest value was evaluated in ra. the highest and the lowest amount of urea were 7 mg/dl and 3 mg/dl, which were observed in ba and am larvae, respectively. the uric acid amount had the maximum value in go population (3.4 mg/dl) and the minimum value in ba population (104 mg/dl). the highest and the lowest value of cholesterol were observes in go and ba larvae, which were measured 35 mg/dl and 16 mg/dl, respectively. finally, the amount of glucose was the maximum in ba population (341 mg/dl) and minimum in am population (131 mg/dl). hierarchical agglomerative clustering table 2 shows genetic distances in five groups of larvae based on enzymatic activity levels. as it shows, genetic distance between ba and sh larvae 23 table 1 enzyme activity and non-enzymatic compounds amount of 4th instar larvae of stripped stem borer biochemical compounds no. range mean f value c. v. aspartate aminotrasferase (iu/l) 30 1,420-2,580 1,973.79 1.90 13.28 alanine aminotransferase (iu/l) 30 1,600-2460 2,414.82 0.87 114.90 alkaline phosphatase (iu/l) 30 261-926 633.20 89.83 10.48 alpha-amylase (iu/l) 30 21-40 33.13 13.02 10.11 lactate dehydrogenase (iu/l) 30 249-1040 616.06 187.43 7.88 total protein (g/dl) 30 1-2.6 1.56 5.78 16.60 cholestrol (mg/dl) 30 16-35 22.68 10.25 15.93 glucose (mg/dl) 30 131-341 261.75 32.9 8.39 urea (mg/dl) 30 3-7 4.51 4.39 19.18 uric acid (mg/dl) 30 1.4-5 2.48 7.36 23.69 table 2 genetic distance of five groups of rice striped stem borer based on enzyme activity levels populations go sh ra ba am gourabzarmikh (go) 0.0000 sheikhmahale (sh) 12.864 0.0000 rasht (ra) 6.1312 9.0180 0.0000 baboul (ba) 12.785 15.186 10.314 0.0000 amoul (am) 13.149 6.0702 7.5441 6.9349 0.0000 table 3 genetic distance among five different populations of rice striped stem borer based on non-enzymatic compounds populations go sh ra ba am gourabzarmikh (go) 0.0000 sheikhmahale (sh) 5.8316 0.0000 rasht (ra) 6.8027 79.599 0.0000 baboul (ba) 20.219 9.0972 8.1452 0.0000 amoul (am) 16.682 6.4529 5.9860 18.100 0.0000 table 4 genetic distance among five different populations of rice striped stem borer based on all biochemical parameters populations go sh ra ba am gourabzarmikh (go) 0.0000 sheikhmahale (sh) 10.8316 0.0000 rasht (ra) 6.1027 7.599 0.0000 baboul (ba) 18.269 12.100 8.1552 0.0000 amoul (am) 12.722 8.2548 6.6560 14.1020 0.0000 was maximum (15.86) and the most similarity was measured 6.1312 between ra and go larvae. on the basis of these data, hierarchical clustering was divided into two groups, ba population was in one part and the rest of the populations were placed in another cluster. in current group, go population was separated from others (fig. 3). genetic distances in five groups of larvae based on non-enzymatic compounds are shown in table 3. the highest value, observed between ba and go groups, was 20.219, while the least value was between sh and go groups, 5.8316. hierarchical clustering on the basis of non-enzymatic compounds is similar to enzyme activity figure (fig. 4). 24 fig. 3 hierarchical cluster of five populations of rice striped stem borer based on enzymatic characteristics. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). in the table 4, genetic distance of groups on the basis of both enzyme levels and non-enzymatic compounds are shown. on the basis of these data, genetic distance of ba and go larvae with 34.904 was the highest and the least genetic distance was between ra and sh groups. the nearest group to them was ra and go whose genetic distance was 12.933. figure 5 demonstrates hierarchical analysis among these five groups on the basis of all biochemical parameters. on the basis of this dendrogram and a transaction in 50 %, larvae were divided into two distinct clusters; ba was in one part and the rest were placed in another part. in the second group, go population was separated from others. sh and ra population had the least distance. discussion in this study, the activities of two aminotransferases presented in the larval body were evaluated. the aminotransferases are important components of amino acid catabolism and they are mainly involved in transferring an amino group from one amino acid to another keto acid. the ast and alt serve as a strategic linkage between the carbohydrates and protein metabolism that are known to be altered during various physiological and pathological conditions (etebari et al., 2005). horie and nakamura (1986) figured out that activity of alt in the silk gland of silkworm larvae was much and demonstrated it to be more than the midgut and fat body, while maximum activity of ast was reported from the fat body. therefore, activity levels of these enzymes are different in various tissues. scaraffia et al. (2005) showed that when females of aedes aegypti ate a blood meal, activity level of these enzymes increased in fat body and midgut. researches have shown that isoenzyme pattern of ast is easily able to differentiate between the two species of stem borers chilo sp. (kioko et al., 1995). in this study, the amount of these enzymes didn’t demonstrate a significant difference among various groups. etebari et al. (2005) showed that the activity levels of aminotransferases have a significant difference among eight groups of silkworm. several factors are effective on amount of these enzymes. the increase in temperature causes the enhancement of alt and ast activity (reddy and benchmain, 1992), the diet and type of food also have high impact on activity levels of enzymes (gogoi and yadav, 1995). for this reason, rearing conditions such as variety of rice, temperature, relative humidity and etc were uniform for each population in this study. the sampling time and biochemical analysis were also similar for all populations so that results had the least side effects. but as it was said, no significant differences were measured among different populations. in the present study, a significant difference between activity levels of alp were observed in various groups. the alp is a set of hydrolytic enzymes that hydrolyze phosphomonoesters under the alkaline condition (miao, 1988). the production of this enzyme has a clear relationship with feeding behavior. in addition, activity of it depends on larval status, absorption, digestion and transportation of nutrients in midgut (eguchi and iwamoto, 1975; yoshitake et al., 1966). toxic chemicals in food decrease nutrition efficiency and alp activity. nathan et al. (2005) showed that treatment of rice plants with neem limonoids and melia azedarach extracts decreased the activity level of alp in cnaphalcrocis 25 fig. 4 hierarchical cluster of five populations of rice striped stem borer based on nonenzymatic characteristics. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). medinalis. they also showed that feeding of spodoptera litura on ricinus communis treated with azadirachtin decreased the amount of this enzyme in midgut. present results demonstrated that, because of suitable conditions, am group had been widely distributed, but go group was vice versa. the larvae of am, ba and ra regions have been sprayed with diazinon for more than 30 years. because of alp role in hydrolyzing phosphomonoesters, it could be concluded that there were some degrees of resistance in these populations. saeb (2002b) reported no chemical resistance in his field collected specimens however, we found that rice striped stem borer in ra, ba, am and sh had resistance ratio of 12.88, 12.81, 8.8 and 4.4 to diazinon, respectively in 2006 (unpublished data). ldh is an important glycolytic enzyme being present in virtually all tissues (kaplan and pesce, 1996); it is also involved in carbohydrate metabolism and has been used as an indicative criterion of exposure to chemical stress (wu and lam, 1997; diamantino, amadeu and soaresa, 2001). and it is used as an index of anaerobic metabolism (chamberlin and king, 1998). in this study, activity level of ldh in five groups of larvae showed a significant difference. nathan et al. (2005) showed that feeding of s. litura on r. communis treated with azadirachtin and nucleopolyhedrovirus decreased the amount of this enzyme in midgut that demonstrated low nutritional efficiency of the larvae. similar results were also observed on effectiveness of m. azedarach on rice leaffolder (nathan, 2006). kim et al. (2002) showed that feeding on different varieties of mulberry affect the amount of ldh and cholesterol in longicorn beetle. therefore, different factors such as feeding, growth stages and even type of tissues affect on quantitative changes of this enzyme in insect bodies. also, smith and collier (2001) showed that activity level of ldh among different population of orthopsyche embriata and acanthophlebia cruentata have no significant differences. in current study, all populations are affected by diazinon except for go larvae. because of stress caused by this chemical, the amount of ldh in go population was the highest among others. alpha-amylase is one of the midgut enzymes that is involved in starch and other carbohydrates metabolism. the activity level of this enzyme depends on feeding diet is different. in insects feeding on wool this enzyme is in the lowest amount while in phytophagous insects, it is the highest, especially in clethrophagous insects (chapman, 1998). in this study, amount of this enzyme was significantly different among various groups of larvae. go population had the maximum level of alpha-amylase that showed suitable feeding habitat. hirano and ishi (1961) showed that starch in rice stem was very suitable for nutrition of rice striped stem borer which higher amount of alpha-amylase in go population confirmed this idea. because in go area those varieties were planted that was more susceptible to rice stem borer than those in ba, hence, type of planted variety caused differences among these five different groups. 26 fig. 5 hierarchical cluster of five populations of rice striped stem borer based on all biochemical parameters. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). considerable differences were observed in the amount of five non-enzymatic compounds, including glucose, cholesterol, urea, uric acid and protein. on the basis of glucose amount, ba group was differentiated from others. etebari et al. (2005) showed that amount of glucose differentiates line 104, x2 and 107 from others in silkworm. etebari and matindoost (2004b) reported when the feeding activity was appropriate, glucose and cholesterol of the silkworm hemolymph increased and in these larvae a considerable improvement was observed in production characteristics. in contrast, when the feeding of larvae was interrupted, the amount of this compound severely decreased (etebari and matindoost, 2004a). several activities of insects depend on carbohydrates metabolism. the amount of glucose demonstrates the available sugar for cells that could represent the metabolism of carbohydrates (satake et al., 2000). the quality of consumed food and starvation affect the hemolymph sugar (etebari and matindoost, 2004b). satake et al. (2000) observed when the fifth instar larvae were under starvation, the glucose of hemolymph decreased immediately. etebari (2002) showed that the type of food could affect the amount of cholesterol in larval hemolymph. therefore, in the present study, larvae of each group were feed upon the same rice variety and the enhancement of cholesterol and glucose level could be relative to the efficiency of absorption system for each group of larvae. etebari and matindoost (2004b) demonstrated that adding vitamin b3 increased the amount of protein and cholesterol in silkworm hemolymph. in current study, enhancement of protein and cholesterol in ra and am larvae showed appropriate status of feeding. mosavi (1979) showed that there was a direct relationship between larval weight and fertilization in adults. the weight of insects depends on amount of carbohydrates, proteins, lipids and other substances. in this study, the weight of go larvae was 10-14 mg and ba larvae was 9-11 mg and amount of total protein, cholesterol, urea, uric acid and glucose were the highest in go population. these data showed that approximate digestibility (ad) of this population was better than ba population. on the basis of hierarchical clustering in different groups of rice stripped stem borer, all the studied populations were divided into two groups, ba in one and the rest in another group. this showed that ba group based on biochemical markers was different from others. in the second cluster, go population has been separated from others and sh and ra groups have the least genetic distance on the basis of biochemical parameters. chattarijee and data (1992) utilized the biochemical markers to classify 54 silkworm strains with different geographical origins. they also obtained similar results on some strains with different origin in one group and also strains with the same origin in different groups. identification of different biotypes in rice stripped stem borer is the most important for adoption of integrated pest management procedures. each biotype has a significant difference depending on environmental conditions such as temperature, humidity, food, chemicals and natural enemies. these factors cause changes in behavioral characteristics and damage level to plants. acknowledgments this study was supported by the university of guilan and rice research institute of iran (rrii). 27 we really appreciate dr a shadparvar, dr f majidi, dr m fazeli-dinan for their comments and mr z hashemi for his technical assistance and anonymous reviewers for their comments. references bartlett ac. the genetics of morphological and biochemical markers in two heliothis species. acta. phytopathol. entomol. hungarica, 24: 4953, 1989. chamberlin me, king me. changes in midgut active ion transport and metabolism during the fifth instar of the tobacco hornworm (manduca sexta). j. exper. zool. 280: 135-141, 1998. chapman,rf. the insect: structure and function. 4th edition. cambridge university press, 1998. chatterjee sn, datta,rk. hierarchical clustering of 54 races and strains of mulberry silkworm, bombyx mori: significance of biochemical parameters. theoret. appl. genet. 85: 394-402, 1999. chatterjee sn, rao c, chatterjee gk, ashwath sk, patnaik ak. correlation between yield and biochemical parameters in the mulberry silk mulberry silkworm, bombyx mori. theoret. appl. genet. 87: 385-391, 1993. dezfoulian a, moustofipoor p. rice striped stem borer. agricultural organization of iran publishing, 1972. diamantino tc, amadeu e, soaresa mv, guilherminoc. lactate dehydrogenase activity as an effect criterion in toxicity tests with daphnia magna, straus. chemosphere 45: 553-560, 2001. eguchi m, iwamoto a. changes in protease, esrerase and phosphatase in the alimentary canal of the silkworm during metamorphosis. insect. biochem. 5: 495-507, 1975. eguchi m. alkaline phosphatase isozymes in insects and comparison with mammalian enzyme. comp. biochem. physiol. 111b: 151-162, 1995. etebari k. effect of enrichment of mulberry leaves (morus alba) with some vitamins and nitrogenous compounds on some economic traits and physiological characters of silkworm bombyx mori (lep., bombycidae). ms thesis, isfahan university of technology, 2002. etebari k, matindoost l. effects of hypervitaminosis of vitamin b3 on silkworm biology. j. biosci. 29: 417-422, 2004a. etebari k, matindoost l. the study on effects of larval age and starvation stress on biochemical macromolecules abundance of hemolymph in silkworm bombyx mori. proceedings of the 16th iranian plant protection congress, general entomology symposium, august 28th-september 1st, 2004, the university of tabriz, iran, 2004b. etebari k, mirhoseini sz, matindoost l. a study on intraspecific biodiversity of eight groups of silkworm (bombyx mori) by biochemical markers. insect sci. 12: 87-94, 2005. gogoi r, yadav r. effect of host plants on some biochemical parameters of eri silkworm, philosamia ricini, during its development. indian j. exp. biol. 33: 372-374, 1995. hirano c, ishii s. utilization of dietary carbohydrates and nitrogen by rice stem borer larvae, under axenic conditions. entomol. exp. appl. 5: 5359, 1961. horie y, nakamura m. effect of dietary pyridoxine on alanine and aspartate aminotransferases in the silkworm, bombyx mori (lepidoptera: bombycidae). appl. entomol. zool. 21: 164170, 1986. kaplan la, pesce, aj. clinical chemistry-theory analysis and correlation. mosby-year book, mo, 1996. khanjani m. crop pests of iran. boali sina university press, 2006. khosroshahi m, dezfoulian a, nikjo f. a study on effect of granules and soluble pesticides on rice striped stem borer. j. plant pests dis. 40: 1626, 1979. kim hb, park kj, seuk ys, kim sl. morphological characteristics and physiological effects of mulberry leaves and fruits with wild varieties. korean j. sericul. sci. 44: 4-8, 2002. king j. the dehydrogenases or oxidoreductases. lactate dehydrogenase, in: van nostrand d (ed), practical clinical enzymology, london, pp 83-93, 1965. kioko en, overholt wa, omwega co, mueke jm. taxonomic significance of isoenzymes in two stem borers (lepidoptera: pyralidae) of maize and sorghum in kenya. african entomol. 3: 167-171, 1995. kondo h, shiraishi t, nagata k, tomita k. an enzymatic method for the α-amylase assay which comprices a new procedure for eliminatingh glucose and maltose in biological fluids. clin. chim. acta 172: 131139, 1989. loxdale hd, brooks, cp. temporal genetic stability within and restricted migration (gene flow) between local populations of the blackberry grain aphid sitobium avenae in southeast england. j. anim. ecol. 59: 497-514, 1990. miao y. study on the alkaline phosphatase in the midgut of domestic silkworm, bombyx mori l. acta sericol. sinica 14: 154-158, 1988. mihara y, saito a, koga k, sakaguchi b. changes in alkaline phosphatase activity during embryogenesis. j. sericol. sci. japan 52: 6267, 1988. moghaddas h, nasiri m. (1995). identification of rice striped stem borer in isfahan, study of its biology and distribution. 12th iranian plant protection congress, iran, 1995. mosavi, m. rice striped stem borer in iran. j. pest dis. inst. 47: 179-197, 1979. nathan ss, kalaivani k, chung pg. the effects of azadirachtin and nucleopolyhedrovirus on midgut enzymatic profile of spodoptera litura fab. (lepidoptera: noctuidae). pest. biochem. physiol. 83: 46-57, 2005. nathan ss. effects of melia azedarach on nutritional physiology and enzyme activities of the rice leaffolder cnaphalcrocis medinalis (guenee) (lepidoptera: pyralidae). pest. biochem. physiol. 84: 98-108, 2006. 28 reddy kv, benchaim kv. heat shock effect on testicular composition: a biochemical study in silkworm, bombyx mori. proc. indian natl. sci. acad.-biol. sci. 58: 329-332, 1992. richmond w. preparation and properties of cholesterol oxidase from nocardia sp. and its application to enzymatic assay of total cholesterol in serum. clin. chem. 19: 13501356, 1973. romesburg w. cluster analysis for researchers. lifetime learning publications, belmont, ca, 1973. saeb h, mohammad-salehi ms. study on different variety of rice resistance to rice striped stem borer in field. 13th iranian plant protection congress, iran, 1998. saeb h. study on resistance mechanisms of different rice germplasts to rice striped stem borer in guilan province. phd thesis, azad university, 1999. saeb h, geramy a. a study on spacious distribution of immature stages of rice striped stem borer in field. 14th iranian plant protection congress, iran, 2000. saeb h, tabrizian m, najafi-navaee a. study on attractance level of rice stem borer synthetic pheromones in field. 15th iranian plant protection congress, iran, 2002a. saeb h. study on susceptibility of different population of rice striped stem borer to sprayed pesticides in fields. 15th iranian plant protection congress, iran, 2002b. sas institute. sas/stat user’s guide for personal computers, sas institute, cary, nc, 1997. satake s, kawabe y, mizoguchi a. carbohydrate metabolism during starvation in the silkworm bombyx mori l. archiv. insect biochem. physiol. 44: 90-98, 2000. scaraffia p, isoe j, murillo a, wells, ma. ammonia metabolism in aedes aegypti. insect biochem. mol. biol. 35: 491-503, 2005. siegert kj. carbohydrate metabolism in manduca sexta during late larval development. j. insect physiol. 33: 421-427, 1987. smith pj, collier kj. allozyme diversity and population genetic structureof the caddisfly orthopsyche embriata and the mayfly acanthophlebia cruentata in new zealand streams. freshwater biol. 46: 795-805, 2001. stoikova t, popov p, grekov d, panayotov m. genetic control of nonspecific esterase in mulberry silkworm (bombyx mori) silkglans during ontogenesis. sericologia 38: 237-242, 1998. thomas l. clinical laboratory diagnostic. 1st ed. th books verlasgesellschaft, frankfurt, 1998. valovage wd, brooks ma. uric acid quantities in the fat body of normal and aposymbiotic german cockroaches blatella germanica. ann. entomol. soc. am. 72: 687-689, 1979. wu rss, lam pks. glucose-6-phosphate dehydrogenase and lactate dehydrogenase in the green-lipped mussel (perna viridis): possible biomarkers for hypoxia in the marine environment. water res. 11: 2797-2801, 1997. yoshitake n, eguchi m, akiyama a. genetic control on the alkaline phosphatase of 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice gene function and cellular pathways in higher vertebrates, including humans, have increasingly shown to be highly conserved thr isj 6: 7-14, 2009 issn 1824-307x review immunorecognition and immunoreceptors in the cnidaria sr dunn center for marine studies, university of queensland, australia accepted january 29, 2009 abstract recent studies that are focused on cnidarians as model systems for cell biology are offering key insight into the complexities of higher metazoan biology. the innate immune system of these basal invertebrates is one of the cellular processes that until recently, was undescribed. the knowledge regarding both innate immunity and other cellular processes in cnidarians is far from complete. however, the evidence acquired so far, suggests highly conserved components of these cellular processes are more closely related to vertebrate homologues than more complex, but divergent invertebrate model systems. this review examines the immunorecognition and receptors that have been identified within the cnidarians so far. the complement of receptors and pathways already identified indicates that these basal invertebrates are far from “simple” in the array of methods they possess for dealing with potential invading microbes and pathogens. key words: cnidaria; immunity; symbiosis; pathogen; symbiodinium; pamp; prr introduction gene function and cellular pathways in higher vertebrates, including humans, have increasingly shown to be highly conserved through metazoan evolution from the discovery of homologues in basal metazoans, such as sponges and cnidarians (kortschak et al., 2003; kuo et al., 2004; kusserow et al., 2005; dunn et al., 2006; hemmerich et al., 2007). the recent use of cnidarians as a metazoan model system (for review see weis et al., 2008), has brought about a wealth of new information that is unravelling the cellular processes involved in symbiosis, immunity regulation, cell death and organism longevity. there are, of course, considerable differences between the lower and higher metazoans: cnidarians lack a vertebrate-like adaptive immune system in so far as immunological memory is absent. furthermore, whilst amoebocytes have been shown to play a role in cnidarian cell defenses (olano and bigger 2000; mullen et al., 2004; mydlarz et al., 2008), specialization of cnidarian cells into components of an adaptive immune system is not evident. the ability of cnidarians to distinguish between self and non-self has previously been shown to occur in anthozoans and hydrozoans (frank et al., ___________________________________________________________________________ corresponding author: simon r dunn center for marine studies university of queensland st lucia, brisbane qld 4072, australia e-mail: s.dunn@cms.uq.edu.au 2001; rinkevich 2004; bosch, 2008). the capacity of allorecognition may vary across and between classes, colonies, such as anthopleura sp. (class: anthozoa) (lubbock, 1980) and solitary individuals, such as hydra sp. (class: hydrozoa) (kuznetsov and bosch, 2003). the molecular triggers that lead to allorecognition in cnidarians may utilize the innate immune response, however at present, the cellular mechanisms remain unknown and beyond the scope of this review. this review focuses upon the complexity of cnidarian immunorecognition. a cellular process that was once thought of as ‘basic’ in these simple metazoans is in fact complemented by an array highly conserved defenses, which offer key insight into the foundation of the higher metazoan innate immunity. one of the first lines of defense of the invertebrate immune system against potential invasive microbes are the pattern recognition receptors (prr’s). the host prr’s detect and bind to highly conserved components of microbe cell walls, such as proteins, lipids, carbohydrates and lipoteichoic acids (gram-positive bacteria) or lipopolysaccharides (lps; gram-negative bacteria), which form a recognisable matrix or pattern, known as pathogen-associated molecular patterns (pamp) or microbe-associated molecular patterns (mamps) (murphy et al., 2008). the activation of signal responses following the binding of host prr’s to pamp’s is rapid and can operate in three ways: firstly, to stimulate microbe ingestion through phagocytosis and enzymatic degradation. secondly, 7 through chemotactic directives for molecules to move to sites of infection, and thirdly, the induction of effector molecules that leads to a cell signal cascade and ultimately an immune response (murphy et al., 2008). at present, the complement of cnidarian prr’s appears to be diverse across classes, although the limited number of descriptive immunoreceptor studies still hinders a full evaluation and requires much more in-depth research. recent extensive cnidarian expressed sequence tag (est) and genome sequencing projects have highlighted the broad range of highly conserved biological processes within all metazoans that are also found in cnidarians, including the innate immune system (miller et al., 2007). across animal evolution, there has also been significant gene loss, and cnidarians are no exception. gene loss and duplication has occurred across all of the cnidarian classes, and suggests that the hydrozoa are divergent from basal anthozoa, and the scyphozoa from hydrozoa (miller et al., 2007; bosch, 2008). several key domains and conserved components of pathways associated with innate immune receptors that have already been identified are; 1) toll-like receptors (tlr’s) containing leucine rich repeats (lrr’s), which recognise pamps, 2) components similar to that of the complement cascade, and 3) lectins (for review see hemmerich et al., 2007; miller et al., 2007; kvennefors et al., 2008). toll-like and lrr receptors toll and toll-like receptors (tlr) are part of a metazoan receptor superfamily that all share a toll interleukin-1 receptor domain (tir). in the cnidarians, tlr-domain conserved proteins vary in number and structure. in the hydrozoan, hydra magnipapillata, there are four tlr-domain proteins described so far. two of which, hmmyd88-1 and hymyd88-2 are related to the downstream myd88, each displaying an additional characteristic death domain. the two remaining hydra tir-domain proteins, hytrr-1 and hytrr2 have a typical transmembrane and short extracellular scaffold, and are likely to be pathway initiator receptors. yet unusually, they lack the typical lrr-domains, and may be devoid of canonical structure (miller et al., 2007). however, this structural variation may not be uncommon or necessary for the same function (sun jin and lee, 2008). recent work by bosch et al. (in press) has revealed additional tlr-related lrr domain proteins in hydra may function synergistically with hytrr-1 and hytrr-2 in pamp recognition, indicating multiple receptor responses to an immune challenge operate in the cnidarians. the anthozoa, in comparison to hydrozoans, have a broader selection of tlr-domain proteins reflecting their basal phylogeny in the group. the estuarine sea anemone, nematostella vectensis have at least 5 tlr-domain proteins from the predicted structures. one of the tlr-domain proteins, nvmyd88 has a similar structure to the hmmyd88-1 and 2 and is thought to function in the same manner to induce expression of immune response genes through activation of the transcription factor, nfkb. a second tlr-domain protein, nvtlr-1, has multiple lrr domains and both carboxy and amino-terminal flanking cysteine rich motifs, which is characteristic of an ancestral domain structure (miller et al., 2007). the remaining identified predicted tlr-domain protein structures all contain immunoglobulin (ig) domains. the ig domains, which may function in cell-cell recognition, form a distinct clade away from the higher vertebrate structures. in comparison to other anthozoan sequences so far screened, n. vectensis has the highest complement of tlrs. at present, only one tlr-domain protein has been identified in the corals, acropora palmata and acropora millepora. both of the coral tlr-domains were similar to the n. vectensis ig-domain tlr, nvil1r1. however, no extracellular domains were detected in the coral receptors (miller et al., 2007). in addition to the tlr-domain structures described by (miller et al., 2007), downstream components of the toll/tlr pathways were also described from the est/ genome analysis, such as those associated with the c-jun n-terminal kinases (jnk)/mitogenactivated protein kinase (mapk) pathway and nfkb transcription that can lead to cell death. the depth of the known pathway homology indicates that it is not just toll/tlr receptors that are highly conserved, but a complete representative prr immune response pathway. antimicrobial peptides and metabolites an important contribution to the cnidarian immune response is the array of anti-microbial peptides. peptides are small signalling molecules that control a variety of processes, such as development, muscle contraction and the control of target gene expression (for review see bosch and fujisawa, 2001). antimicrobial peptides secreted by microbes within the mucus of corals are known to be a potent inter-specific microbial regulator of the epiphytic coral microbial community (ritchie, 2006). the composition of the cnidarian microbial community structure is important to host health (ritchie 2006; fraune and bosch, 2007), yet the roles of host anti-microbial peptides in host immunity until recently were unknown. host peptides have a stable structure that allows translocation to different areas around the cnidarian diploblastic body structure via the interepithelial space or mesoglea (fraune and bosch, 2007). the antimicrobial properties of peptides such as, aurelin, from the scyphozoan, aurelia aurita may focus activity on removing invasive gram-positive or gram-negative bacteria (ovchinnikova et al., 2006). host peptides may play a role in regulating associated microbial community populations to benefit the host, such as observed in the hydrozoan, hydra olgactis (fraune and bosch, 2007). in hydra, the induction of antimicrobial peptides is mediated by the interaction of a lrr domain protein with a tir-domain protein lacking lrr’s. the antimicrobial peptides from hydra, such as hydramacin-1 (jung et al., 2009), which is upregulated in the presence of lps and the peptide, pereculin-1, upregulated in the presence of lps and flagellin, are important components of the 8 microbe and stress host response. these particular peptides have been shown to have important therapeutic qualities as potent antibiotics against drug resistant human pathogens (bosch et al., in press). the initial descriptions of the antimicrobial peptide structures indicate that they are unique, but also, as is the case with aurelin, have similarity to defensins and k+-channel blocking toxins (ovchinnikova et al., 2006). a current rapidly expanding area of research is that of biodiscovery or bioprospecting. one target of this research are peptides, the other are secondary metabolites and their important roles in cellular homeostasis and defense against predation, parasites and disease (newman and hill, 2006; dunlap et al., 2007). the roles of many peptides and secondary metabolites as part of the cnidarian innate immune system is now being explored and have been shown to function as antioxidants and as antimicrobial compounds (mydlarz and jacobs 2004, shapo et al., 2007). however, the pathways associated with the synthesis and receptor mediation of these metabolites remain unknown. complement and lectins the complement signalling cascade is a major part of the vertebrate innate immune system, whereby microbes and foreign cells that are detected undergo opsonisation, phagocytosis and lysis. complement is composed of four pathways. three of the pathways are involved in activation and contain a thiolester c3 component, which leads to the fourth membrane attack complex (mac) lytic pathway (murphy et al., 2008). in vertebrates the primary function of c3, c4 and other members of the alpha-2-macroglobulin (a2m) paralogous gene family is opsonisation of microbes or immune complexes (armstrong and quigley, 1999). there are complement or precursors of the complement pathways, in the form of c3-like thiolester-containing proteins (tep) that predate the protostome-deuterostome split identified in cnidarians (dishaw et al., 2005; miller et al., 2007). the first c3-like, tep cdna to be identified from the gorgonian coral, swiftia exserta, had high conservation to vertebrate c3. there was an overall similarity with mammalian c3, c4 and c5 sequences, with a characteristic anaphylatoxin region which is absent in other a2m proteins, and cleavage sites of vertebrate c4 (dishaw et al., 2005). this basal, multiple ‘attribute’ domain protein, which may predate a later divergence into a larger protein family with individual domains and specific function, has previously been suggested to be a feature of cnidarians, such as caspases and bcl-2 family members of the apoptotic pathway (dunn et al., 2006). a similar c3-like protein has also identified in the anthozoans, acropora and nematostella, and although the counterpart in hydra was absent, hydra was shown to contain an a2m-like protein. it is interesting to note that all forms were restricted to the endoderm/gastrodermal tissues respectively (miller et al., 2007). although they have different structures, all forms may play a role in opsonisation fig. 1 a laser confocal microscopy image of the surface of a cultured symbiodinum sp. labelled with fitc-labelled concanavalin-a lectin (green), which binds specifically to glycans, including α-mannose. (fc: 100 μg/ml). sample prepared by d logan and s davy, university of wellington, new zealand in accordance with wood-charlson et al. (2006). image taken by srd. key: red = chlorophyll autofluorescence. bar = 5 μm. and therefore, their presence in cells associated with food particle /microbe selection and uptake is not surprising. in addition, miller et al. (2007) described a suite of predicted proteins with a membrane attack complex (mac) and perforin domains associated with the final phase of the complement cascade, indicating that multiple components from different stages of the complement cascade pathways exist in the cnidarians. activation of complement-like pathways and opsonization through the formation of a lectinbinding complex has been indicated in cnidarians during the onset of the complement-like cascade. the lectin-binding complex may also be of particular relevance in the onset and specificity of the prolific symbiosis with the dinoflagellate, symbiodinium sp., found in many cnidarians. the role of lectins in cell surface recognition of potential pathogens or symbionts is the important first step in cnidarian cell surface recognition. in previous studies using glycosides and concanavalin-a lectin to bind and mask the surface sugars of symbiodinium sp. (fig. 1) prior to infection of aposymbiotic hosts, have shown differential uptake and onset of symbioses (jimbo et al., 2000; lin et al., 2000; koike et al., 2004; wood-charlson et al., 2006). at present, only one cnidarian lectin has been characterised, millectin from the scleractinian anthozoan, a. millepora (kvennefors et al., 2008). millectin has sequence homology to the lectin domain of a range of c-type lectins and is a relative of both the collectins, mannose binding lectin (mbl) and the surfactant, sp-a. in addition, millectin has the unusual characteristic of having extensive variability in the substrate binding region, indicating 9 a potential broad range of pamp’s that may be recognized and bound by millectin/s, and in part, may be due to a dual role in pathogen/potential symbiont recognition of this receptor (kvennefors et al., 2008). cnidarian cells detect previously ‘undetectable’ or persistent invading microbes are only now being identified and may also operate to remove dysfunctional symbionts under stress (for review see weis, 2008). two key components of this intracellular innate immune repertoire can lead to cell death activation: firstly, the upregulation of nitric oxide (no) in the anthozoan, aiptasia pallida in response to lps, and hyperthermic stress (perez and weis, 2006). although inducible nitric oxide synthase is still yet to be identified in cnidarians, nitric oxide up-regulation has been shown to play an active role in the removal of symbiont symbiodinium sp. under symbiosis stress through cell death pathway activation (trapido-rosenthal et al., 2001; perez and weis, 2006). intracellular immune receptors so far, this review has covered receptors associated with intercellular-mediated innate immune recognition, which act as a first line of defense and gateway to phagocytosis and entry into the host. cnidarian innate immunorecognition is not limited to the cell surface or just the ‘gate’ into the cell. the cnidarian cell is well equipped with intracellular receptors and pathways that act as a second line of defense to recognise and remove the “trojan horse” that has managed to slip through the first line of defense and is now inside the walls. this multilevel approach to microbe recognition for removal of the microbe/pathogen and/or retention of the symbiotic ‘rent payer’ is key to the onset and specificity of symbiosis known as the ‘winnowing process’ that was first described in the squid, euprymna scolpes and vibrio fischeri bacterium, symbiosis (nyholm and mcfall-ngai, 2004). secondly, expression of a member of the cd36 family, the scavenger receptor sr-b1, is upregulated in the symbiotic anthozoan, anthopleura elegantissima compared to aposymbiotic individuals, (rodriguez-lanetty et al., 2006). cd36 family members including srb-1, act in host defense through the lipid metabolism. members of the cd36 family are known to be manipulated by invasive pathogens to gain entry into the host cell, (stafford et al., 2002) and may be controlled through bridging molecules, such as thrombospondin-1, c1q collectins and β2 glycoproteins (for review see májai, 2006). although est’s to homologs of a number of bridging molecules have been identified in a.millepora (meyer et al., 2007), a direct link between either lipids or bridging molecules and cd36 in host defense is yet to be shown in cnidarians. one of the intracellular recognition immune response found in cnidarians is the increased enzyme driven production of melanin (petes et al., 2003; mydlarz et al., 2008; palmer et al., 2008). the phenoloxidase (po) cascade that leads to melanin production, is known to play an active associated role with phagocytic aggregation and microbe/pathogen removal in many other invertebrates (johansson and söderhäll, 1996). initiation of the po cascade through pro-form cleavage in other invertebrate systems may vary according to substrate, suggesting a functioning role in recognition. in arthropods, activation occurs through contact with specific polysaccharides such as β-1,3-glucan (β13g), lps and peptidoglycan, in urochordates the inducers are lps and β13g, and in echinoderms only β13g has been shown to activate the cascade (johansson and söderhäll, 1996). in cnidarians, differential po activity was shown by experimental substrate addition to samples of both a healthy and compromised branching acroporid coral species, and to a lesser degree in the massive porites spp. (palmer et al., 2008). however, although previous studies have detected increased melanin within coral tissues (petes et al., 2003; mydlarz et al., 2008; palmer et al., 2008), it is important to note that similar host pigmentation occurs in response to a multitude of different stimuli (roff et al., 2008), and quite normally is often more visible in areas of healthy new growth or in areas of varying symbiotic dinoflagellate density. therefore, defining and attributing a cause to the different areas of host pigmentation is important to resolving the extent to which the po-melanin pathway is activated and associated with inflammation and immunity across a broad spectrum of cnidarian hosts. in all metazoans the destruction or removal of microbes/pathogens through host cell apoptosis, autophagy or induced microbe programmed cell death (pcd) can occur at initial contact stage to prevent infection, or at an intracellular level to mitigate damage (james and green, 2004). the initiation of cnidarian host apoptosis, autophagy and / or in situ symbiont pcd /necrosis, is known to occur during development (cikala et al., 1999), allorecognition (seipp et al., 2001; kuznetsov and bosch, 2003), in response to disease (ainsworth et al., 2007), in response to hyperthermic oxidative stress (dunn et al., 2002, 2004, 2007; franklin et al., 2004; richier et al., 2006), and as a postphagocytic removal mechanism of symbiodinium sp. during the onset of symbiosis (dunn and weis, 2009). the success of symbiosis in cnidarians appears to stem from the ability to restrain or prevent autophagy/apoptotic cell death pathways (rodriguez-lanetty et al., 2006; dunn et al., 2007), and corresponds with similar cell death inhibition in other invertebrate symbioses (pannebakker et al., 2007). intracellular parasites (the “trojan horse”) of vertebrates host cells, such as leishmania donovani and mycobacterium tuberculosis, also avoid immuno-detection by retarding apoptotic and autophagic pathways (for reviews see dermine et al., 2000; koul, 2004; gutierrez et al., 2004). the pathogenic control over these pathway occurs by manipulating ca2+ signalling, phosphoinositide metabolism, phosphatidylinositol 3-kinase signalling the intracellular immune receptors by which 10 fig. 2 a schematic representation of the innate immunoreceptors and associated protein domains, and peptides found in cnidarians that are prosed to contribute to the innate immune response. some receptors are part of the primary response at the cell surface leading to increased secretion of peptides and lectins or to a secondary intracellular response, gene expression and pathway activation. key to abbreviations are reported in table 1. cascade and inhibiting pro-cell death molecular triggers (fratti et al., 2001; chua et al., 2004; koul et al., 2004; hilbi 2006). whether the cnidariandinoflagellate symbiosis is controlled by the host, or if there is a symbiont directed manipulation of host immunity and associated cell-death pathways at present remains unresolved. in conclusion, there is now strong evidence that cnidarian innate immunorecognition is far from simple and members of all classes display a diverse armoury of innate immune receptors that operate at both an inter-cellular and intra-cellular level. the interaction between tlr-domain and lrr domain proteins and the production of peptides (possibly with the addition of secondary metabolites) is complemented with lectin secretion that may interact with a c3-like complement phagocytic signalling cascade (fig. 2). this multiple attribute defense system leading to an immune response, offers a key insight into the basal function of more complex innate immune pathways found in vertebrates. there is no doubt that the discovery of homologous, highly conserved cell pathways, and their functioning molecular components within cnidarians, will continue to promote members of the phylum as an ideal model system for the study of not only innate immunity, but much broader areas of higher metazoan cell biology. table 1 different classes of receptors retrieved in cnidarians and their conserved domains abbreviation domain name lrr leucine rich repeat tmd trans membrane domain tir (toll-like) toll interleukin-1 receptor ig immunoglobulin a2m alpha-2-macroglobulin sr-b1 scavenger receptor b1 cell surface receptor signalling pro-po prophenoloxidase tir/ death tir (as above) /death domain po phenoloxidase no nitric oxide intracellular signalling ros reactive oxygen species 11 acknowledgements i would like to thank c kvennefors, m pernice and s dove for their comments and views, and t bosch for the communications provided in advance of publications. references ainsworth td, kvennefors ec, blackall ll, fine m, hoegh-guldberg o. disease and cell death in white syndrome of acroporid corals on the great barrier reef. mar. biol. 151: 19-29, 2007. armstrong pb, quigley jp. alpha-2 macroglobulin: an evolutionary conserved arm of the innate immune system dev. comp. immunol. 23: 375390, 1999. bosch tcg. the path less explored: innate immune reactions in cnidarians. in: heine h (ed), innate immunity of plants, animals, and humans (vol. 21), springer-verlag, berlin, pp 27-42, 2008. bosch tcg, augustin r, anton-erxleben f, fraune s, hemmerich g, zill h, et al. uncovering the evolutionary history of innate immunity: the simple metazoan hydra uses epithelial cells for host defence. dev. comp. immunol. [in press]. bosch tcg, fujisawa t. polyps, peptides and patterning. bioessays 23: 420-427, 2001. chua j, vergne i, master ss, deretic v. a tale of two lipids: mycobacterium tuberculosis phagosome maturation arrest. curr. opin. microbiol. 7: 71-77, 2004. cikala m, wilm b, hobmayer e, böttger a, david cn. identification of caspases and apoptosis in the simple metazoan hydra. curr. biol. 9: 959962,1999. dermine j-f, scianimanico s, privé c, descoteaux a, desjardins m. leishmania promastigotes require lipophosphoglycan to actively modulate the fusion properties of phagosomes at an early step of phagocytosis. cell. microbiol. 2: 115126, 2000. dishaw lj, smith sl, bigger ch. characterization of a c3-like cdna in a coral: phylogenetic implications. immunogenetics 57: 535-548, 2005. dunlap wc, battershill cn, liptrot ch, cobb re, bourne dg, jaspars m, long pf, newman dj. biomedicinals from the phytosymbionts of marine invertebrates: a molecular approach. methods 42: 358-376, 2007. dunn sr, weis vm. apoptosis as a post-phagocytic winnowing mechanism in a coral-dinoflagellate mutualism. environ. microbiol. 11: 268-276, 2009. dunn sr, schnitzler ce, weis vm. apoptosis and autophagy as mechanisms of dinoflagellate symbiont release: every which way you loose. proc. r. soc. b. 274: 3079-3085, 2007. dunn sr, phillips ws, spatafora jw, green dr, weis vm. highly conserved caspase and bcl-2 homologues from the sea anemone aiptasia pallida: lower metazoans as models for the study of apoptosis evolution. j. mol. evol. 63: 95-107, 2006. dunn sr, thomason jc, le tissier mda, bythell jc. heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration. cell. death differ. 11: 1213-1222, 2004. dunn sr, bythell jc, le tissier mda, burnett wj, thomason jc. programmed cell death and cell necrosis activity during hyperthermic stressinduced bleaching of the symbiotic sea anemone aiptasia sp. j exp. mar. biol. ecol. 272: 29-53, 2002. frank u, leitz t, müller weg. the hydroid hydractinia: a versatile, informative cnidarian representative. bioessays 23: 10, 963-971, 2001. franklin dj, hoegh-guldberg o, jones rj, berges ja. cell death and degeneration in the symbiotic dinoflagellates of the coral stylophora pistillata during bleaching. mar. ecol. prog. ser. 272: 117-130, 2004. fratti ra, backer jm, gruenberg j, corvera s, deretic v. the role of phosphatidylinositol 3kinase and rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. j. cell. biol. 154: 631-643, 2001. fraune s, bosch tcg. long-term maintainance of species-specific bacterial microbiota in the basal metazoan hydra. proc. natl. acad. sci. usa 104: 32, 2007. gutierrez mg, master ss, singh sb, taylor ga, colombo mi, deretic v. autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages. cell 119: 753-766, 2004. hemmerich g, miller dj, bosch tcg. the evolution of immunity: a low-life perspective. trends immunol. 28: 449-454, 2007. hilbi h. modulation of phosphoinositide metabolism by pathogenic bacteria. cell. microbiol. 8: 16971706, 2006. james er, green dr. manipulation of apoptosis in the host-parasite interaction. trends parasitol. 20: 280-287, 2004. jimbo m, yanohara t, koike k, koike k, sakai r, muramoto k, et al. the d-galactose-binding lectin of the octocoral, sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates. comp. biochem. physiol. 124a: 227-236, 2000. johansson mw, söderhäll k. the prophenoloxidase activating system and associated proteins in invertebrates. in rinkevich b, muller weg (eds), invertebrate immunology, springerverlag, berlin, pp 46-66, 1996. jung s, dingley aj, augustin r, anton-erxleben f, stanisak m, gelhaus c, et al. hydramacin-1: structure and antibacterial activity of a protein from the basal metazoan hydra. j. biol. chem. 284: 1896-905, 2009. koike k, jimbo m, sakai r, kaeriyama m, muramoto k, ogata t, et al.. octocoral chemical signalling selects and controls dinoflagellate symbionts. biol. bull. 207: 80-86, 2004. kortschak dr, samuel g, saint r, miller dj. est analysis of the cnidarian acropora millepora reveals extensive gene loss and rapid sequence divergence in the model invertebrates. curr. biol.13: 2190-2195, 2003. 12 koul a, herget t, klebl b, ullrich a. interplay between mycobacteria and host signalling pathways. nat. rev. microbiol. 2: 189-202, 2004. kuo j, chen m-c, lin c-h, fang ls. comparative gene expression in the symbiotic and aposymbiotic aiptasia pulchella by expressed sequence tag analysis. biochem. biophys. res. comm. 318: 176-186, 2004. kusserow a, pang k, sturm c, hrouda m, lentfar j, schmidt h, et al. unexpected complexity of the wnt gene family in a sea anemone. nature 433: 156-160, 2005. kuznetsov sg, bosch tcg. self/nonself recognition in cnidaria: contact to allogeneic tissue does not result in elimination of nonself cells in hydra vulgaris. zoology 106: 109-116, 2003. kvennefors ece, leggat w, hoegh-guldberg o, degnan bm, barnes ac. an ancient and variable mannose-binding lectin from the coral acropora millepora binds both pathogens and symbionts. dev. comp. immunol. 32: 15821592, 2008. lin kl, wang jt, fang ls. participation of glycoproteins on zooxanthellal cell walls in the establishment of a symbiotic relationship with the sea anemone, aiptasia pulchella. zool. stud. 39: 172-178, 2000. lubbock r. 1980. clone-specific cellular recognition in a sea anemone. proc. natl. acad. sci. usa 77: 6667-6669, 1980. májai g, petrovski g, fésüs l. inflammation and the apopto-phagocytic system, immunol. lett. 104: 94-101, 2006. meyer e, aglyanova g, buchannan-carter j, colbourne j, willis bl, matz m. (www.bio.utexas.edu/research/matz%5flab/mat zlab/454.html), 2007. miller dj, hemmerich g, ball e, hayward dc, khalturin k, funayama n, agata k, bosch tcg. the innate immune repertoire in cnidaria ancestral complexity and stochastic gene loss. gen. biol. 8: r59, 2007. mullen km, peters ec, harvell cd. 2004. coral resistance to disease. in rosenberg e, loya y, (eds), coral health and disease, springerverlag, new york, pp 377-399, 2004. murphy k, travers p, walport m. janeway's immunobiology (7th ed), garland science, new york, 2008. mydlarz ld, jacobs rs. comparison of an inducible oxidative burst in free-living and symbiotic dinoflagellates reveals properties of pseudopterosins. phytochemistry 65: 32313241, 2004. mydlarz ld, holthouse sf, peters ec, harvell cd. cellular responses in sea fan corals: granular amoebocytes react to pathogen and climate stressors. plos one 3(3):e1811, 2008. newman dj, hill rt. new drugs from marine microbes. j. ind. microbiol. biotech. 33: 539544, 2006. nyholm sv, mcfall-ngai mj. the winnowing: establishing the squid-vibrio symbiosis. nat. rev. microbiol. 2: 632-642,2004. olano ct, bigger ch. phagocytic activities of the gorgonian coral, swiftia exserta. j. invert. path. 76: 176-184, 2000. ovchinnikova tv, baladin sv, aleshina gm, tagaev aa, leonova yf, krasnodembsky ed, et al. aurelin, a novel antimicrobial peptide from jellyfish aurelia aurita with structural features of defensins and channel-blocking toxins. biochem. biophys. res. comm. 348: 514-523, 2006. palmer cv, mydlarz ld, willis bl. evidence of an inflammatory-like response in non-normally pigmented tissues of two scleractinian corals. proc. r. soc. b 275: 2687-2693, 2008. pannebakker ba, loppin b, elemans cph, humblot l, vavre f. parasitic inhibition of cell death facilitates symbiosis. proc. natl. acad. sci. usa 104: 213-215, 2007. perez s, weis vm. nitric oxide and cnidarian bleaching: an eviction notice mediates breakdown of a symbiosis. j. exp. biol. 209: 2804-2810, 2006. petes le, harvell cd, peers ec, webb mah, mullen km. pathogens compromise reproduction and induce melanization in caribbean sea fans. mar. ecol. prog. ser. 264: 167-171, 2003. richier s, sabourault c, courtiade j, zucchini n, allemand d, furla p. oxidative stress and apoptotic events during thermal stress in the symbiotic sea anemone, anemonia viridis. febs j. 273: 4186-4198, 2006. rinkevich b. allorecognition and xenorecognition in reef corals: a decade of interactions. hydrobiology 531: 443-450, 2004. ritchie kb. regulation of microbial populations by coral surface mucus and mucus-associated bacteria mar. ecol. prog. ser. 322: 1-14, 2006. rodriguez-lanetty m, phillips ws, weis vm. transcriptome analysis of a cnidariandinoflagellate mutualism reveals complex modulation of host gene expression. bmc gen. 7: 23, 2006. roff g, ulstrup ke, fine m, ralph pj, hoeghguldberg o. spatial heterogeneity of photosynthetic activity within diseased corals from the corals from the great barrier reef. j. phycol. 44: 526-538, 2008. seipp s, schmich j, leitz t. apoptosis a deathinducing mechanism tightly linked with morphogenesis in hydractinia echinata (cnidaria, hydrozoa). development 128: 48914898, 2001. shapo jl, moeller pd, galloway sv. antimicrobial activity in the common seawhip, leptogorgia virgulata (cnidaria: gorgonaceae). comp. biochem. physiol. b 148: 65-73, 2007. stafford jl, neumann nf, belosevic m. macrophage-mediated innate host defence against protozoan parasites. crit. rev. microbiol. 28: 187-248, 2002. sun jin m, lee j-o. structure of tlr-ligand complexes. curr. opin. immunol. 20: 414-419, 2008. trapido-rosenthal hg, sharp kh, galloway ts, morrall ce. nitric oxide and cnidariandinoflagellate symbiosis: pieces of a puzzle. amer. zool. 41: 247-257, 2001. weis vm, davy sk, hoegh-guldberg o, rodriguezlanetty m, pringle jr. cell biology in model systems as the key to understanding corals. trends ecol. evol. 23: 369-376, 2008. 13 http://www.bio.utexas.edu/research/matz%5flab/matzlab/454.html http://www.bio.utexas.edu/research/matz%5flab/matzlab/454.html weis vm. cellular mechanisms of cnidarian bleaching: stress causes the collapse of symbiosis. j. exp. biol. 211: 3059-3066, 2008. wood-charlson em, hollingsworth ll, krupp da, weis vm. lectin/glycan interactions play a role in recognition in a coral/dinoflagellate symbiosis. cell. microbiol. 8: 1985-1993, 2006. 14 ii scientific meeting of the italian ascidiologists, 30 june – 1 july 2008, department of isj 6: s1-s2, 2009 issn 1824-307x editorial ii° scientific meeting of the italian ascidiologists: dedicated to professor giuseppe reverberi n parrinello department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 periodically the italian ascidiologists are engaged in a meeting on ascidian biology. the 2008 meeting has been dedicated to the memory of prof giuseppe reverberi who, in the second half of the past century, was devoted to study of ascidian developmental biology. he held (1948-1971) the chair of zoology at the university of palermo (italy) where, with valid collaborators, started and strengthened the main scientific route leading to significant improvements, internationally appreciated, in embryology with an experimental approach. he dedicated his life to research and was a master for numerous scholar generations who attained the frame of the scientific method and achieved keenness for research in animal biology. a lot of scholars and scientists, inside and outside his schooling place, appreciated his rigor in research and the valuable teaching method that also improved their human and scientific personality. prof f de bernardi (university of milan, italy), ascidian embryologist, validly retraced the reverberi scientific activity that still nowadays is a milestone in ascidian development study. the meeting broadened diverse fields of ascidian biology and appreciated lectures and oral communications have been presented. due to their phylogenetic key position, ascidians have attained importance for evolutionary studies. the genome of some solitary species (ciona intestinalis, ciona savignyi, halocynthia roretzi) has been fully or partially sequenced, analyzed and annotated and can be validated by gene expression patterns for several specific biological properties and activities. the first session was rich in reports on development and morphogenesis including differentiation of adult sensory organs and larval papillae, developmental expression and gene organization of synapses, distribution of larval neural phenotypes, meiotic progression and fertilization, musculature differentiation and gene expression, evolution of anterior hox regulatory elements, larval metamorphic and juvenile phases, role of nitric oxide ___________________________________________________________________________ corresponding author: nicolò parrinello department of animal biology university of palermo via archirafi 18, 90123 palermo, italy e-mail: nicpar@unipa.it during the development, pigment organ formation during the embryogenesis, angiogenetic mechanism in the colonial circulatory system, and the effects of a xenobiotic on development and metamorphosis. the next session on phylogeny and microevolution concerned the homologies between chordate invertebrates and vertebrates as revealed by the neurophysiology of swimming tadpoles, the fast evolutionary dynamics of mitochondrial genome including new data and forward genetic that unveil and characterize two ciona cryptic species, and the phylogenetic conservation of csf-related genes. since this ascidian is a cosmopolitan representative model, cryptic species identification is of great relevance for research and comparative analysis of results involving several laboratories. at the second day, after a plenary lecture on stem cells and chimerism in colonial ascidians (a voskoboynik, stanford university, usa), the lectures were mainly devoted to immunity and inflammatory response. bioinformatic results, disclosed est and extensive in silico searches have concerned immunorelevant molecules, gene expression patterns and some specific immune characteristics. phenoloxidases, variable domaincontaining chitin-binding proteins (vcbps) genes and their expression, anaphylotoxin and specific receptors, immunomodulatory factors, hemocyte cytotoxic activity, collectin and cytokine-like cloning and genes expression, hemocytes provided with acetylcholinesterasis, were the topics of the reports. finally, some communications concerned with environmental stressors effects on embryos and hemocytes. several italian research groups, that esteemed the prof reverberi research, attended the meeting. besides the ascidiologists from the university of palermo who organized the event, scientists from the universities of padua, genoa, milan, bari, from the stazione zoologica “anton dohrn” of naples, and from palermo cnr, contributed in the success of the scientific happening. in particular, we were honoured for the interest and presence of the prof. armando sabbadin emeritus at the padua university and a founder of the research field on colonial ascidian allorecogniton. moreover prof e ottaviani, president in charge of the italian association of developmental and comparative immunology and editor in chief of the open access s1 journal “invertebrate survival journal”, was an appreciated guest. the abstracts of the scientific contributions have been published in inv. surv. j. 5: 83-96, 2008. acknowledgements we are grateful to prof g silvestri rector of the university of palermo, and to the department of animal biology of the university of palermo for their helpful support and sponsorship. thanks are due to prof e ottaviani, editor in chief, for his contribution in publishing the inv. surv. j. special issue. miur-prin cofin 2006-2008 grant to n parrinello also contributed to the meeting success. s2 61 isj 15: 61-65, 2018 issn 1824-307x visions and perspectives going beyond a static picture: the apple snail pomacea canaliculata can tell us the life history of molluscan hemocytes d malagoli department of life sciences, university of modena and reggio emilia, italy accepted february 27, 2018 abstract more than 40 years of studies on molluscan immunity have revealed a complex and dynamic immune system endowed with multifunctional circulating cells, i.e., hemocytes that are regulated by diverse signaling molecules. however, very little is known about the dynamic processes that drive hemocyte proliferation, differentiation, maturation, and senescence. evidence reported here highlights how the apple snail pomacea canaliculata is an extremely promising research organism that will provide answers to the numerous questions regarding the life-history of molluscan and lophotrochozoan hemocytes. key words: ampullariidae; gastropoda; hemocyte maturation; immunity; invertebrate hematopoiesis; pomacea canaliculata metazoans base their survival and reproductive success on the simultaneous functioning of different organ systems. while many organ systems have a well-defined location, clear anatomical borders, and a recognized physiological role, the immune system does not have a fixed anatomy, and its primary function is maintaining the equilibrium between the organism and all the microorganisms that live in or around it (bosch, 2014; bachère et al., 2015). this structure and function make it extremely difficult to identify and dissect the many activities that the immune system plays during homeostasis. most researchers have focused on mechanisms that are activated when the organism is challenged by pathogens or stressful events (malagoli et al., 2017). since the 1980s, numerous scientists have studied the immune system of invertebrates. they were principally attracted by its relative simplicity and the lack of components related to acquired immunity, such as lymphocytes and immunoglobulins. the main rationale for these analyses was to both clarify the functional basis of the innate component of the human immune system and to improve our understanding of the many pathologies resulting from the malfunctioning of the innate immune system (notarangelo et al., 2009). surprisingly, the invertebrate immune system has been revealed to be much more complex than ___________________________________________________________________________ corresponding author: davide malagoli department of life sciences university of modena and reggio emilia via campi 213/d, 41125, modena italy e-mail: davide.malagoli@unimore.it expected, changing experimental approaches and revising our current knowledge of the comparative immunology field. one of the main changes that has occurred in the last decade was the publication of numerous papers that revealed the high degree of complexity in the invertebrate immune system, in addition to the presence of highly variable molecules (armitage and brites, 2016; doolittle, 2016; oren et al., 2016). the involvement of these molecules in the immune response of invertebrates has yet to be understood, but it is undeniable that these molecules are strikingly similar to vertebrate antibodies (immunoglobulins), which are characterized by hypervariable regions. among invertebrates, the most important and commonly studied model is drosophila melanogaster (buchon et al., 2014). however, several other research organisms are available and well-studied, mainly because of their economic relevance (such as crustaceans and bivalves [smith et al., 2016; bachère et al., 2015]) or their importance as vectors of parasitic diseases (e.g., mosquitoes and some gastropods) (choi et al., 2012; adema et al., 2017). over the past few years, our research group has used a molluscan gastropod, pomacea canaliculata, as a research organism. this organism has great potential in the comparative immunology field because it gathers together many point of interest mentioned above, and many biological features make it easy to maintain in the lab and work with (fig. 1a). p. canaliculata (aka, the golden apple snail), is indexed among the most invasive species in the world (www.issg.org/worst100_species.html), and its fast and vast spread raised concerns for several reasons. in economic terms, p. canaliculata is a 62 fig. 1 a) the apple snail p. canaliculata laying eggs outside the water onto the wall of the tank. the eggs are bright pink because of the neurotoxic perivitellin fluid contained inside the egg shell. every egg clutch consists of hundreds of eggs. b) p. canaliculata hemocytes. the hemolymph has been collected and cytocentrifuged onto a slide. hemocytes have been stained with diff quik kit. a small blast-like cell (high nuclear/cytoplasmic ratio, white arrow), many large hyalinocytes (agranular blue cytoplasm), and a large granulocyte (granular cytoplasm, black arrows) are present. bar in a = 2 cm; bar in b = 20 um. voracious grazer of crops, and this problem is particularly relevant in asia (gilioli et al., 2017; lei et al., 2017) where this species was introduced as source of food. unfortunately, p. canaliculata was demonstrated to be potentially neurotoxic (sun et al., 2010) and not edible, and, thanks to the small number of predators, it freely eats and ultimately kills young rice plants (horgan, 2018; http://www.knowledgebank.irri.org/step-by-stepproduction/growth/pests-and-diseases/goldenapple-snails). aware of its impact on the environment and economy, both the eu and some states in the usa implemented tight regulations that forbid circulation and commercialization of the freshwater snails belonging to the genus pomacea (commission implementing decision, 2012; united states department of agriculture, animal and plant health inspection service [usda_aphis]; lei et al., 2017). pomacea and its relationship to the environment have also been studied, suggesting that this snail is a potential bio-indicator as a result of its ability to accumulate heavy metals (hoang et al., 2011). apple snails are also interesting from a biomedical perspective because they can regenerate complex organs de novo, such as the eyes and the tentacles (important tactile and chemosensory organs). recent studies have highlighted common aspects and mechanisms shared between adult regeneration and embryonic development (accorsi et al., 2017b). advances in the understanding of both regeneration and immune responses are especially interesting, and in the past few years, many researchers have been trying to elucidate the deep relationships between regeneration abilities and characteristics of the immune system by comparing different vertebrate and invertebrate model systems with varying regenerative abilities and types of immune systems (godwin et al., 2017; neves et al., 2016; tasiemski and salzet, 2017). the adaptability to different external conditions, the immune-tolerance towards a parasite (song et al., 2016), and the astonishing regenerative capacity of p. canaliculata are possible thanks to the role played by a common and fundamental component that is the immune system. as a consequence, the study of the immune system of p. canaliculata becomes of wide interest. the characterization of the cellular component of p. canaliculata immune system, aka the circulating hemocytes, demonstrated that p. canaliculata is not significantly different from other gastropods, since small, blastlike cells (aka pro-hemocytes) and larger hemocytes have been identified (accorsi et al., 2013; smith et al., 2016). among the larger hemocytes, hyalinocytes (agranular) and granulocytes (granular cytoplasm) were distinguished, with the former endowed with phagocytic activity (fig. 1b). direct observation of the organ structures and the comparison with other gastropod anatomical descriptions allow us to define the pericardial fluid a plausible candidate for the hematopoietic tissue. this tissue has a gel-like texture in younger animals, and it is more fluid in older adults. however, in both cases, it fills the pericardial 63 chamber in which the heart and the ampulla are housed (accorsi et al., 2014). after performing experiments involving repeated hemolymph withdrawal and immunostaining with a mitotic marker, we confirmed the presence of dividing cells in the pericardial fluid, and we hypothesized the involvement of the ampulla as a hemocyte reservoir. the tight functional and anatomical connections between the hemolymph, the heart, the ampulla and the pericardial fluid intrigued us, prompting us to perform further studies on hematopoiesis in the apple snail. real-time pcr experiments demonstrated the expression of a prokineticin-like protein in the apple snail pericardial fluid, supporting our model of hemocyte replication in this freshwater snail (accorsi et al., 2017a). these significant advances represent a solid foundation for studying the dynamic nature of the immune system of mollusks, particularly with respect to hemocyte turnover, maturation, and senescence. despite the characterization of hemocytes in mollusks (smith et al., 2016) and the identification and localization of a hematopoietic tissue/organ (accorsi et al., 2014; smith et al., 2016), hemocytes maturation still remains to be described, traced, and mechanistically understood. which anatomical sites are involved in this process? what are the stages of hemocyte differentiation and maturation? when does an hemocyte start to express the receptors that characterize it as a functional immune cell and when it becomes an old and no more functional cell? can this process be influenced by external events, such as environmental cues or pathogen exposure? (fig. 2). fig. 2 schematic representation of the hypothesis described in this paper about the turnover and maturation of p. canaliculata hemocytes. the hematopoietic cells (pink cells) proliferate in the hematopoietic organ(s) where they probably both self-renew and give rise to a population of immature hemocytes (orange cells) that differentiate. the differentiating cells mature into the hemocyte stage (green cells), which are functional until they become senescent hemocytes (grey cells) and eventually die. this process has not been carefully dissected in any mollusc. the hypothesis presented here suggests that mature and senescent hemocytes that are already functional can interact with maturing cells (blue cells), influencing their maturation process. this mature/maturing hemocyte interaction would drive the maturation of the new hemocytes towards specific and already encountered targets, enriching for a pool of cells active against the immune challenges that they are likely to face. besides hemocyte/hemocyte interactions, i hypothesize that also the environmental cues can influence the hematopoietic cell replication rate and hemocyte differentiation process, influencing the compositon of circulating cells on the basis of changes in environmental stimuli. this is, to the best of my knowledge, a new and dynamic perspective for considering the main cellular component of the molluscan immune system, consisting of the circulating hemocytes. the hemocytes have a short lifespan and constant turnover and this might provide to p. canaliculata its ability to overcome both persistent and new immune challenges during their long lifespan. 64 in accordance with observations performed also in other molluscan models (ottaviani et al., 2013; tascedda et al., 2015; malagoli and ottaviani, 2010), the main hypothesis of my laboratory is that the immune system of p. canaliculata is constantly refined on the basis of the immune stimulations encountered during an individual organism’s lifetime. this would also explain the remarkable capacity of these animals to quickly adapt and begin reproducing in new environments (accorsi, personal communication; gilioli et al., 2017; lei et al., 2017). to answer these and many other questions regarding the biology of p. canaliculata, several tools are now available thanks to the efforts of several laboratories around the world. recently, a few organ-specific transcriptomes have been published (yang et al., 2017; zhou et al., 2016), and the sánchez alvarado group at the stowers institute for medical research (kansas city, mo, usa) is developing an extensive database that includes organ-specific transcriptomes of many adult organs and the transcriptomes of many embryonic stages (accorsi, personal communication). proteomic studies have also been performed, which identified the presence of a neurotoxin in the perivitellin fluid of their eggs that likely evolved as defense against predators, expanding the scientific community’s interest in this model (sun et al., 2010, 2012; mu et al., 2017). at present, more than one research group is sequencing the genome of p. canaliculata in order to obtain a reliable genome database. the first assembly of the genome will soon be published and made available to the community working on apple snails (accorsi, personal communication; zhou et al., 2016; guo et al., 2018). the availability of these databases will provide our community the opportunity to efficiently search for genes, transcripts, and proteins of interest for both descriptive and functional studies (malagoli et al., 2011; tascedda et al., 2015; malagoli et al., 2017). altogether, this increasing body of evidence and the exponential growth and refining of molecular databases will allow a deeper understanding of the biology of p. canaliculata such that a wider set of experiments can be performed. while the data collected on p. canaliculata are increasing, as well as the number of papers published per year on this snail, it is important to emphasize how frequent species misidentification can be among apple snails. in this field, light has been shed by hayes et al. (2009), who described different apple snail species and their phylogenetic relationships, providing a detailed list of approaches to correctly identify each species (hayes et al., 2009, 2015; guo et al., 2018). the evidence recapitulated in this vision and perspectives show that p. canaliculata is a versatile model for several studies in the biological field and single it out among mollusks as one of the best candidate to study the process of replication, maturation, and senescence of immune-competent cells in lophotrochozoans. acknowledgments the author wishes to thank drs. a. accorsi and a. sánchez alvarado (stowers institute for medical research, kansas city, mo, usa) for helpful discussion and essential input. the author would also like to thank dr. s elliott for the careful linguistic revision of the manuscript. references accorsi a, benatti s, ross e, nasi m, malagoli d. a prokineticin-like protein responds to immune challenges in the gastropod pest pomacea canaliculata. dev. comp. immunol. 72: 37-43, 2017. accorsi a, bucci l, de eguileor m, ottaviani e, malagoli d. comparative analysis of circulating hemocytes of the freshwater snail pomacea canaliculata. fish shellfish immunol. 34: 12601268, 2013. accorsi a, ottaviani e, malagoli d. effects of repeated hemolymph withdrawals on the hemocyte populations and hematopoiesis in pomacea canaliculata. fish shellfish immunol. 38: 56-64, 2014. accorsi a, ross e, ottaviani e, sánchez alvarado a. pomacea canaliculata: a new model system for studying development and regeneration of complex eyes. in: proceedings of the 63rd congress of the italian embryological group (gei). eur. j. histochem. 61: 3 ,2017. adema cm, hillier lw, jones cs, loker es, knight m, minx p, et al. whole genome analysis of a schistosomiasis-transmitting freshwater snail. nat. commun. 8: 15451, 2017. armitage sao, brites d. the immune-related roles and the evolutionary history of dscam in arthropods. in: malagoli d (ed), the evolution of the immune system: conservation and diversification, academic press, pp 241-274, 2016. bachère e, rosa rd, schmitt p, poirier ac, merou n, charrière gm, et al. the new insights into the oyster antimicrobial defense: cellular, molecular and genetic view. fish shellfish immunol. 46: 50-64, 2015. bosch tc. rethinking the role of immunity: lessons from hydra. trends immunol. 35: 495-502, 2014. buchon n, silverman n, cherry s. immunity in drosophila melanogaster--from microbial recognition to whole-organism physiology. nat. rev. immunol. 14: 796-810, 2014. choi yj, fuchs jf, mayhew gf, yu he, christensen bm. tissue-enriched expression profiles in aedes aegypti identify hemocytespecific transcriptome responses to infection. insect biochem. mol. biol. 42: 729-738, 2012. commission implementing decision. as regards measures to prevent the introduction into and the spread within the union of the genus pomacea (perry), notified under document c. off. j. eur. union 7803 (2012/697/eu), l311/14e15, 2012. doolittle rf. structural and functional diversity of fibrinogen-related domains. in: malagoli d (ed), the evolution of the immune system: conservation and diversification, academic press, pp 275-294, 2016. gilioli g, pasquali s, martín pr, carlsson n, mariani l. a temperature-dependent physiologically based model for the invasive http://www.ncbi.nlm.nih.gov/pubmed?term=accorsi%20a%5bauthor%5d&cauthor=true&cauthor_uid=24636857 http://www.ncbi.nlm.nih.gov/pubmed?term=ottaviani%20e%5bauthor%5d&cauthor=true&cauthor_uid=24636857 http://www.ncbi.nlm.nih.gov/pubmed?term=malagoli%20d%5bauthor%5d&cauthor=true&cauthor_uid=24636857 http://www.ncbi.nlm.nih.gov/pubmed/24636857 65 apple snail pomacea canaliculata. int. j. biometeorol. 61: 1899-1911, 2017. godwin jw, pinto ar, rosenthal na. chasing the recipe for a pro-regenerative immune system. semin. cell dev. biol. 61: 71-79, 2017. guo l, accorsi a, he s, guerrero-hernández c, sivagnanam s, mckinney s, et al. an adaptable chromosome preparation methodology for use in invertebrate research organisms. bmc biol. 16: 25, 2018. hayes ka, burks rl, castro-vazquez a, darby pc, heras h, martín pr, et al. insights from an integrated view of the biology of apple snails (caenogastropoda: ampullariidae). malacologia 58: 245-302, 2015. hayes ka, cowie rh, jørgensen a, schultheiß r, albrecht c, thiengo sc. molluscan models in evolutionary biology: apple snails (gastropoda: ampullariidae) as a system for addressing fundamental questions. am. malacol. bull. 27: 47-58, 2009. hoang tc, pryor rl, rand gm, frakes ra. bioaccumulation and toxicity of copper in outdoor freshwater microcosms. ecotoxicol. environ. saf. 74: 1011-1020, 2011. horgan fg. the ecophysiology of apple snails in rice: implications for crop management and policy. ann. appl. biol.: 2018. lei j, chen l, li h. using ensemble forecasting to examine how climate change promotes worldwide invasion of the golden apple snail (pomacea canaliculata). environ. monit. assess. 189: 404, 2017. malagoli d, accorsi a, ottaviani e. the evolution of pro-opiomelanocortin: looking for the invertebrate fingerprints. peptides 32: 21372140, 2011. malagoli d, mandrioli m, tascedda f, ottaviani e. circulating phagocytes: the ancient and conserved interface between immune and neuroendocrine function. biol. rev. camb. philos. soc. 92: 369-377, 2017. malagoli d, ottaviani e. discrepant effects of mammalian factors on molluscan cell motility, chemotaxis and phagocytosis: divergent evolution or finely tuned contingency? cell biol. int. 34: 1091-1094, 2010. mu h, sun j, heras h, chu kh, qiu jw. an integrated proteomic and transcriptomic analysis of perivitelline fluid proteins in a freshwater gastropod laying aerial eggs. j. proteomics 155: 22-30, 2017. neves j, zhu j, sousa-victor p, konjikusic m, riley r, chew s, et al. immune modulation by manf promotes tissue repair and regenerative success in the retina. science 353: aaf3646, 2016. notarangelo ld, fischer a, geha rs, casanova jl, chapel h, conley me, et al. primary immunodeficiencies: 2009 update: the international union of immunological societies (iuis) primary immunodeficiencies (pid) expert committee. j. allergy clin. immunol. 124: 11611178, 2009. oren m, barela hudgell ma, golconda p, man lun c, smith lc. genomic instability and shared mechanisms for gene diversification in two distant immune gene families: the plant nbslrr genes and the echinoid 185/333 genes. in: malagoli d (ed), the evolution of the immune system: conservation and diversification, academic press, pp 295-310, 2016. ottaviani e, accorsi a, rigillo g, malagoli d, blom jm, tascedda f. epigenetic modification in neurons of the mollusc pomacea canaliculata after immune challenge. brain res. 1537: 1826, 2013. tascedda f, malagoli d, accorsi a, rigillo g, blom jm, ottaviani e. molluscs as models for translational medicine. med. sci. monit. basic res. 21: 96-99, 2015. tasiemski a, salzet m. neuro-immune lessons from an annelid: the medicinal leech. dev. comp. immunol. 66: 33-42, 2017. smith v, accorsi a, malagoli d. hematopoiesis and hemocytes in pancrustacean and molluscan models. in: malagoli d (ed.), the evolution of the immune system: conservation and diversification, academic press, pp 1-28, 2016. song l, wang x, yang z, lv z, wu z. angiostrongylus cantonensis in the vector snails pomacea canaliculata and achatina fulica in china: a meta-analysis. parasitol. res. 115: 913-923, 2016. sun j, zhang h, wang h, heras h, dreon ms, ituarte s, et al. first proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition. j. proteome res. 11: 42404248, 2012. sun j, zhang y, thiyagarajan v, qian py, qiu jw. protein expression during the embryonic development of a gastropod. proteomics 10: 2701-2711, 2010. united states department of agricuture, animal and plant health inspection service (usda_aphis). regulated organism and soil permits, snails and slugs. https://www.aphis.usda.gov/aphis/ourfocus/plan thealth/import-information/permits/regulatedorganism-and-soilpermits/sa_snails_slugs/ct_snails_slugs. yang l, cheng ty, zhao fy. comparative profiling of hepatopancreas transcriptomes in satiated and starving pomacea canaliculata. bmc genet. 18: 18, 2017. zhou x, chen y, zhu s, xu h, liu y, chen l. the complete mitochondrial genome of pomacea canaliculata (gastropoda: ampullariidae). mitochondrial dna 27a: 884-885, 2016. 32 isj 14: 32-43, 2017 issn 1824-307x research report effect of heavy metals on four different earthworm’s species specific autofluorescing eleocytes a chatterjee, r thilagaraj, m gobi department of biotechnology, school of bioengineering, srm university potheri 603203, tamilnadu, india accepted january 11, 2017 abstract earthworm is key bio-indicator of soil milieu to assess heavy metal contaminations. the celomic fluid of earthworm plays a significant role in the storage of riboflavin within the celomic cavity thereby maintaining its homeostasis. so the measurement of these autofluorescent ‘self-marking’ eleocytes will give more information about chloragocyte derived cells and species. the present study is to investigate the percentage of autofluorescing cells, riboflavin content, elemental and heavy analysis of immunologically significant eleocytes. the present study is focused on four different earthworms namely lampito mauritii, octochaetona serrata, eudrilus eugeniae and eisenia fetida, to characterize immune factors such as cellular and riboflavin content exposed to various heavy metal concentrations under laboratory conditions. four different earthworms were exposed to three different concentrations (63.5, 112.4 and 207.2 µg/ml) of heavy metals (cu, cd, and pb) over 96 h. the celomic fluid were subjected for facs analysis to find out the percentage difference of autofluorescent eleocytes cells between control and exposed species. the discrepancies between riboflavin content of control and heavy metal exposed worms were analyzed by spectrofluorescence. the low and negligible percentage of autofluorescent cells were recorded in e. eugeniae and l. mauritii but large numbers of autofluorescent cells were recorded in e. fetida and o. serrata. the experimental results show that riboflavin content and autofluroscence cells of the heavy metal exposed worms displays significant decrease in the celomic fluid. the present study clearly demonstrated that investigated species possess the significant population of celomocytes, but they differ considerably in the number of cells per body mass and by species. this non-invasive technique proves stable cellular biomarkers in earthworm for toxicological and biological soil monitoring studies. key words: earthworms; autofluorescence; heavy metals; riboflavin; facs introduction earthworms are ubiquitous and used as a model organism for many eco-toxicological studies. the interaction between earthworm and soil leads to increase in soil fertility and mobilization of heavy metals from soil to terrestrial ecosystem. the homeostasis properties of earthworm play a vital role/ significant role in maintaining the elemental concentration in the coelomic fluid. when the heavy metal concentration increases in the soil, it affects the homeostasis of the earthworm which leads to the excretion of heavy metals in the celomic fluid. the riboflavin storage is universal in earthworm ___________________________________________________________________________ corresponding author: muthukaruppan gobi department of biotechnology school of bioengineering srm university potheri 603203 tamilnadu, india e-mail: gobicc@gmail.com species, as it was detected both in attached chloragocytes forming the chloragogen tissue and in chloragocyte-derived eleocytes (mazur et al., 2011). according to (ottaviani, 2011), the celomocytes of all earthworm species contain amebocytes. immune system parameters may be used as a sensitive sublethal endpoint to assess the toxicity of atmospheric deposition to earthworms. the earthworm immune system is composed mainly of celomocytes, i.e., cells found within the fluids in the worm celomic cavity (stein et al., 1977). it has been demonstrated that many chemicals, including various trace elements, can adversely affect the immune system (fournier et al., 2000). the immunodeficiency of exposed species is interpreted as an indication of toxic effects of environmental contaminants (dales and kalac, 1992; fournier et al., 2000). the celomic fluid exhibits “molecules of many biological functions” (antimicrobial peptides and lysozymes) in annelids which provides effective 33 protection mechanisms against invaders. the composition of celomocytes in earthworm are species-specific, particularly in regard to autofluorescent eleocytes (koziol et al., 2006, plytycz et al., 2006). the chloragocyte and riboflavin inclusion in the earthworm are species specific and depends on the soil metal quality in lumbricidae (verdrengh and tarkowski, 2005; iwanaga et al., 2007). there was no previous report for celomocytes community of lampito, octochaetona and eudrilus worms. the aim of this work was to characterize the selected immune factors like cellular and riboflavin content. it was of interest to address the differences between immunological features of these four species, because although they share many similarities, their natural environment varies considerably. therefore, it was expected to discover some discrepancies between the immunological features of the four species. the differences of cellular levels and riboflavin content of control and heavy metal exposed worms were analyzed by facs and spectrofluorescence. furthermore, the variations of elements were quantitively measured by aas. materials and methods collection and identification of earthworm four different species of earthworms lampito mauritii, octochaetona serrata,(two anecics worms) eudrilus eugeniae and eisenia fetida (two epigeic)were mass cultured at our vermiary (400 800 lux light) and then these earthworms were identified based on the morphological characters. gut cleaning collected earthworms were washed with distilled water after which 50 ml jars were filled with 30 ml of 1.5 % agar gel prepared with deionised water. after getting cooled and solidified in the jars, the gel was taken out and cut into small pieces. after which earthworms were transferred separately into jars and kept for 96hrs at room temperature (400 800 lux light) to remove all the soil from their gut (pokarzherskii et al., 2000). harvesting of celomic fluid after removing the gut contents, diverse species of earthworms were cranked for 30 sec with a 4.5 v electric current to expel celomic fluid with suspended celomocytes through the dorsal pores, as described previously (plytycz et al., 2006). briefly, earthworms were placed individually in petri dishes containing 3ml of extrusion fluid (phosphatebuffered saline, pbs, supplemented with 2.5 g/l ethylenediamine tetraacetic acid, edta). freshly prepared 2 ml suspensions were used for spectrofluorimetry and flow cytometry analysis. further, celomic fluid was collected by above said method without pbs and edta. cell counting and viability cells viability were assessed by trypan blue exclusion test, mixing an equal volume of celomic fluid and 0.4 % trypan blue solution. cell viability should always exceed 90 %. collected celomic fluid was smeared on a clean glass slide, to observe the autofluorescence of celomic fluid cells. flow cytometric measurements and analysis a thin smear was prepared on a clean glass slide using collected celomic fluids and examined under olympus inverted fluorescence microscope. samples of celomocytes were analyzed with a bd facscalibur flow cytometer system. during scientific experiments, 10,000 threshold events per worm sample were collected and analyzed by their forward scatter (fs) (for cell size) and sideward scatter (ss) (cell complexity) properties. fluorescence fl1-h (emission 530 nm; excitation 488 nm) was recorded and resulting data were analyzed using winmdi 2.8 software, by producing dot plots of cell size versus fl1 autofluorescence. table 1 shows the species-specific characteristics of celomocytes in four earthworm species species n bw (g) tc (x 105) tc/bw (x 105/g) % afc afc/ bw (x 105/g) eisenia fetida 4 0.16 ± 0.00 3.4 ± 0.4 21.2 55.1 ± 0.07 344.3 lampito mauritii 4 0.54 ± 0.02 3.06 ± 0.3 5.6 18.8 ± 1.25 34.8 octochaetona serrata 4 0.51 ± 0.01 4.4 ±0.23 8.6 42.4 ± 0.97 83.1 eudrilus eugeniae 4 1.2 ± 0.07 1.6± 0.3 1.3 0.18 ± 0.09 0.15 means ± sd; n = number of individuals; bw = body weight; tc = total celomocytes; tc/bw = celomocytes /gram of body weight; % afc = percentage of autofluorescent celomocytes; afc/bw = percentage of autofluorescent celomocytes/gram body weight. 34 estimation of riboflavin by spectrofluorometer spectrofluorometric analyses were performed using the celomocyte lysate. different species of the earthworm celomocyte suspension lysates with 2 % triton. the supernatant was collected, and it was subjected to spectrofluorometric analyses with standard riboflavin (himedia laboratories). excitation spectra were recorded at 300 and 520 nm (excitation at λ = 525 nm) while emission spectra were recorded at 380 and 700 nm using excitation at λ = 370 nm. the spectrofluorometric signatures of unbound riboflavin were characterized by two maxima (at 370 nm and 450 nm) in the excitation spectrum, and an emission spectrum maximum at 525 nm. arbitrary units (au) of fluorescence were recorded using microsoft excel v. 2007. metal exposure metal chlorides (cu, cd, pb) were dissolved in double distilled water to a final 1mm metal concentration being equivalent of 63.5, 112.4 and 207.2 µg/ml respectively. dissolved metal chlorides were incorporated in 1.5 % agar gel, after cooling and solidifying this gel in the jars, it was taken out and cut into small pieces. the earthworms were then transferred to jars containing agar pieces and kept for 96 h and these agar pieces were made possible to consume easily by the earthworm. one group served as a control, and other three groups were exposed to a concentration of 63.5, 112.4 and 207.2 µg/ml, respectively. each level was tested in three replicates using ten animals. heavy metal analysis the concentration of heavy metals was analyzed in acid digested samples of earthworm by atomic absorption spectrometer as described by aqac (1999). heavy metal bioavailability to earthworm evaluated both in terms of relative toxicity and bioaccumulation factor (baf) (saxe et al., 2001). the baf was calculated as per equation; baf = [metal] earthworm/[metal] agar, where [metal] earthworm represents the total metal concentration of earthworm (mg kg), and [metal] agar represents the total metal concentration of sludge (mg kg-1). statistical analysis results were expressed as mean ± standard deviations. further data were analyzed using the one-way anova with post hoc tukey’s test. the level of statistical significance was set at p ˂ 0.05. results celomocytes the celomycytes of four different earthworms were calculated as per its body weight as shown in table 1. the low and negligible percentage of autofluorescent cells were recorded in e. eugeniae and l. mauritii. the high rate and the large numbers of autofluorescent cells were recorded in e. fetida and o. serrata. the celomocytes number per gram of body weight was found to be maximum in e. fetida (4.6x105/g), and it was recorded as 2.2x105/g in o. serrata, but it was 1.27x105/g, 3.0x105/g in l. mauritii and e. eugeniae respectively. it indicates the absence of a simple correlation between the body size/ weight and the number of celomocytes inhabiting the celomic cavity. flow cytometric analysis of eleocyte fluorescence microscopic observation of celomocytes from representative adults of four different species of earthworm revealed autofluorescent eleocytes and some adherent amebocytes which are devoid of autofluorescence. moreover, flow cytometry and fluorescent microscopy revealed the significant proportion of eleocytes in octochaetanea and e. fetida, while autofluorescent cells were very lower in lampito and eudrilus. flow cytometric analysis showed the high proportion of amebocytes in lampito and eudrilus. figure 1 shows the percentage of granular fig. 1 percentage of granular and eleocytes in four different species of earthworm at flow cytometric analysis. mean ± sd; significant p < 0.01. 35 figs 2 (a, b) riboflavin derived fluorescence spectra of celomocyte lysates from four different species of earthworm, au = arbitrary units of fluorescence intensity. mean ± sd; significant p < 0.01. and eleocytes of four different earthworms species. the forward scatter is an indicator of particle size (cell volume), whereas side scatter is an indicator of particle granularity (internal complexity) in the flow cytometer dot plot analysis. both measurements are obtained uniformly in the absence of fluorochrome molecule. the fluorochrome molecule is bound to the particles and get excited in to higher energy state and followed by the release of energy as a photon of light at a longer wavelength (lower energy state) which is known as stokes shift. density plots of the cell size versus cell complexity show that all the four different species of earthworm possess two distinct celomocyte populations i.e., agranular amebocytes and eleocytes. density plots of ssc-h versus fl1-h show that the latter exhibit very distinct fl1-h autofluorescence, arbitrarily assessed as that more intensive than 102 units on x-axis. the percentage of eleocytes are differ from species to species, and the ratio of eleocytes is directly proportional to granular cells. riboflavin content measurement by spectrofluorometry all earthworms e. fetida, e. eugeniae, l. maruitii and o. serrata possess riboflavin in celomocytes lysates. figure 2a shows emission spectra of four different species of earthworm celomic fluid and the comparison of the emission spectra of evocative samples of 1x105 celomocytes. the riboflavin content in the eleocytes is proportional to the peak fluorescence at 525 nm. a) b) 36 figs 3 (a, b, c, d) riboflavin-derived fluorescence spectra of control and heavy metal treated worm’s celomocyte lysates. au = arbitrary units of fluorescence intensity. emission spectra (kex = 370 nm) with peaks at 525 nm. the peaks at 525 nm is evident in each of them, albeit of different height in the order of e. fetida>> lampito spp>> octochatenea spp. >> eudrilus spp. it confirms, the richness of the riboflavin content in the eleocytes of eisenia, lampito and octochatenea inhabiting the unpolluted soil. the amount of riboflavin is proportional to the intensity of the emission band measured at 525 nm (expressed in arbitrary units, au). when compared to the control, the intenstity of riboflavin fluorescence found to be drastically reduced in eudrilus celomocytes. figures 3a, b, c, d displays the comparative riboflavin emission spectra of celomic fluids of four different species of earthworms. the experimental results show that that the emission spectrum occurs very high in e. fetida followed by lampito and octochaeta. the fluorescence spectrum observed from e. eugeniae is almost nonexistent, compared to control. the fluorescence emission spectrum of fluorophore characterizes the electron distribution of the molecule in the ground state. thus, it helps to identify the structure or the nature of the emitting molecule. a) b) c) d) 37 fig. 4 analysis of celomocytes of four different earthworms after 3 days exposure to heavy metal: a) comparative total riboflavin content; b) biomass of four different species of earthworm. mean ± sd; significant p < 0.01. figures 4a, b shows body weights of four different species were unaffected by the experiment days, and the mean biomass of four different worms was slightly decreased. one-way anova revealed that significant effect of metals on total number of celomocytes f value = 181.4; p-value = 0.000; p < 0.01), percentage of eleocytes set up by flow cytometry (p < 0.01) and eleocytes number (p < 0.01) and also riboflavin content in arbitrary units (au) established by spectrofluorometry (p < 0.01). results of post hoc t-tukey’s test revealed that all investigated parameters coelomocytes, eleocytes and riboflavin were consistently lowest in celomocytes from exposed worms. quantitative analysis of celomocytes flow cytometer analysis offers excellent information on the nature of the cells present in the earthworm celomic fluid and allows the identification of different cells structure which is capable of interacting with metal ions. all the earthworm was retaining two distinct population i.e., granular amebocytes (green) and autofluorescent eleocytes (red). predisposed eleocytes population were observed in the right of 102 units (figs 5 a, b, c, d) than in control. the autofluorescent intensity increases from left to right indicating a more intensive population of eleocytes in control than in heavy metal treated worms. figures 6, 7 shows the autofluorescent eleocytes of octochaetona was more biased towards the right than in control. but the eleocyte population is influenced toward left in heavy metal exposed worms indicating a reduction in the percentage of eleocytes. the dot plot intensity of granulocytes is more in control than in exposed worms. similarly, flow cytometry of lampito confirmed the presence of amebocytes and eleocytes, the latter exhibiting strong autofluorescence. the earthworms from control possessed a significantly higher percentage of autofluorescence value than their counterparts from the metalliferous soil. dot plot analysis of eudrilus revealed a significant reduction in the amoebocytes population in the exposed worms when compared to control. a) b) 38 control exposed figs 5 (a, b, c, d) analysis by flow cytometry in respect of percentages of celomocytes extruded from four different species of earthworms a) eisenia b) octochaeta c) lampito d) eudrilus. a) b) d) c) 39 fig. 6 influence of heavy metals on eleocytes in four different species of earthworm. mean ± sd; significant p < 0.01. figures 6, 7 depicts the influence of heavy metals on eleocytes and granulocytes in four different species of earthworm. heavy metal could, of course, have affected the riboflavin content, granulocytes and eleocytes percentage and viability of cells, which in turn could have affected the growth and reproduction indirectly. the present study also proves, cells variability depends on species-specific, and the individual differences in riboflavin content may reflect species-specific, nutritional preferences and vitamin b12 availability of the local resources. further, present study concludes that metal contamination of the soil caused their accumulation in the earthworm body including their coelomocytes which in turn affects their health and viability. figure 8 depicts the accumulated heavy metal contents of four different species of earthworm. the cr concentration was 0.36 ± 0.01, 0.34 ± 0.15, 0.27 ± 0.02, 0.23 ± 0.01 and cd concentration was 0.76 ± 0.02, 0.56 ± 0.02, 0.63 ± 0.15, 0.0.47 ± 0.20 in eisenia, lampito, octocheta and eudrilus respectively. their bioaccumlation factor (baf) values were 0.135, 0.168, 0.14 and 0.11. so the present study reveals that earthworm subjected to heavy metal (cd, cr and cu) exposure exhibited a higher accumulation of the heavy metals in the coelomic fluid. fig. 7 influence of heavy metals on granulocytes in four different species of earthworm. mean ± sd; significant p < 0.01. 40 fig. 8 bioaccumulation and baf of heavy metals in earthworm celomic fluid. mean ± sd; significant p < 0.01. discussion in the present study, celomocytes are collected from celomic fluid retrieved non-invasively from four different species of earthworm by electrostimulation was analyzed by spectrofluorimetry and flow cytometry. an astonishing divergence was detected in the four earthworm species with autofluorescent cells (eleocytes), and high number of celomocytes per body weight. plytycz et al. (2006) concluded that amebocytes with phagocytic properties are present in all species, but the presence of autofluorescent eleocytes, derived from the chloragogenous tissue, is species specific. as koziol et al. (2006) showed, the partial source of fluorescence in eleocytes is riboflavin, which is involved in immune responses in vertebrates (verdrengh and tarkowski, 2005), and is also considered responsible for balancing between earthworms and microorganisms present in soil (homa et al., 2010). content and composition of celomocytes, as well as riboflavin content, may vary not only between species but also within them depending on the presence of stress factors such as heavy metals (plytycz and morgan, 2011). in the present study, percentages of autofluorescent eleocytes were relatively stable, as well as the amounts of riboflavin measured in celomocyte lysates samples (plytycz et al., 2012). it is well established that riboflavin is essential for the proper functioning of the innate immunity of both animals (powers, 2003; verdrengh and tarkowski, 2005) and plants (dong and beer, 2000; asai et al., 2010; yoshioka et al., 2011), as well as being a signaling molecule in bacterial quorum sensing (rajamani et al., 2008; atkinson and williams 2009). riboflavin is considered as the chemoattractant for immunocytes in earthworms (plytycz et al., 2011) that putatively mobilizes the defense response during a disrupted balance of host and microbes/parasites induced by toxic factors (plytycz et al., 2006). the present work clearly showed that there is no correlation between body weight and riboflavin content. furthermore, a maximum decrease of riboflavin was recorded in the e. eugeniae than other species while exposed to heavy metals. and also it was observed that the physical nature of the celomic fluid of the four different species shows remarkably different in color. the celomic fluid of the e. eugeniae is color less where as other three species are brilliant yellow in color due to the presence phosphorescence pigments. this adaptation might be connected with very efficient riboflavin metabolism, because its amount in celomocytes of eisenia sp. is much higher than in another earthworm species (plytycz et al., 2006). from experimental results, it was observed that the body weight and the coelomic fluid does not correlate with respect to immunological defense point of view. fluorescence data shown in the present study indicates clearly that e. fetida, l. maruitii, o. serreta and e. eugeniae, display different molecular composition in their celomic fluid and thus have different metabolisms. therefore, interpretations of toxicological and immunological studies obtained on earthworms could be significantly different depending upon the different and the species interactions. the purpose of studying this parameter was to determine if the contaminant exposure reduced the cell defense system regarding cell survival. eisenia lampito octo eudrilus 0.168 0.135 0.14 0.11 41 many studies concerned the effects of environmental pollution, including heavy metals on earthworm immune functions mediated by celomocytes (scott-fordsmand and week, 2000; kurek et al., 2002). fugere et al. (1996) found that inhibition of the phagocytic activity of coelomocytes exposed to the heavy metal solution. plytycz et al. (2009) discovered that riboflavin content was reduced in the eleocytes of worms transferred from unpolluted to the metal-polluted soil. recent findings indicate that the riboflavin/lipofuscin balance may be a sensitive indicator of soil pollution intensity (cygal et al., 2007). according to plytycz et al. (2009), states that the heavy metal pollution strongly influences the riboflavin content in the immune competent cells of d. rubidus. the reduction of riboflavin content in the eleocytes cells leads to decrease in the fluorescence signal (riboflavin quenching) due to heavy metal binding. indeed, earthworms living in stress conditions are obligated to manage their energy, because they are constantly exposed to the high cost of maintaining homeostasis. these results are concordant with previous in vitro observations (sauve´ et al., 1998) showing that the immune function of celomocytes exposed to trace elements is impaired before the onset of cell death (brousseau, 1997). heavy metal accumulation depends on the exposure duration whereas the accumulation of cu, cd and cr is dependent upon the metabolic turnover (gobi, 2015). the use of celomic fluid for elemental analysis in earthworm offers many advantages on the commonly used whole-body measure, particularly for in field application. conclusion the present study clearly demonstrated that investigated species possess the significant population of celomocytes, but they differ considerably in the number of cells per body mass and by species. percentage of eleocytes and riboflavin were found to be reduced drastically in the earthworms exposed to the heavy metal containing a medium and these two markers playing an important role in earthworm biology. this noninvasive technique proves stable cellular biomarkers in earthworm for toxicological and biological soil monitoring studies. references andersen c, laursen j. distribution of heavy metals in lumbricus terrestris, aporrectodea longa and a. rosea measured by atomic absorption and xray fluorescence spectrometry. pedobiologia 24: 347-356, 1982. asai s, mase k, yoshioka h. a key enzyme for flavin synthesis is required for nitric oxide and reactive oxygen species production in disease resistance. plant j. 62: 911-924, 2010. atkinson s, williams p. quorum sensing and social networking in the microbial world. j. r. soc. interface 6: 959-978, 2009. brousseau p, fugere n, bernier j, coderre d, nadeau d, poirier g, et al. evaluation of earthworm exposure to contaminated soil by cytometric assay of ceolomocytes phagocytosis in lumbricus terrestris (oligochaeta). soil biol. biochem. 29: 681-684, 1997. chuparina ev, gunicheva tn. non-destructive xray fluorescence determination of some elements in plant materials. j. anal. chem. 58: 856-861, 2003. cygal m, lis u, kruk j, plytycz b. coelomocytes and fluorophores of the earthworm dendrobaena veneta raised at different ambient temperatures. acta biol. crac. ser. zool. 49: 511, 2007. dales rp, kalac y. phagocytotic defense by the earthworm eisenia fetida against certain pathogenic bacteria. comp. biochem. physiol. 101a: 487-490, 1992. descotes j, choquet-kastylevsky g, van ganse e, vial t. responses of the immune system to injury. toxicol. pathol. 28: 479-481, 2000. dong h, beer sv. riboflavin induces disease resistance in plants activating a novel signal transduction pathway. phytopathology 90: 801 811, 2000. fournier m, cyr d, blakley b, boermans h, brousseau p. phagocytosis as a biomarker of immunotoxicity in wildlife species exposed to environmental xenobiotics. am. zool. 40: 412420, 2000. frouin h, lebeuf m, saint-louis r, hammill m, pelletier e, fournier m. toxic effects of tributyltin and its metabolites on harbor seal (phoca vitulina) immune cells in vitro. aquat. toxicol. 90: 243-251, 2008. fugere n, brousseau p, krzystyniak k, coderre d, fournier m. heavy metal specific inhibition of phagocytosis and different in vitro sensitivity of heterogenous coelomocytes from lumbricus terrestris (oligochaeta). toxicology 109: 157166, 1996. gobi m. heavy metal induced biomolecule and genotoxic changes in earthworm eisenia fetida. inv. surv. j. 12: 237-245, 2015. gratão pl, monteiro cc, antunes am, peres lep, azevedo ra. acquired tolerance of tomato (lycopersicon esculentum cv. micro-tom) plants to cadmium-induced stress. ann. appl. biol. 153: 321-333, 2008. homa j, klimek m, kruk j, cocquerelle c, vandenbulcke f, plytycz b. metal-pecific effects on metallothionein gene induction and riboflavin content in coelomocytes of allolobophora chlorotica. ecotoxicol. environ. saf. 73: 1937-1943, 2010. ireland mp. metal accumulation by the earthworms lumbricus rubellus, dendrobaena veneta and eiseniella tetraedra living in heavy-metal polluted sites. environ. poll. 19: 201-206, 1979. iwanaga k, hasegawa t, hultquist de, harada h, yoshikawa y, yanamadala s, et al. riboflavinmediated reduction of oxidant injury, rejection, and vasculopathy after cardiac allotransplantation. transplantation 83: 747753, 2007. kauschke e, mohrig w, cooper el. coelomic fluid proteins as basic components of innate immunity in earthworms. eur. j. soil biol. 43: s110-s115, 2007. 42 kielbas e, grebosz a, duchnowski m, morgan aj. riboflavin mobilization from eleocyte stores in the earthworm dendrodrilus rubidus inhabiting aerially-contaminated ni smelter soil. chemosphere 8: 199-205,2010. koziol b, markowicz m, kruk j, plytycz b. riboflavin as a source of autofluorescence in eisenia fetida coelomocytes. photochem. photobiol. 82: 570-573, 2006. kurek a, homa j, plytycz b. earthworm coelomocytes: convenient model for basic and applied sciences. in: beschin a, bilej m, cooper el (eds) a new model for analyzing antimicrobial peptides with biomedical applications, ios press, ohmsha, pp. 38-46, 2002. maity jp, kar s, chakraborty a, sudershan m, santra sc. study on trace elements (using energy dispersive x-ray fluorescence technique) of edible seed from cicer arietinum l. plants developed from gamma irradiated seeds and variation of yielding capacity. j. radioanal. nucl. chem. 283: 225-230, 2010. marguí e, queralt i, carvalho ml, hidalgo m. comparison of edxrf and icp-oes after microwave digestion for element determination in plant specimens from an abandoned mining area. anal. chimica acta 549: 197-204, 2005. marguí e, queralt i, hidalgo m. application of x-ray fluorescence spectrometry to determination and quantification of metals in vegetal material. trends anal. chem. 28: 362-372, 2009. mazur ai, klimek m, morgan aj, plytycz b. riboflavin storage in earthworm chloragocytes and chloragocyte-derived eleocytes and its putative role as chemoattractant for immunocompetent cells. pedobiologia doi:10.1016/j.pedobi.2011.09.008, 2011. morgan je, morgan aj. the distribution of cadmium, copper, lead, zinc, and calcium in the tissues of the earthworm lumbricus rubellus sampled from one uncontaminated and four polluted soils. oecologia 84: 559-566, 1990. morris b, morgan aj. calcium-lead interactions in earthworms: observations on lumbricus terrestris l. sampled from a calcareous abandoned lead mine site. bull. environ. contam. toxicol. 37: 226-233, 1986. necemer m, kump p, scancar j, jacimovic r, simcic j, pelicon p, et al. application of x-ray fluorescence analytical techniques in phytoremediation and plant biology studies. spectrochimica acta part b 63: 1240-1247, 2008. noda t, tsuda s, mori m, takigawa s, matsuuraendo c, kim sj, hashimoto n, yamauchi h. determination of the phosphorus content in potato starch using an energy dispersive x-ray fluorescence method. food chem. 95: 632-637, 2006. oliveira al, almeida e, silva fbr, nascimento filho vf. elemental contents in exotic brazilian tropical fruits evaluated by energy dispersive xray fluorescence. sci agric. 63: 82-84, 2006. orescanin v, katunar a, kutle a, valkovic v. heavy metals in soil, grape and wine. j. trace microprobe tech. 21: 171-180, 2003. ottaviani e. immunocyte: the invertebrate counterpart of the vertebrate macrophage. inv. surv. j. 8: 1-4, 2011. plytycz b, homa j, koziol b, rozanowska m, morgan jm. riboflavin content in autofluorescent earthworm coelomocytes is species-specific. folia histochem. cytobiol. 44: 275-280, 2006. plytycz b, molenda ul, cygal m, kielbasa e, grebosz a, duchnowski m, et al. riboflavin content of coelomocytes in earthworm (dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution. environ. poll. 157: 3042-3050, 2009. plytycz b, kielbasa e, grebosz a, duchnowski m, morgan aj. riboflavin mobilization from eleocyte stores in the earthworm dendrodrilus rubidus inhabiting aeriallycontaminated ni smelter soil. chemosphere 81:199-205, 2010. plytycz b, morgan j. riboflavin storage in earthworm chloragocytes/eleocytes in an ecoimmunology perspective. j. inv. surv. 8: 199209, 2011. pokarzhevskii ad, nico m, straalen v, semenov am. agar as a medium for removing soil from earthworm guts. soil biol. biochem. 32: 13151317, 2000. powers hj. riboflavin (vitamin b-2) and health. am. j. clin. nutr. 77: 1352-1360, 2003. rajamani s, bauer wd, robinson jb, farrow jm, pesci ec, teplitski m. the vitamin riboflavin and its derivative lumichrome activate the lasr bacterial quorum-sensing receptor. mol. plant microbe interact. 21: 1184-1192, 2008. rozman kk, lebofsky m, pinson dm. chronic toxicity and carcinogenicity of 1, 2, 3, 4, 6, 7, 8heptachlorodibenzo-pdioxin displays a distinct dose/time toxicity threshold (c xt = k) and a lifeprolonging subthreshold effect. food chem. toxicol. 43: 729-740, 2005. sauvé s, dumestre a, mcbride m, hendershot w. derivation of soil quality criteria using predicted chemical speciation of pb2+ and cu2+. environ. toxicol. chem. 17: 1481-1489, 1998. scott-fordsmand jj, week jm. biomarkers in earthworms. environ. cont. toxicol. 165: 17-59, 2000. spurgeon dj, hopkin sp. comparisons of metal accumulation and excretion kinetics in earthworm (eisenia fetida) exposed to contaminated field and laboratory soils. appl. soil ecol. 11: 227-243, 1999. stein e, avtalion rr, cooper el. the coelomocytes of the earthworm lumbricus terrestris: morphology and phagocytic properties. j. morphol. 153: 467-477, 1977. sulik p, klimek m, talik p, kruk j, morgan aj, plytycz b. searching for external sources of the riboflavin stored in earthworm eleocytes. inv. surv. j. 9: 169-177, 2012. verdrengh m, tarkowski a. riboflavin in innate and acquired immune responses. inflamm. res. 54: 390-393, 2005. vernile p, fornelli f, bari g, spagnuolo m, minervini f, de lillo e. bioavailability and toxicity of 43 pentachlorophenol in contaminated soil evaluated on coelomocytes of eisenia andrei (annelida : lumbricidae). toxicol. in vitro 21: 302-307, 2007. wang c, sun z, liu y, zhang x, xu g. a novel antimicrobial vermipeptide family from earthworm eisenia fetida. eur. j. soil biol. 43: s127-s134, 2007. yoshioka h, mase k, yoshioka m, kobayashi m, asai s. regulatory mechanisms of nitric oxide and reactive oxygen species generation and their role in plant immunity. nitric oxide 25: 216-221, 2011. zapponi ga, marcello i. low-dose risk, hormesis, analogical and logical thinking. ann. ny acad. sci. 1076: 839-857, 2006. review isj 8: 143-152, 2011 issn 1824-307x review the neuroimmunoendocrine network during worm helminth infections k nava-castro1, s muñiz-hernández2, r hernández-bello3, j morales-montor3 1departamento de inmunología e infectología perinatal, instituto nacional de perinatología, secretaría de salud, méxico, d.f. cp 11000. méxico 2subdirección de investigación básica, instituto nacional de cancerología, secretaría de salud, méxico, d.f. cp 14080. méxico 3departamento de inmunología, instituto de investigaciones biomédicas, universidad nacional autónoma de méxico, ap 70228, méxico, d.f. 04510, méxico accepted july 25, 2011 abstract the physiological interactions during the course of the immune response to helminthes are complex. as our understanding of the neuroendocrine system grows, it has become increasingly clear that this complex network of neurotransmitters, hormones, and cytokines plays an important role in mediating immunity, in general, but in the case of helminthes this interaction among different systems is crucial. helminthes present a complex relationship in the host’s physiological systems, with neuro and hormonally dependent host factors such as sex, age, and the host physiological status correlated with parasite success. on top of the effect that this particular type of parasites may have on the invaded host, recent experimental evidence suggest that helminth parasites not only actively evade immune response, but are also able to exploit the hormonal microenvironment within their host to favor their establishment, growth and reproduction. the close interaction of the worm with the host’s homeostatic systems, the molecules produced by them, and the activation of immune mediated mechanisms to eliminate it, activate a complex neuroendocrine network, that produces strong behavioral changes in the infected host. understanding how the host’s neuroendocrine system can under certain circumstances favor the establishment of a parasitic infection opens interesting perspectives into the host parasite relationship field. this review focuses on the host-parasite neuroendocrine network activated by parasite worm infections. key words: neuroendocrine network; helminthes; worm; immunity; endocrine host-parasite relationship introduction the interaction of the nervous, endocrine and immune systems is crucial in the maintenance of homeostasis in vertebrates, and is absolutely vital in mammals. the capacity of the immune system to discriminate between self and non-self is based on a wide spectrum of specificity expressed by the immune system cells. this feature of the immune system implies that it can perceive an internal image of the organism’s components and react to the distortions of this image (such as transformed cells of the self). the immune response, as a homeostatic response under physiological control, ___________________________________________________________________________ corresponding author: jorge morales-montor departamento de inmunología instituto de investigaciones biomédicas u.n.a.m., ap 70228, méxico d.f. 04510 e-mail: jmontor66@biomedicas.unam.mx contributes to maintain the integrity of the body cells and tissues. hormones and neurotransmitters present in the immune cell microenvironment can restrict its autonomy, probably by acting on the receptors of these neuroendocrine factors. efficient communication among these three systems implies the existence of afferent and efferent pathways, constituting a complex feedback system. the alterations of this network trigger pathologies that involve its components (bottasso and moralesmontor, 2009; perez et al., 2009). in recent years, information on the multiple functions of the immune system has expanded remarkably. one of these functions has been biological adaptation through pathogens, such as helminthes, and its elimination from the organism. immune functions, in turn, require delicate control of the involved cells, which allow adaptation of the organism to the different physiological and 143 mailto:jmontor66@biomedicas.unam.mx pathological situations that it will face along life. to meet this end, interaction with the nervous and endocrine systems of the organism is necessary. this interaction is constant and makes the merged functioning of the three systems possible. this communication involves common messengers and receptors, simultaneously participating in a complex feedback system. alterations in communication among the three systems, lead to different pathologies. this is the case with the neuropsychiatric disorders that cause immunosuppression, such as depression (blume et al., 2011), and immune disorders that cause endocrinological problems such as hashimoto’s thyroiditis (tomer and huber, 2009) and diabetes mellitus type i (lehuen et al., 2010), both examples of the functional interaction between the immune and the neuroendocrine systems (wilder, 1995). numerous experimental data show that, as with other body cells, the cells of the immune system are influenced by the neuroendocrine system, which displays various control levels, from metabolism to cell division, regulated by hormones and neurotransmitters (jacobs et al., 2010; muñoz-cruz et al., 2011). the immune response is possibly the only physiological phenomenon in which the amplification of the response is based on cell proliferation and the specific transformation of its components. this process requires metabolic changes and growth factors, which make the immune response dependent on neuroendocrine control (fig. 1). it is well known that cd4+ t cells play an important role on the adaptive immunity against pathogens as well as on autoimmune diseases. also they are a crucial key for immunological memory. the activation phase of non-differentiated cd4+ t lymphocytes is determined by the specific recognition of antigenic determinants, which appear in the context of major histocompatibility complex class ii molecules (mhc-ii) and are expressed on professional antigen-presenting cells, such as macrophages, b or dendritic cells (zhu et al., 2010). the specificity of the immune response determined by cd4+ t lymphocytes is modulated by selective expansion of the clones capable of recognizing these antigenic determinants and, thus, of differentiating into helper cells (th1, th2, th17 and tregs) which contribute to the protection of the organism against infectious diseases (zhu et al., 2010). the classification of th1 and th2 was based on the specific pattern of cytokines they produce: th1 lymphocytes produce cytokines such as interleukin 2 (il-2), gamma interferon (ifn-γ) and the tumor necrosis factor alpha (tnf-α) are mainly involved in protection against intracellular pathogens through cell-mediated immunity, macrophage activation, and also in delayed hypersensitivity (cox et al., 2011). on the other hand, th2 lymphocytes produce interleukins il-4, il-5, il-6, il-10 and il-13, and regulate the humoral immune response through the proliferation of b lymphocytes and the change of the specific antibody isotype, beside promoting eosinophil and mastocyte differentiation (reviewed in wan and bramson (2001)). th17 lymphocytes, the third effector population of cd4 t cell, are characterized by producing il-17, il-21 and il-22 principally (nurieva et al., 2007). the t regulatory cells (tregs) are implicated on the control and regulation of particular subsets of cd4 t cells (zhu et al., 2010). however, both hormones and neurotransmitters have influence on immune cells, since they affect the production of several cytokines, and various products of the immune response, both th1 and th2, have a regulatory effect on the neuroendocrine system (fig. 1). the host-parasite neuroimmunoendocrine network the relationship between parasites (p), particularly helminthes, and their hosts (h), implies biochemical co-evolution and communication between their complex physiological and metabolic systems among themselves and with the environment, at all levels of biological organization (derijk and berkenbosch, 1991; grossman et al., 1991). hormones regulate a variety of cellular and physiological functions of organisms such as growth, reproduction and differentiation. hormones and immune actors are prominent in h-p relationships (klein, 2004). the comparatively sophisticated immune systems of vertebrates add complexity to h-p interactions. mammals sense and react with their innate and acquired immunological systems to the presence of a parasite and the parasite is also sensitive and reactive to the host’s immune systems effectors. host’s hormones are also involved in the modulation of the immune system’s protective or pathogenic functions and also on the parasite’s metabolism and reproduction (escobedo et al., 2005). host’s adrenal hormones are well known as immune modulators (loria et al., 1996), whilst sex steroids (estradiol, progesterone and testosterone) are recognized to also significantly affect the immune system’s functions (hughes and randolph, 2001; roberts et al., 2001). more recently, the ability of hormones to affect the immunological response directed against pathogenic agents, particularly helminthes, has gained attention (bottasso and morales-montor, 2009). this is clearly evident during various parasitic diseases including malaria (cernetich et al., 2006), schistosomiasis (morales-montor et al., 2008), toxoplasmosis (henriquez et al., 2009), cysticercosis (larralde et al., 1995), trypanosomiasis (brazão et al., 2009), leishmaniasis (snider et al., 2009), where strong hormonal regulation of the immune response has been described. however, other factors than the immunoendocrine response affect the course of a parasitic infection. a striking example of exploitation of host molecules is the ability of a number of parasites to use host-synthesized cytokines as indirect growth factors for the parasite (damian, 1997). the case of helminthes helminthes are estimated to include 18,000 to 24,000 species, and are divided into two subclasses 144 fig. 1 proposed neuroimmunological interactions that occur in higher vertebrates. in physiological conditions there is a crosstalk between neurological and immune systems of the host. external stimuli, such as infections, results in a th1/th2 systemic cytokine production of the immune response. in addition to, central nervous system (cns) is able to actively induce cytokines expression, which may affect the cns function. (touassem et al., 1992). nearly all trematodes are parasites of molluscs and vertebrates. the smaller aspidogastrea, comprising about 100 species, are obligated parasites of molluscs and may also infect turtles and fishes, including cartilaginous fishes (rosas-valdez and leon, 2011). the digenea, which constitute the majority of trematode diversity, are obligate parasites of both molluscs and vertebrates, but rarely occur in cartilaginous fishes. one-quarter of a billion people are infected with parasitic trematode worms worldwide (razomendivil and perez-ponce de leon, 2011). diseaseassociated symptoms occur in 120 million people, and 20 million people suffer from severe morbidity (cribb et al., 2002). cestoda is the class of parasitic flatworms, commonly called tapeworms that live in the digestive tract of vertebrates as adults and often in the bodies of various animals as juveniles (olson and caira, 1999). there are two subclasses in class cestoda, the cestodaria and the eucestoda. by far the most common and widespread are the eucestoda, with only a few species of unusual worms in subclass cestodaria. the cyclophyllideans are the most important to humans because they infect people and livestock (hoberg, et al., 1999). two important tapeworms are the pork tapeworm taenia solium, and the beef tapeworm t. saginata (levron et al., 2010). taennids, particularly taenia solium (causal agent of porcine cysticercosis and human neurocysticercosis) and taenia crassiceps (causal agent of murine cysticercosis) are highly evolved parasites that have developed diverse mechanisms of survival within the host that facilitate their establishment (hoberg, 2006). these mechanisms can be roughly grouped into two types. the first is evasion of the immune response by molecular mimicry or by inactivating effector immune processes (i.e., complement inhibition) (ludin et al., 2011). in the second mechanism, the parasite exploits the host system to its benefit in its establishment, growth or reproduction (long and boots, 2011). this exploitation mechanism provides 145 parasites with a dual benefit: first, obtaining amino acids for metabolism, and second preventing the surface-bound antibody from interfering with cytotoxic cells interacting with the parasite (long et al., 2011). effect of steroid hormones on helminth infections in last years, research has proved the influence of sex hormones in the immune system regulation (arteaga et al., 2002), and the idea of a neuroimmunoendocrine network was released. since then, investigations focused in the role of these hormones and their possible mechanism to intervene in the host susceptibility or resistance have grown (klein, 2000). it is well-known that males of vertebrate species tend to exhibit higher rates of parasites than females, and sex-associated hormones may influence immunocompetence. thus, sex hormones are hypothezised to lead to this bias (hoby et al., 2006). in this point, females have also been shown to have higher susceptibility to many parasitic infections, a finding particularly striking in helminth infections such as those produced by taenia solium (morales-montor et al., 2004) and trichinella spiralis (hernandez-bello et al., 2011). there are enough evidence about corticoids and their influence in the regulation of the immune response involved in parasitic infections (aly et al., 2010). however, there is recent data about their direct influence on the growth of the parasite, without an immune regulation. for instance, in a moderately resistant strain of mice, cysts of e. multilocularis developed into hydatid cysts in cortisone-treated mice (barnard et al., 1998). cortisone treatment significantly increased the average number of cysts, the average area of each cyst, and the total surface area occupied by cysts when compared with the untreated mice. collectively, the cysts in the treated mice occupied more of the surface area of the liver but less of the same area in the untreated mice (barnard et al., 1998). treatment of a e. multilocularis resistant mouse strain with cortisone drastically increased both the number of cysts and the average size of each cyst if the treatment occurred early in the infection. consequently, this treatment increases the susceptibility of mice to primary infections with e. multilocularis. based on these results, it could not be determined whether the increased susceptibility results from physiological or immunological effects caused by the cortisone treatment (hildreth et al., 2003). 146 these results are in agreement with studies of the nematode heligmosomoides polygyrus. in this infection, peripheral immune responsiveness in male laboratory mice was reduced by infection with the parasite. responsiveness was also lower among high-rankers or aggressive males regardless of infection status. reduced responsiveness on infected animals and high rankers was associated with elevated serum corticosterone concentration among high-ranking males (perkins et al., 2008). although glucocorticoids have a stimulatory effect on the initial cell proliferation phase of t lymphocytes, and thus some elevation might have been expected on this account, the change in corticosterone concentration during the infection phase was the best hormone-measure predictor of eventual worm burden. the negative relationship between the immune status and high corticosteroids levels is more in keeping with the later impact of glucocorticoids on the secretion of th2 cytokines and thus depression of the th2 arm of the immune response. this is consistent with effects of glucocorticoids in prolonging intestinal nematode infections, increasing the susceptibility of rodent hosts to h. polygyrus and depressing the expression of acquired resistance to h. polygyrus. there was no testosterone-dependent increase in parasite burden among high rankers in this experiment, perhaps because resistance to the parasite relies on a different emphasis on the th1 and th2 arms of the acquired immune response (barnard et al., 1998). schistosomiasis is another example in which steroids play an important role in the host susceptibility (kurtis et al., 2006). the disproportionately high intensity and prevalence of schistosome infection in children, compared with adults, has been documented for decades, so understanding the mechanisms of this naturally occurring protection may guide efforts to develop a vaccine for schistosomiasis (kurtis et al., 2006). then, the importance of the hormonal changes during pubertal development, including increases in the levels of the adrenal hormones dhea-s (dehydroepiandrosterone sulfate) and dhea (dehydroepiandrosterone) may have part of responsibility for the dramatic reduction in age susceptibility to schistosome infection. another evidences that points out to the relationship between increasing pubertal development, dhea-s levels, and resistance to schistosome infection are: (1) in mice, exogenous administration of dhea-s leads to decreased schistosome worm burdens after challenge infection, thus dehydroepiandrosterone sulfate treatment of mice modulates infection with schistosoma mansoni; (2) dhea-s kills larval and adult parasites in culture at physiologic concentrations; (3) in another 2 cross-sectional studies increased dhea-s levels are associated with decreased intensity of infection in humans; and (4) increased dhea-s levels are associated with decreased intensity of re-infection after treatment with praziquantel (pzq) (kurtis et al., 2006). dhea-s is known to have potent immunomodulatory activities, including upregulation of th2-driven antibody isotypes and down-regulation of pro-inflammatory cytokines. finally dhea-s could mediate resistance through a direct anti-parasite effect. but also via innate immune mechanisms, such as host skin thickness or fat deposition, then capitalizing on these mechanisms for vaccine development will be difficult. however, if dhea-s mediates resistance via enhancement of acquired protective immune responses, then vaccine strategies designed to induce and augment these protective acquired immune responses, including hormonal adjuvants, may be promising (kurtis et al., 2006) (fig. 2). fig. 2 regulation of cytokine production in th1 and th2 lymphocytes by dhea. the differentiation from th0 towards th1 or th2 is simetric, each one controls a unique type of immune response and increases the development of cells of the same subclass while suppress the expansion and effector functions of the other subtype. dhea can control th1/th2 balance by inducing the development of one subtype and inhibit the other subtype. dhea effects could depend on concentration, having a dual effect. also, dhea has been demonstrated to have direct helminticidal effects on different parasites, without mediation of immune response. finally, the prediction of male biased parasitism was tested in free ranging chamois (rupicapra rupicapra), which are infested intensely by gastrointestinal and lung helminthes. male chamois had a higher output of gastrointestinal eggs and lungworm larvae when compared to females. male biased parasitism originating in sex related hormone levels was confirmed for the elevated output of lungworm larvae, but not for the gastrointestinal nematodes. the faecal output of lungworm larvae was significantly correlated with androgen and cortisol metabolite levels. the immunosuppressant effects of these hormones may explain the greater susceptibility of males to infection by parasites and developing disease. the stress of the rutting season with elevated glucocorticoid levels is hypothesized to reduce humoral antibodies, and to enhance larval output of nematodes in males. however, it should be considered that the subset samples were collected predominantly around the period when androgen levels between sexes differ most significantly. in contrast to the output of lungworm larvae, the male bias in quantitative gastrointestinal nematode output was not significantly correlated with sex differences in steroid levels (hoby et al., 2006). for instance, a strong negative correlation was found between sex and adrenal steroid hormone circulating levels and some proinflammatory cytokines in microfilaremic women. plasma samples from amicrofilaremic women contained higher concentrations of testosterone and estradiol than those from microfilaremic ones. testosterone was also negatively correlated with il6 and estradiol with ifn-γ. the fact that cortisol concentrations were not elevated in women with filariasis may be related to the chronic nature of the disease (mavoungou, et al., 2005). another research that focused on the immuneendocrine system relationship in an helminth infection, is a study designed in rodents infected with strongiloides ratti, in which a sex-related differences in host susceptibility was previously known. in this infection, male mice were more susceptible to s. ratti and the difference was seen in migrating larvae. it has been shown that natural immunity against migrating larvae of strongyloides ratti is regulated by macrophages. in the small intestine, host mast cells were related to adult worm expulsion (watanabe et al., 1999). according to this study, the sex-related difference are clearly mediated by testosterone during the migration of larvae, suggesting that testosterone renders mice 147 susceptible to migrating larvae by modulating their natural defense mechanisms (watanabe et al., 1999). so, steroid hormones produced by the adrenal cortex, such as dhea-s and cortisol, influence the intensity of the immune response during s. mansoni infections and have been implicated among the most important host factors controlling the onset, establishment, and pathogenesis of schistosomiasis. these hormones inhibit oviposition by s. mansoni both in vitro and in vivo. in vivo, the increased numbers of worms, larger number of eggs and more vigorous hepatic granulomas can be related to the lack of circulating glucocorticoids, whose presence in some way ameliorates the inflammatory immune response in the liver. the effect of adrenalectomy produced high levels of infection and more severe pathology, a fact that can be related to the well-known glucocorticoid antiinflammatory effect. low levels of cortisol could promote vigorous granuloma formation and the production of cytokines necessary for schistosome reproduction, such as tnf-α. the immunosuppresive effects of hydrocortisone and dexamethasone are counteracted by dhea, suggesting a tightly controlled balance in the secretion of these hormones to regulate the inflammatory response. an intriguing question is how the lack of adrenal hormones affects the infection in the parasitized host. host derived candidates that could possibly be affected by adrenalectomy are the interleukins (il’s), which are known to be altered during schistosomiasis. potential endocrinological-immunological mediators of this process are il-1, il-6, tnf-α and macrophage migratory inhibitory factor, all known regulated by adrenal steroids. the changes produced in the infected adrenalectomized host could thus be cytokine mediated. further work could elucidate the mechanism by which adrenalectomy induces changes in the immune function during disease progression and could establish causal links, if indeed they exist (morales-montor et al., 2004). trichuris muris infection is an ideal model for defining t-cell-driven immunity, and also provides essential insights that may impact on potential helminth therapies currently in development. the female-associated hormone 17-β estradiol (e2) significantly enhanced the generation of a th2 response in vitro (hepworth et al., 2010); however, this stimulatory effect was found to be dispensable for the generation of immunity to trichuris in the gender-biased il-4 ko mouse model (hepworth, et al., 2010). these mice are compromised in their ability to generate an efficient th2 response, necessary for parasite expulsion, as they lack il-4 a key th2 polarizing cytokine. in addition, female il-4 ko balb/c mice mount an unusually delayed th2 response, associated with t-cell and accessory nk cell-derived il-13 and an associated decrease in the levels of the pro-inflammatory cytokines tnf-α and il-6, which combine to mediate worm expulsion (hepworth et al., 2010). conversely, male littermates are unable to expel the parasite and retain high adult worm burdens, a phenotype found to be dependent on il-18. in contrast, the maleassociated hormone dihydrotestosterone (dht), significantly inhibited the t-cell stimulatory capacity of dc and directly suppressed the immune response of male il-4 ko mice, with worm expulsion restored following castration (hepworth et al., 2010). this finding was associated to a dramatically reduced il-18 mrna expression, suggesting that androgens may act via this cytokine to suppress th2 immunity to trichuris muris infection (hepworth et al., 2010). behavioral changes in the infected host several behavioral changes that are induced by infections with parasites have been described. for instance, there are sexual changes in body morphology as well as sex-related behavioral changes in crabs when parasitized with a rhizocefalan, through mechanisms that are still obscure but could involve changes in the hormonal pattern of the host (cited in larralde et al., 1995). taenia taeniformis is also known to alter reproduction in rats by interfering with sex-steroids (lin et al., 1990). perhaps the most studied helminth that induces strong hormonal, and behavioral changes is the helminth parasite taenia crassiceps. male mice infected with t. crassiceps show remarkable changes in sexual behavior, characterized by a complete loss of the ejaculation response early at the infection (six weeks), followed by a gradual decrease in the number of mounts and intromissions, and their latencies increased, until none of the parasitized mice showed any sexual response toward female mice (morales et al., 1996). moreover, it was demonstrated that alterations in sexual behavior were due to the change in the normal production of sex-steroids by the mouse, since the testosterone or dihydrotestosterone restitution of infected male mice, showed a complete restoration of their sexual behavior (morales-montor et al., 2002). since c-fos and progesterone receptor (pr), both are key estradiolregulated genes involved in the regulation of sexual behavior, we studied possible changes of c-fos and pr expression in the central nervous system (cns) of infected male mice. indeed, c-fos and pr expression oscillated with time of infection and to different magnitudes in hypothalamus, brain cortex and preoptic area but neither in other areas of the brain nor in several other organs of the host (morales-montor et al., 1999, 2004). furthermore, infection disrupts the dominantsubordinate status (gourbal et al., 2002). in infected male mice strong perturbations in territorial behavior and aggressiveness were found. in addition, during confrontation between naive infected and healthy mice, infected animals more often assumed a subordinate status than healthy ones. the effects of the infection by t. crassiceps were more likely to prevent adult male mice from becoming behaviorally dominant than to reverse existing dominance relationships (gourbal et al., 2002). significant cns changes in c-fos, and progesterone receptor (pr) expression during infection signifies the brain senses the infection 148 fig. 3 chart of the proposed host-parasite neuroimmunoendocrine network. sex steroids may act directly upon the parasite whereby progesterone (p4), and oestradiol (e2) favor its reproduction. also, sex steroids act upon parasite via the immune system, e2 favoring a permissive th2 response that inhibits the restrictive th1. the brain of the host shows changes in pr, that could be the result of the high oestrogens levels that reveal that it senses and may react to the infection. also, the outcome of the feminization process is shown, that directly affects the sexual behavior of the infected male mice, through out the binding of sex steroids and/or neurosteroids to pr. the arrows show the interconnection among all system components. (+) positive stimulation; (-) negative stimulation. episode and may be involved in the ensuing behavioral changes of the infected mice, as well as, through its connectivity, extend the effects of infection to other physiological systems under its influence. that these changes in cns are beneficial to the host or parasite remains speculative (morales-montor et al., 1999, 2004). one could argue that feminization of male hosts favors the parasite by allowing its reproduction, however it is equally arguable to consider feminization of the male host as deleterious to the parasite’s completion of its cycle since there is a reduction of male exposure to its predators, the definitive hosts. other similar mutually conflicting statements may be elaborated with the above premises, the true ones remain to be identified and could perhaps vary with each different host-parasite relationship (fig. 3). not only male mice are behaviorally affected by cysticercosis, female mice also suffer perturbations in their sexual behavior, i.e., receptivity to the male, as well as disruption of the estrous cycle (arteaga et al., 2010). concluding remarks until some years ago, the immune system was perceived as a system isolated from other body systems. the present review makes evident that the immune and neuroendocrine systems share numerous ligands and receptors, which results in a constant bidirectional communication. in fact, it has been postulated that an important function of the immune system is to serve as a sensory organ for cognitive stimuli that pass unnoticed to the nervous system, as could be infectious agents, such as parasites. our present proposal is to reintegrate an important system to the physiological context of the whole organism. this will doubtlessly lead to an improved understanding of physiology, and generate changes in modern medical practice. for further understanding of the bidirectional communication process between the immune and neuroendocrine systems, it will be necessary to continue the search for ligands and receptors common to both systems, and to examine in depth 149 the similarities and differences in their functional regulation. in addition, it will also constitute a challenge for physiologists to integrate this information to the context of the whole organism during helminth infections. on the other hand, new information about immunoneuroendocrine interactions in infected hosts will help us to design novel therapies for the treatment and diagnosis of human diseases of apparently immune or endocrine origin. we have documented here that a complex interactive network involving the immune, endocrine and nervous systems, is in control of the parasite growth, reproduction and establishment. if such complex a management of the parasite loads, as that we shown here between different hosts and worms, extends to other parasite diseases of mammals, as current research seems to indicate in a number of helminthes infections, their means of exploration, fuller understandings and forms of control must be reviewed and approached with designs matching in complexity and plasticity that of the infections. the evidence presented above illustrates the complexity and importance of neuroimmunoendocrine interactions during cysticercosis and provides clues to the many other possible mechanisms of parasite establishment, growth and reproduction in an immunocompetent host. further, strong neuroimmunoendocrine interactions may have implications in the control of transmission and treatment of this parasitic disease in porcines and humans. in practical importance, the complexity of the cysticerci-host relationship suggests that all physiological factors (i.e., sex, age) should be taken into account in the design of vaccines and new drugs. acknowledgements financial support was provided by grant # in 214011-3 from programa de apoyo a proyectos de innovación tecnológica, dirección general de asuntos del personal académico, (papiit, dgapa), universidad nacional autónoma de méxico to j morales-montor. romel hernándezbello has a posdoctoral fellowship from red farmed from conacyt. references aly ir, hendawy ma, ali e, hassan e, nosseir mm. immunological and parasitological parameters after treatment with dexamethasone in murine schistosoma mansoni. mem. inst. oswaldo cruz 105: 729-735, 2010. arteaga m, chavarria a, morales-montor j. immunoneuroendocrine communication network and homeostasis regulation: the use of hormones and neurohormones as immunotherapy. rev. invest. clin. 54: 542-549, 2002. barnard cj, behnke jm, gage ar, brown h, smithurst pr. the role of parasite-induced immunodepression, rank and social environment in the modulation of behaviour and hormone concentration in male laboratory mice (mus musculus). proc. biol. sci. 265: 693-701, 1998. 150 blume j, douglas sd, evans dl. immune suppression and immune activation in depression. brain behav. immun. 25: 221-229, 2011. bottasso o, morales-montor j. neuroimmunomodulation during infectious diseases: mechanisms, causes and consequences for the host. neuroimmunomodulation 16: 65-67, 2009. brazão v, santello fh, caetano lc, del vecchio filipin m, toldo mp, do prado jc jr. immunomodulatory effects of zinc and dhea on the th-1 immune response in rats infected with trypanosoma cruzi. immunobiology 215: 427-34, 2010. cernetich a, garver ls, jedlicka ae, klein pw, kumar n, scott al, et al. involvement of gonadal steroids and gamma interferon in sex differences in response to blood-stage malaria infection. infect. immun. 74: 31903203, 2006. cox ma, harrington le, zajac aj. cytokines and the inception of cd8 t cell responses. trends immunol. 32: 180-186, 2011 cribb th, bray ra, wright t, pichelin s. the trematodes of groupers (serranidae: epinephelinae): knowledge, nature and evolution. parasitology 124: s23-42, 2002. damian rt. parasite immune evasion and exploitation: reflections and projections. parasitology 115: s169-175, 1997. derijk r, berkenbosch f. the immunehypothalamo-pituitary-adrenal axis and autoimmunity. int. j. neurosci. 59: 91-100, 1991. escobedo g, roberts cw, carrero jc, moralesmontor j. parasite regulation by host hormones: an old mechanism of host exploitation? trends parasitol. 21: 588-593, 2005. gourbal be, lacroix a, gabrion c. behavioural dominance and taenia crassiceps parasitism in balb/c male mice. parasitol. res. 88: 912-917, 2002. grossman cj, roselle ga, mendenhall cl. sex steroid regulation of autoimmunity. j. steroid biochem. mol. biol. 40: 649-659, 1991. henríquez sa, brett r, alexander j, pratt j, roberts cw. neuropsychiatric disease and toxoplasma gondii infection. neuroimmunomodulation 16: 122-133, 2009. hepworth mr, hardman mj, grencis rk. the role of sex hormones in the development of th2 immunity in a gender-biased model of trichuris muris infection. eur. j. immunol. 40: 406-416, 2010. hernández-bello r, ramírez-nieto r, sánchezacosta ag, muñiz-hernández s, nava-castro k, pavón l, et al. sex steroids effects on the molting process of the helminth human parasite trichinella spiralis [submitted]. hildreth mb, granholm nh. effect of mouse strain variations and cortisone treatment on the establishment and growth of primary echinococcus multilocularis hydatid cysts. j. parasitol. 89: 493-495, 2003. hoberg ep. phylogeny of taenia: species definitions and origins of human parasites. parasitol. int. 55: s23-30, 2006. hoberg ep, gardner sl, campbell ra. systematics of the eucestoda: advances toward a new phylogenetic paradigm, and observations on the early diversification of tapeworms and vertebrates. syst. parasitol. 42:1-12, 1999. hoby s, schwarzenberger f, doherr mg, robert n, walzer c. steroid hormone related male biased parasitism in chamois, rupicapra rupicapra rupicapra. vet. parasitol. 138: 337-348, 2006. hughes vl, randolph se. testosterone increases the transmission potential of tick-borne parasites. parasitology 123: 365-371, 2001. jacobs b, papewalis c, ehlers m, schott m. endocrine organs under the control of the immune system: potential implications for cellular therapies. horm. metab. res. 42: 9127, 2010. klein sl. the effects of hormones on sex differences in infection: from genes to behavior. neurosci. biobehav. rev. 24: 627-638, 2000. klein sl. hormonal and immunological mechanisms mediating sex differences in parasite infection. parasite immunol. 26: 247-264, 2004. kurtis jd, friedman jf, leenstra t, langdon gc, wu hw, manalo dl, et al. pubertal development predicts resistance to infection and reinfection with schistosoma japonicum. clin. infect. dis. 42: 1692-1698, 2006. larralde c, morales j, terrazas i, govezensky t, romano mc. sex hormone changes induced by the parasite lead to feminization of the male host in murine taenia crassiceps cysticercosis. j. steroid biochem. mol. biol. 52: 575-580, 1995. lehuen a, diana j, zaccone p, cooke a. immune cell crosstalk in type 1 diabetes. nat. rev. immunol. 10: 501-513, 2010. levron c, miquel j, oros m, scholz t. spermatozoa of tapeworms (platyhelminthes, eucestoda): advances in ultrastructural and phylogenetic studies. biol. rev. camb. philos. soc. 85:523543, 2010. long gh, boots m. how can immunopathology shape the evolution of parasite virulence? trends parasitol. 27: 300-305, 2011. loria rm, padgett da, huynh pn. regulation of the immune response by dehydroepiandrosterone and its metabolites. j. endocrinol. 150: s209220, 1996. ludin p, nilsson d, maser p. genome-wide identification of molecular mimicry candidates in parasites. plos one 6: e17546, 2011. mavoungou d, poaty-mavoungou v, ongali b, akoume my, maka g, mavoungou e. hypothalamic-pituitary gonadal axis and immune response imbalance during chronic filarial infections. trop. med. int. health 10: 1180-1186, 2005. morales j, larralde c, arteaga m, govezensky t, romano mc, morali g. inhibition of sexual behavior in male mice infected with taenia crassiceps cysticerci. j. parasitol. 82: 689-693, 1996. 151 morales-montor j, arrieta i, del castillo li, rodriguez-dorantes m, cerbon ma, larralde c. remote sensing of intraperitoneal parasitism by the host's brain: regional changes of c-fos gene expression in the brain of feminized cysticercotic male mice. parasitology 128: 343351, 2004. morales-montor j, baig s, hallal-calleros c, damian rt. taenia crassiceps: androgen reconstitution of the host leads to protection during cysticercosis. exp. parasitol. 100: 209216, 2002. morales-montor j, chavarria a, de leon ma, del castillo li, escobedo eg, sanchez en, et al. host gender in parasitic infections of mammals: an evaluation of the female host supremacy paradigm. j. parasitol. 90: 531-546, 2004. morales-montor j, escobedo g, vargasvillavicencio ja, larralde c. the neuroimmunoendocrine network in the complex host-parasite relationship during murine cysticercosis. curr. top. med. chem. 8: 400407, 2008. morales-montor j, mohamed f, damian rt. schistosoma mansoni: the effect of adrenalectomy on the murine model. microbes infect. 6: 475-480, 2004. morales-montor j, rodriguez-dorantes m, cerbon ma. modified expression of steroid 5 alphareductase as well as aromatase, but not cholesterol side-chain cleavage enzyme, in the reproductive system of male mice during (taenia crassiceps) cysticercosis. parasitol. res. 85: 393-398, 1999. muñoz-cruz s, togno-pierce c, morales-montor j. non-reproductive effects of sex steroids: their immunoregulatory role. curr. top. med. chem. 2011 [epub ahead of print]. nurieva r, yang xo, martinez g, zhang y, panopoulos ad, ma l, et al. essential autocrine regulation by il-21 in the generation of inflammatory t cells. nature 448: 480-483, 2007. olson pd, caira jn. evolution of the major lineages of tapeworms (platyhelminthes: cestoidea) inferred from 18s ribosomal dna and elongation factor-1alpha. j. parasitol. 85: 11341159, 1999. perez ar, bottasso o, savino w. the impact of infectious diseases upon neuroendocrine circuits. neuroimmunomodulation 16: 96-105, 2009. perkins se, ferrari mf, hudson pj. the effects of social structure and sex-biased transmission on macroparasite infection. parasitology 135: 1561-1569, 2008. roberts cw, walker w, alexander j. sexassociated hormones and immunity to protozoan parasites. clin. microbiol. rev. 14: 476-488, 2001. snider h, lezama-davila c, alexander j, satoskar ar. sex hormones and modulation of immunity against leishmaniasis. neuroimmunomodulation 16: 106-113, 2009. tomer y, huber a. the etiology of autoimmune thyroid disease: a story of genes and environment. j. autoimmun. 32: 231-239, 2009. touassem r, cabaret j, combes c. identification of schistosoma mansoni, s. rodhaini, s. bovis, s. haematobium, and s. intercalatum cercariae using segmentation analysis. trop. med. parasitol. 43: 159-161, 1992. wan y, bramson j. role of dendritic cell-derived cytokines in immune regulation. curr. pharm. des. 7: 977-992, 2001. watanabe k, hamano s, noda k, koga m, tada i. strongyloides ratti: additive effect of testosterone implantation and carbon injection on the susceptibility of female mice. parasitol. res. 85: 522-526, 1999. wilder rl. neuroendocrine-immune system interactions and autoimmunity. annu. rev. immunol. 13: 307-338, 1995. zhu j, yamane h, paul we. differentiation of effector cd4 t cell populations. annu. rev. immunol. 28: 445-489, 2010. 152 impacts of pollution on mitochondria activity and lipids store in feralm mya arenaria clams isj 7: 22-31, 2010 issn 1824-307x research report change in metallothionein phosphorylation state in mya arenaria clams: implication in metal metabolism and oxidative stress f gagné, m gélinas, c gagnon, c andré, c blaise fluvial ecosystem research, aquatic ecosystem protection research division, water science and technology, environment canada, 105 mcgill street, montréal, quebec, canada h2y 2e7 accepted january 4, 2010 abstract the contamination of the benthic environment poses a threat to long-lived sessile organisms such as clams. the purpose of this study was to investigate metal contamination in tissues and changes in metallothioneins (mt) in respect to its redox status in mya arenaria clams collected at three polluted sites. the phosphorylation state of mt was also investigated to determine whether this state is changed in clams collected at heavy-metal contaminated site and its involvement in cytoprotective signaling during stress contamination. the results show that clams collected at least one of the three polluted sites presented significantly higher concentrations of silver (ag), arsenic (as), cobalt (co), copper (cu), mercury (hg), nickel (ni), tin (sn) and lead (pb) in tissues. in the visceral tissue, total mt levels and the reduced, metal-binding form of the protein were significantly induced at the sites. the phosphorylation of mt and mitochondrial activity, as determined by electron transport and cytochrome c oxidase activities, were also significantly reduced at the contaminated sites. reduced phosphate levels in mt were negatively correlated with total mt levels, suggesting that decreased phosphorylation was involved in kinase-mediated signaling during cellular stress and could possibly alter the protein’s affinity to confer cytoprotection against heavy metal contamination. these preliminary investigations revealed that the phosphorylation state could change in polluted environment and provide some clues on the modulation of binding affinities during heavy-metal and oxidative stress in clams. key words: phosphorylated metallothioneins; mitochondrial electron transport activity; cytochrome c oxidase; heavy metals; mya arenaria introduction metallothioneins (mt) are low-molecular-weight (6–7 kda) metal-binding proteins. they are ubiquitous in life forms, from bacteria to mammals, as well as being rich in cysteine (25–30%) and heatstable owing to a lack of aromatic amino acids and hydrophobic regions (klaassen et al., 1999). these characteristics make them efficient transitional metal-binding proteins that are capable of binding ag(i), au(i), cd (ii), co(ii), cu(ii), hg(ii), pb(ii) and zn(ii). moreover, it was demonstrated that mt not only binds metals but also has the capacity to sequester reactive oxygen in the oyster (andersen et al., 1999) and nitrogen species such as superoxide anion and nitric oxide (atif et al., 2006). ___________________________________________________________________________ corresponding author: françois gagné fluvial ecosystem research section environment canada 105 mcgill st., montreal, quebec, canada h2y 2e7 e-mail: francois.gagne@ec.gc.ca upon reacting with these radicals, the metal thiolate clusters of mt oxidize, liberating metals from the protein (kang, 2006; gagné et al., 2008). hence, mt exists in both a reduced metal-binding form and in an oxidized metal-releasing form in the cytoplasm of cells. in contaminated mya arenaria populations exhibiting lower clam-bed density, growth and increased mean age values, marked responses in mt, oxidative stress and gonad size were observed (blaise et al., 2003). the same was also found in freshwater mussels, where cd concentrations in the high-molecular-weight protein fraction, where mt intervenes to reduce the metal content of these fractions, was the biomarker response that was most frequently and strongly correlated with the population variables (perceval et al., 2004). the mt biomarker is therefore considered a relevant biomarker of stress and has predictive value at higher levels of biological organization. recent studies suggest that ser/thr protein kinases (protein kinase c, or pkc) can phosphorylate 22 fig. 1 clams were collected at four sites (identified by stars) in june 2007. the site baie du moulin à baude (bau-r) was considered as the reference site; anse saint-jean (asj-e), baie sainte-catherine (bsc-t) and baie éternité (be-m) were the pollution-impacted sites. mt in neuronal cells (aras et al., 2009). pkc is a family of enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of aliphatic ser and thr amino acids. indeed, zn-induced metalresponsive element activation was significantly decreased in neuronal cells expressing a recombinant mt-1 devoid of its phosphorylation site, while cells expressing normal mt-1 had enhanced expression (aras et al., 2009). this suggests that pkc signaling could influence the phosphorylation of mt, which could, in turn, change its affinity for zn. however, it was recognized that the phosphorylation of proteins causes them to be degraded by the atpdependent ubiquitin/proteasome pathway. these target proteins become substrates for particular e3 ubiquitin ligases only when they are phosphorylated. it is interesting to observe that ubiquitin is activated by the formation of a thioester bond between the cterminal carboxyl group of ubiquitin and a (metal sensitive?) cys residue of the e1 enzyme, a process requiring atp as an energy source. at present, the function of mt phosphorylation is unclear despite its apparent involvement in pkc signaling and possible ubiquitinylation proteasome tagging. in another study, mt was seemingly involved in protein phosphorylation signaling: the re-activation of cysteine proteinase like caspases or other phosphatases. to show this, mt-iii prevented the activation of caspase-3 and -9 and the release of mitochondrial cytochrome c to the cytoplasm in neuronal cells (kim et al., 2009). mt-iii also increased the activation of akt, a ser/thr protein kinase, the phosphorylation (and degradation) of ikappa b, which can activate either an inflammatory or immune response, a cell survival response or cellular proliferation. these studies reveal that the phosphorylation state of mt might provide some clues about its involvement in cell signaling during cellular stress. this study represents the first attempt to track changes in mt-phosphate levels in m. arenaria clam populations obtained at polluted sites. the levels of total phosphate bound to mt and the proportion of oxidized/reduced mt collected at three sites contaminated by heavy metals were determined. the implication of mitochrondrial respiration (metabolic activity) was also examined by tracking mitochondrial electron transport and cytochrome c oxidase activities, the terminal enzyme 23 http://en.wikipedia.org/wiki/ubiquitin http://en.wikipedia.org/wiki/proteasome http://en.wikipedia.org/wiki/ubiquitin fig. 2 change in mt characteristics in clams from the st. lawrence estuary and saguenay fjord. clams were analyzed for mt in the visceral mass tissues. the total and phosphate-bound mt (a) and the redox status of mt (b) were determined. before the formation of atp, which is the main source of reactive-oxygen species formation in cells. an attempt was made to examine the influence of mt phosphate level, total mt and redox status on clam’s condition status. materials and methods spatial survey and clam collection soft-shell clams (mya arenaria) were handcollected at low tide in the morning from three pollution-impacted sites and one reference (i.e., under no direct source of pollution) site located in the saguenay fjord and the st. lawrence estuary (figure 1). in the saguenay fjord, the anse-saintjean site is located 40 km upstream from the estuary and receives the primary-treated (screened) effluent of about 2,000 residents (asj-e). the baie éternité site, which is located 15 km farther upstream, is historically recognized to be contaminated by heavy metals (be-m) (blaise et al., 2002). in the st. lawrence estuary, the baie saintecatherine site was chosen because it is heavily impacted by local commercial and pleasure-boat traffic from sightseeing and whale-watching operations, and has displayed a history of contamination by organotin (bsc-t) compounds and heavy metals (gagné et al., 2005). the baie du moulin à baude site is located 3 km downstream along the north shore of the st. lawrence estuary; it was selected as the reference site because of the absence of any direct source of pollution (bau-r). a total number of 30 clams were collected from clam beds at each site during early morning low tide. the clams were processed for biomarker analysis. clam age was estimated by the number of major grooves on the shell. clam weights and maximum longitudinal shell length were determined. the soft and gonad tissues were dissected out at 4 oc to determine the gonadosomatic index (gsi: wet weight of gonad/wet weight of soft tissues), condition factor (cf: clam weight/shell length) and growth index (gi: shell length/age). gender was determined by microscopic examination of gonad smears at 400 times magnification. parasitism in gonad tissues was seldom observed; any infected clams were discarded when present. the clam tissue samples were then frozen using dry ice for transportation to the laboratory and stored at -85 oc until analysis. after thawing on ice, the visceral tissues containing the gonad were crushed with a teflon pestle tissue grinder and homogenized in a 50 mm hepes-naoh buffer, ph 7.4, containing 150 mm nacl, 10 µg/ml apoprotinin and 0.5 mm dithiothreitol at a 1:5 volume/volume ratio (five passes). the dithiothreitol concentration was selected to stabilize the homogenates against mt oxidation during tissue processing (minkel et al., 1980). samples (100 µl) of each homogenate were collected for total protein determinations (bradford, 1976). the remaining homogenates were centrifuged at 1,500 x g (20 min at 2 oc) and the supernatant was centrifuged at 10,000 x g for 20 min at 2 oc to isolate the mitochondria in pellet. the mitochondria were resuspended in two volumes of the homogenization buffer. the remaining supernatant was centrifuged at 15,000 x g (20 min at 2 oc) for mt characterization, as described below. all biomarkers were normalized with total protein levels in the corresponding fractions. heavy metals and metalloids (i.e., ag, al, as, cd, co, cr, cu, hg, ni, pb, sn, and zn) were analyzed in whole soft tissues (three pools of ten individuals per site) according to standard methods of the national laboratory for environmental testing (nlet, 1994). the data were expressed as ng of metals or metalloids/g of dry weight. metallothionein characterization the levels of total, oxidized and reduced mt were determined using a modified version of the spectrophotometric method of viarengo et al. (1997), as described elsewhere (gagné et al., 2008). the original methodology uses a series of 24 solvent fractionation steps to isolate the mt fraction from high-molecular-weight proteins and peptides such as glutathione. the modification includes a step for the complete reduction of mt (with the highly potent reducer tris[2carboxyethyl]phosphine) in the s15 fraction, which was stabilized at the homogenization step using a small amount of dithiothreitol, which was not sufficient to further reduce mt in the homogenates (minkel et al., 1980). the s15 samples were divided between two tubes for total mt and metallic (reduced) mt evaluations. for the former, 200 µl of the s15 fraction was pre-treated with 50 µl of 50 mm tris(2-carboxyethyl)phosphine (sigma chemical company, mo, usa) for 30 min before the addition of an acidic ethanol/chloroform solution and subsequent ethanol fractionation steps to obtain the total level (reduced and oxidized) of mt. for the metal-binding (reduced) mt form, 200 µl of the s15 fraction was mixed with 50 µl of water alone with no reduction step. a sample of the mt pellet was set aside for total phosphate determinations according to the phosphomolybdate methodology of stanton (1968) after treating the mt pellet in 1 m naoh for 30 min to liberate inorganic phosphate. the data were expressed as µmol of thiol (gsh) equivalents/mg total protein for total and reduced mt or as µg phosphates/mg total proteins in the s15 fraction for phosphate mt. the oxidized fraction of mt was calculated as follows: mtoxidized = mttotal (phosphine treated) mtmetallic. analysis of the fractionated mt samples by high-resolution polyacrylamide gel electrophoresis with coomassie blue staining revealed a major band around 10 kda that is characteristic of mt, with no contaminating high-molecular-weight protein bands apparent. mitochondrial activity energy expenditures were determined by measuring mitochondrial electron transport (met) activity (smolders et al., 2004). met activity was determined in the mitochondrial fraction (resuspended 10,000 x g pellet) using the piodonitrotetrazolium dye method (king and packard, 1975; smolders et al., 2004). briefly, 100 µl of the resuspended mitochondrial pellet was mixed with 100 mm tris-hcl, ph 8.5, containing 100 µm mgso4, 0.1% triton x-100 and 5% polyvinylpyrrolidone for 1 min on ice. the reaction mixture was mixed with 1 mm and 0.2 mm nadh and nadph, respectively, on ice. the reaction was initiated with the addition of 50 µl of 5 mm piodonitrotetrazolium for 30 min at 20 oc. absorbance readings were measured at 15-min intervals at 520 nm. the data were expressed as the loss of absorbance at 20 oc/30 min/mg total proteins in the mitochondria. cytochrome c oxidase (ccox) was determined by a spectrophotometric cytochrome c oxidation assay (sakai et al., 1988). data analysis a total of 12 clams were analyzed for each site. the homogeneity of variances was determined using bartlett’s test. where the data proved to be heterogeneous they were log-transformed. the data were subjected to an analysis of variance using the benferroni t test for comparison of the biomarker data against the reference site (i.e., bau-r). a pearson product-moment analysis was performed to determine if there were any correlations within the metal and biomarker of effects data. factorial and discriminant function analyses were also performed to examine the interrelatedness among the data, the biomarkers and the metal loads in tissues in order to discriminate among and to examine the sites. significance was set at p<0.05. all statistical tests were performed using statistica software (version 8). results based on the investigation on the primary sequences of mt from various species in public medline database (pubmed), the sequences reveal the substantial presence of serine (ser) and threonine (thr), which can be potential aliphatic phosphorylation sites (table 1). the proportion of {ser+thr} amino acids represents about 17% of the amino acids in various species, ranging from protozoans to mammals. in bivalves, the proportion of {ser+thr} represents between 14% (m. edulis) to 18% (dreissena polymorpha) of the amino acids of the mt protein. the highest proportion was found in rainbow trout (oncorhynchus mykiss) mt-1 with 23% of the amino acids as either ser or thr residues. the proportion of {ser+thr} residues was significantly correlated with the proportion of cys (r=0.78; p<0.01) and independent of the total number of amino acids of mt. moreover, ser and thr residues were often located in the vicinity of cys residues, frequently forming -cys-ser-cysor –cysthr-cyssequences. this suggests that mt could undergo phosphorylation and perhaps influence the protein’s capacity to bind heavy metals and/or reactive oxygen species, which is well-established in mts. the investigation of mt sequences also revealed that these proteins contains lysine residues which are possible ubiquitinylation sites. indeed, mt typically has 10% lysine residues, which suggests possible ubiquitinylation sites (table 1). the proportion of lysine appears to be independent (i.e., not correlated) of the proportion of ser+thr, of cys and of the total number of amino acids in the various species of mt. clams were harvested at one reference and one polluted site in the st. lawrence estuary (baur and bsc-t, respectively) and two sites located upstream in the saguenay fjord (asj-e and be-m, respectively). in an attempt to characterize the quality of each site, total heavy-metal contents were determined in m. arenaria clams (table 2). the data revealed that as and co were significantly increased at the bsc-t sites while cu was elevated at be-m sites (p<0.05 level). cr was significantly higher at the asj-e and be-m sites. ni loads in the clam tissues were significantly higher at site asj-e. pb was significantly higher at all sites relative to the reference site bau-r. mercury, pb and sn were significantly higher at the bsc-t site, already known to be contaminated by organotin compounds (viglino et al., 2006). zn was significantly reduced at this site. a correlation analysis revealed that ag was significantly correlated with cd (r=0.67; p<0.001), cu (r=0.68; p<0.001) and hg (r=0.89; p<0.001). tissue 25 table 1. characteristics of mt in different species. organism species proportion of ser and thr amino acids1 proportion of lysine residues2 proportion of cysteine (%) total amino acids protist tetrahymena pyriformis 16 14 29 107 mussels mytilus galloprovincialis 15 7 26 72 psychotria viridis 16 5 28 75 mytilus edulis 14 10 25 73 clams dreissena polymorpha 18 8 28 73 musca lusoria 14 12 28 76 gastropods helix pomatia 16 12 24 67 nematodes caenorhabditis elegans 11 13 25 63 fish oncorhynchus mykiss 23 12 31 61 perca fluviatilis 20 10 33 60 human homo sapiens 20 11 31 61 mean±sd 16.6±3.4 10.4±2.7 28±3 71±13 1 potential phosphorylation sites as estimated by the percentage of number (ser+thr)*100/total number of amino acids of mt. 2 potential ubiquitinylation sites as calculated by the percentage of lysine residues: number lys (*100)/total number of amino acids of mt. al content was significantly correlated with as (r=0.44; p<0.05), pb (r=0.4; p=0.05) and sn (r=0.83; p<0.001). tissue as levels were significantly correlated with co (r=0.61; p=0.001), cr (r=0.51; p=0.01), cu (r=0.52; p<0.01) and pb (r=0.66; p<0.001). cd levels in tissues were significantly correlated with cr (r=0.53; p<0.01), cu (r=0.53; p<0.01), zn (r=0.72; p<0.001) and hg (r=0.66; p<0.001). co tissue levels were significantly correlated with pb (r=0.78; p<0.001) only. cr levels were significantly correlated with cu (r=0.47; p<0.05) and zn (r=0.47; p<0.05). tissue cu levels were significantly correlated with sn (r=-0.44; p<0.05), zn (r=0.66; p<0.001) and hg (r=0.63; p=0.001). tissue sn levels were significantly correlated with hg (r=-0.50; p=0.01). zn tissue levels were significantly correlated with hg (r=0.44; p<0.05). it is noteworthy that most correlations were positive, with the exception of sn levels in tissue, which were negatively correlated with metals such as ag, cu and hg. clam condition factor (cf) varied significantly across all sites (anova p<0.001). the cf was significantly reduced (1.3-fold) at the bsc-t and asj-e (1.5-fold) sites. no significant change was observed in cf at the be-m site (table 3). the gi was significantly reduced by 1.2 at only one site (bsc-t). gsi also significantly affected (anova p<0.001) at one site and was significantly increased 1.4-fold at the be-m site. a correlation analysis revealed that cf and gsi were significantly correlated (r=0.31; p<0.05). correlations between biomarkers of effects were included in table 4 while correlations with the heavy metal tissue loadings were cited in the text. clam cf was significantly correlated with tissue ag (r=0.51; p=0.01), cu (r=0.51; p=0.01) and hg (r=0.57; p<0.05). the growth index was negatively correlated with tissue co (r=-0.42; p<0.05) and sn (r=-0.4; p=0.05) levels and positively correlated with tissue cu (r=0.48; p<0.05) and zn (r=0.50; p=0.01) levels. the gsi was not correlated with any of the metal burdens in tissue. the levels of mt in the clam gonad/visceral mass were determined (fig. 2a and b). the results revealed that the total levels of mt were significantly affected at the polluted sites (fig. 2a). total mt levels decreased at bsc-t while they were significantly elevated at the be-m site. at the asj-e site, only a marginal increase was observed (p=0.07). the phosphate levels of the mt fraction were significantly reduced at the bsc-t and be-m sites, while a marginal decrease was found at site asj-e (p=0.06). a correlation analysis revealed that total mt was negatively correlated with the amount of phosphate bound to mt (r=-0.69; p<0.001) and positively so with as (r=0.50; p=0.05), cu (r=0.83; p<0.001), zn (r=0.79; p<0.001) and hg (r=0.49; p=0.07). the phosphate associated with mt levels was negatively correlated with as (r=0.65; p<0.01), co (r=-0.61; p<0.01), cu (r=-0.50; p<0.05) and pb (r=-0.64; p<0.01). the redox status of mt was also appraised (fig. 2b). the proportion of the reduced and metal-binding forms of mt rose significantly at all three polluted sites. the proportion of reduced metal-binding mt was significantly correlated with phosphorylated mt (r=0.43; p=0.05) and cr (r=0.47; p<0.05). this indicates that mt phosphorylation follows the formation of oxidized (metal-releasing) mt at the expense of the metal-binding (reduced) fraction of mt. 26 table 2. heavy-metal content of m. arenaria clams from the st. lawrence estuary and saguenay fjord. metals (µg/g) bau-r bsc-t asj-e be-m ag 0.212±0.02 0.044±0.006 0.122±0.022 0.36±0.14* al 91±13 122±24 96±6 109±5 as 0.63±0.04 0.83±0.02* 0.77±0.001 0.96±0.1* cd 0.08±0.008 0.06±0.003 0.09±0.002 0.094±0.009 co 0.115±0.02 0.2±0.01* 0.12±0.009 0.18±0.006* cr 0.4±0.09 0.48±0.08 0.8±0.05* 0.74±0.05* cu 1.2±0.05 0.7±0.06* 1.16±0.07 1.65±0.2* ni 0.37±0.04 0.39±0.05 0.51±0.03* 0.46±0.04 pb 0.03±0.001 0.06±0.005* 0.04±0.002 0.05±0.002* sn 0.02±0.02 0.117±0.02 0.043±0.02 0.03±0.02 zn 14±1 10±1* 14±1 15±0.5 hg 0.05±0.005 0.027±0.004* 0.03±0.001 0.064±0.01* * indicates statistical significance from the reference site at p<0.05 (anova and least-square-difference tests). table 3. values of morphometric parameters at four study sites. site bau-r bsc-t asj-e be-m parameter condition factor (cf) 0.67±0.02 0.54±0.01* 0.44±0.01* 0.70±0.02 growth index 9.3±0.3 7.6±0.1* 9.2±0.3 8.8±0.3 gsi 0.08±0.01 0.09±0.01 0.080±0.003 0.110±0.004* sex ratio (1= all males; 2= all females) 1.5±0.1 1.67±0.14 1.75±0.10 1.6±0.2 * indicates significance at p<0.05 level. the influence of metabolic activity and oxidative stress on the expression of mt was determined by following mitochondrial electron transport activity (a measure related to oxygen production in mitochondria) and ccox, the last enzyme complex involved in electron transport activity (fig. 3a and 3b). met activity was readily reduced at the asj-e (2.8-fold) and be-m (2.3-fold) sites. met activity was significantly correlated with phosphates bound to mt (r=0.58; p<0.01), co (r=0.51; p<0.05), cr (r=0.64; p<0.01), cu (r=-0.54, p<0.05) and marginally correlated with ni (r=-0,43; p=0.09). the activity of ccox was also affected in mitochondria (fig. 3b). its activity was significantly decreased at the bsc-t (1.6-fold) and be-m (2.2-fold) sites, significantly correlated with phosphorylated mt (r=0.50; p=0.05) and marginally so with redox mt (r=0.41; p=0.07 for oxidized mt). ccox was not significantly related to any of the measured metals in tissues. the various tissue biomarker and metal loads were analyzed using factorial and discriminant function analyses to identify the major biomarkers that could explain most of the data responses and identify site-specific characteristics (fig. 4a and 4b). a factorial analysis revealed that most of the variance (60%) was explained by three factors. the biomarkers with the highest factorial weights were ag, cu, hg, pb, sn, zn, cf and total mt. these metals formed a cluster close to total mt, cf, growth and gsi (fig. 4a), indicating that these metals were statistically related to diminished clam condition and altered gonad development. phosphorylated mt was located at the opposite side of this cluster and was closely located with ccox activity and met endpoints. a discriminant function analysis revealed that all sites were well discriminated at 100% accuracy (fig. 4b). the first root function was able to discriminate between sites be-m and asj-e, with the following biomarkers having the highest factorial weights: cf, mt-p and total mt. the second root function well discriminated the reference site bau-r from the other sites, with the following biomarkers having the highest factorial weights: cf, mt-p and tissue pb levels. these analyses revealed that the phosphorylation state of mt is an important factor relating to clam morphological status and health condition. 27 table 4 correlation analyses of the measured endpoints. total mt oxidized mt reduced mt mt-p met ccox gsi growth cf total mt 0.25 -0.25 -0.68 -0.17 -0.33 0.51 0.22 0.67 p>0.1 p>0.1 p=0.001 p>0.1 p=0.1 p<0.01 p>0.1 p<0.001 oxidized mt 0.40 0.15 0.44 -0.14 0.04 0.42 p=0.05 p>0.1 p<0.05 p>0.1 p>0.1 p<0.05 reduced mt -0.40 -0.15 -0.44 0.14 -0.05 -0.42 p=0.05 p>0.1 p<0.05 p>0.1 p>0.1 p<0.05 mt-p 0.58 0.50 -0.56 0.05 -0.43 p<0.01 p<0.05 p=0.001 p>0.1 p=0.01 met -0.04 -0.1 -0.19 0.1 p>0.1 p>0.1 p>0.1 p>0.1 ccox -0.41 0.08 -0.32 p<0.05 p>0.1 p=0.09 gsi 0.09 0.31 p>0.1 p<0.05 growth 0.11 p>0.1 discussion the phosphorylation state of mt in clam tissues appears to be related to the catabolism of mt in cells. our data support this hypothesis since mtphosphate was negatively related with the total amount of mt in cells. the increase in mt phosphate was also related to the proportion of oxidized mt in cells. since mitochondria are the principal source of reactive oxygen species in cells (abele et al., 2002), we examined their activity by tracking electron transport and ccox activities. the data revealed that mt-phosphate was closely related to changes in both met and ccox activity in tissues, suggesting that 1) mt phosphorylation is coupled in some way with mitochondrial electron transport activity and/or 2) mt is oxidized by increased mitochondrial respiration rates and then phosphorylated for removal, perhaps through the ubiquitin/proteasome pathway. the latter hypothesis was consistent with the observation that mtphosphate was negatively related to total mt levels and positively related to oxidized mt, met and ccox activities. protein phosphorylation is a prerequisite for tagging proteasome degradation and ubiquitin requires lysine residues for binding to the phosphorylated proteins which are found in various mt sequences of different species (table 1). however, this would have to be verified by more specific experiments (i.e., finding ubiquitylated mt in bivalve tissues by western blot analysis using antiubiquitin antibodies). it is noteworthy that the decrease in mt-phosphate was associated with clams with lower cfs and altered gonad weights. clams contaminated by metals from polluted sites had decreased phosphate bound to mt with a concomitant elevation in the metal-binding form of mt and total mt levels. this indicates that clams from metal-contaminated sites increase the levels of mt for metal sequestration, perhaps at the expense of oxygen radical scavenging. this was corroborated by a previous study in which the metalbinding form of mt was more closely associated to oxidative stress and tissue damage than was the oxidized form of mt (gagné et al., 2008). perhaps this explains, at least in part, why met and ccox activity tended to be lower at the polluted sites. moreover, it has long been established that phosphorylation plays an important role in controlling mitochondrial metabolism, where a delicate balance between electron transport for respiration and protection against the production of reactive oxygen species is required (foster et al., 2009; sokolova et al., 2005). met and related enzyme complexes involved in respiration (ccox) would release reactive oxygen species that are less sequestered by mt and thus likely to do damage. this was consistent with increased lipid peroxidation in the gonadal homogenates of clams at the polluted sites in this study (results not shown). it is noteworthy that sn in tissues were negatively related to ag, cu and hg in clams. according to the factorial analysis, sn tissue loadings were relatively close to the mt-phosphate and ccox cluster. a possible explanation for would be that sn competes with ag and hg to cu (and zn) binding sites. moreover, organotin compounds are well-known to have a high affinity for hemoproteins which could decouple electron flow in mitochondria (simionatto et al., 1984). to the best of our knowledge, this is the first report of the modulation of mt phosphorylation in clam populations from polluted sites as determined by increased heavy-metal tissue loadings. ser and thr protein kinases are also ubiquitous in bivalves (dailianis and kaloyianni, 2004). the external face of 28 fig. 3 mitochondrial electron transport activity in selected clam populations. mitochondrial electron transport (a) and cytochrome c oxidase (b) activities were measured in isolated mitochondria from feral clam populations. asterisks (*) indicate significance at p<0.05 the outer mitochondrial membranes is filled with various intracellular receptors and serves as a site for signals implicated in steroidogenesis, cytoprotection and energy demands, where pkcinteracting proteins are found (smith et al., 2006; poole et al., 2004). this family of protein kinases is also involved in zn signaling in neuronal cells, which confers tolerance towards zn2+ and suggests that pkc acts directly on the intracellular source of zn and the expression of zn-regulated genes such as mt (aras et al., 2009). in mt, a protein kinase c phosphorylation site was identified at serine 32 regardless of the species examined. moreover, mt phosphorylation appears to modulate the zn-binding efficiency of mt, as shown by the reduction in zn2+induced metal-responsive element in cells expressing a mutated mt that is devoid of a phosphorylation site. this was explained by the phosphate-associated mt, which lowers the binding affinity for zn and favoring the subsequent release of zn2+ for the activation of metal-responsive elements and the production of de novo zndependent genes such as mt. our data revealed an inverse relationship between phosphate-bound mt and total mt/metal-binding mt, which suggests that decreased mt phosphorylation might have intervened in the mt forms in handling metal homeostasis in clams from metal-contaminated sites. in another study, copper-induced mt-1 transcription was regulated by pkc and a metal transcription factor (mattie and freedman, 2004). the role of mt in the maintenance of redox homeostatis appears to recall other protein phosphatases such as protein kinase b, leading to ikappa b degradation which, in turn, activates the expression of nf-kappab activity during the inflammatory response and oxidative stress (kim et al., 2009). the inhibition of several intracellular protein kinases such as protein kinase a and c, mapk and calmodulin kinase-ii eliminated the neuroprotective effect of mt, highlighting the interplay between metal-binding capacity, oxidative stress and protein phosphorylation (asmussen et al., 2009). in mytilus galloprovincialis, cd and zn caused an increase in superoxide anion production, with cd being more potent (2.2-fold) than zn (1.5fold) (koutsogiannaki et al., 2006). in addition, the metal effect was reversed in the presence of calphostin, a pkc inhibitor, and an amiloride analogue that blocks na/k-exchanges. conversely, cd induced pyruvate kinase activity and a rise in intracellular ph was augmented by phorbol esters, a potent activator of pkc (dailianis and kaloyianni, 2004). this is in agreement with the increase in metal affinity in less phosphorylated-mt hypothesis, which confers cytoprotection to cd and zn but perhaps at the expense of radical oxygen scavenging. taken together, these studies reveal a hormone-like effect of divalent metals such as cd and zn at the β-adrenergic and pkc signal transduction pathways in m. galloprovincialis. however, gsi was not correlated with any of the biomarkers in this study suggesting perhaps that hormonal signaling involved in gametogenesis (i.e., steroids) had no important effects on the phosphorylation state of mt. we should bear in mind that these sites were not only contaminated by heavy metals but with organic contaminants such as polyaromatic hydrocarbons which could also produce oxidative stress and perhaps influence the phosphorylation state of mts. more research is required to better understand the physiological role of mt phosphorylation in the handling of heavy metals and oxygen radicals in bivalves. in conclusion, m. arenaria clams from heavy metalcontaminated sites show increased total and metalbinding mt with decreased met and ccox activities. total mt and the metal-binding (reduced) form of mt were closely related with levels of ag, cu, hg and zn in tissue. moreover, mt phosphorylation was also reduced at the contaminated sites (less-phosphorylated mt would have a stronger metal-binding capacity), suggesting 29 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22dailianis%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22kaloyianni%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus fig. 4 factorial and discriminant function analyses of the biomarker responses. the factorial analysis was performed with the principal component extraction procedure (a). the discriminant function analysis was also performed to examine the capacity of the biomarkers used in this study to identify the sites (b). the involvement of pkc signaling pathways that can change the metal-binding affinity of mt towards exposure to various metals. the measurement of phosphates bound to the mt fraction might provide some clues on the hormone-like effects of metals and the modulation of binding affinities towards metal contamination of the environment. acknowledgements the authors thank sophie trépanier for her technical support with the biomarker analyses. this work was funded by environment canada under the cepa initiatives. the manuscript was edited by patricia potvin of environment canada. references abele d, heise k, portner ho, puntarulo s. temperature dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam mya arenaria. j. exp. biol. 205: 1831–1841, 2002. anderson sr, patel km, röesijadi g. oyster metallothionein as an oxyradicals scavenger: implication for hemocyte defense responses. dev. comp. immunol 23:443–449, 1999. aras ma, hara h, hartnett k a, kandler k, aizenman e. protein kinase c regulation of neuronal zinc signaling mediates survival during preconditioning j. neurochem. 110:106-117, 2009. asmussen jw, von sperling ml, penkowa m. intraneuronal signaling pathways of metallothionein. j. neurosci. res. 87: 2926– 2936, 2009. atif f, kaur m, yousuf s, raisuddin s. in vitro free radical scavenging activity of hepatic metallothionein induced in an indian freshwater fish, channa punctata bloch. chem. biol. interact. 162: 172–180, 2006. blaise c, gagné f, pellerin j, hansen pd, trottier s. molluscan shellfish biomarker study of the quebec, canada, saguenay fjord with the softshell clam mya arenaria. environ. toxicol. 17: 170–186, 2002. blaise c, gagné f, pellerin j. bivalve population status and biomarker responses in mya arenaria clams (saguenay fjord, québec,canada). fresenius environ. bull. 12: 956–960, 2003. bradford mm. a sensitive method for the total protein determination using the principle of protein-dye binding. anal. biochem. 72: 249– 251, 1976. dailianis s, kaloyianni m. 2004. cadmium induces both pyruvate kinase and na+/h+ exchanger activity through protein kinase c mediated signal transduction, in isolated digestive gland cells of mytilus galloprovincialis (l.). j. exp. biol. 207: 1665–1674, 2004. foster db, van eyk je, marban e, o'rourke b. redox signaling and protein phosphorylation in mitochondria: progress and prospects. j. bioenerg. biomembr. 41: 159-168, 2009. gagné f, blaise c, pellerin j, pelletier e, strand j. health status of mya arenaria bivalves collected from contaminated sites in canada (saguenay fjord) and denmark (odense fjord) during their reproductive period. ecotoxicol. environ. saf. 64: 348–361, 2005. gagné f, andré c, blaise c. the dual nature of metallothioneins in the metabolism of heavy metals and reactive oxygen species in aquatic organisms: implications of use as a biomarker of heavy-metal effects in field investigations. biochem. insights 1: 31–41, 2008. kang yj. metallothionein redox cycle and function. exp. biol. med. 231: 1459–1467, 2006. kim hg, hwang yp, han eh, choi cy, yeo cy, kim jy, et al. metallothionein-iii provides neuronal protection through activation of nuclear factorκb via thetrka/phosphatidylinositol-3 kinase/akt signaling pathway. toxicol. sci. 112: 435-449, 2009. 30 king f, packard tt. respiration and the activity of the respiratory electron transport system in marine zooplankton. limnol. oceanogr. 20: 849–854, 1975. klaassen cd, liu j, choudhuri s. metallothionein: an intracellular protein to protect against cadmium toxicity. ann. rev. pharmacol. toxicol. 39: 267–294, 1999. koutsogiannaki s, evangelinos n, koliakos g, kaloyianni m. cytotoxic mechanisms of zn2+ and cd2+ involve na+/h+ exchanger (nhe) activation by ros. aquat. toxicol. 78: 315– 324, 2006. mattie md, freedman jh. copper-inducible transcription: regulation by metaland oxidative stress-responsive pathways. am. j. physiol.cell physiol. 286: 293–301, 2004. minkel dt, poulsen k, wielgus s, shaw cf, petering dh. on the sensitivity of metallothioneins to oxidation during isolation. biochem j. 191: 475–485, 1980. nlet – national laboratory for environmental testing. 1994. manual of analytical methods. national laboratory for environmental testing, environment canada, burlington, ontario, canada, 1994. perceval o, couillard y, pinel-alloul b, giguère a, campbell pgc. metal-induced stress in bivalves living along a gradient of cd contamination: relating sub-cellular metal distribution to population-level responses. aquat. toxicol. 69: 327–345, 2004. poole aw, pula g, hers i, crosby d, jones ml. pkc-interacting proteins: from function to pharmacology. trends pharmacol. sci. 25: 528–535, 2004. sakai y, tanaka a, ikai i, yamaoka y, ozawa k, orii y. measurement of cytochrome c oxidase activity in human liver specimens obtained by needle biopsy. clini. chim. acta 176: 343–346, 1988. simionatto cs, anderson ke, sassa s, drummond gs, kappas a.. fluorometric measurement of tinprotoporphyrin in biological samples. anal. biochem. 141: 213–219, 1984. smith fd, langeberg lk, scott jd. the where's and when's of kinase anchoring. trends biochem. sci. 31: 316–323, 2006. smolders r, bervoet l, de coen w, blust r. cellular energy allocation in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization. environ. pollut. 129: 99–112, 2004. sokolova im, sokolov ep, ponnappa km. 2005. cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster crassostrea virginica gmelin (bivalvia: ostreidae). aquat. toxicol. 73: 242-255, 2005. stanton mg. 1968. colorimetric determination of inorganic phosphate in the presence of biological material and adenosine triphosphate. anal. biochem. 22: 27-34, 1968. viarengo a, ponzanon e, dondero f, fabbri r. a simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to mediterranean and antarctic molluscs. mar. environ. res. 44: 69–84, 1997. viglino l, pelletier e, lee j. 2006. butyltin species in benthic and pelagic organisms of the saguenay fjord (canada) and imposex occurrence in common whelk (buccinum undatum). arc. env. cont. toxicol. 49: 1–17, 2006. wills ed. evaluation of lipid peroxidation in lipids and biological membranes. in: snell k; mullock b (eds), biochemical toxicology: a practical approach,: irl press, washington dc, usa, pp 127–150, 1987. 31 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22freedman%20jh%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus caenorhabditis elegans minireview isj 9: 58-63, 2012 issn 1824-307x minireview someone like it hot? effects of global warming on insect immunity and microbiota m mandrioli department of biology, university of modena and reggio emilia, modena, italy accepted march 14, 2012 abstract global warming represents a substantial challenge on a broad range of organisms with diverse life-history traits and geographical distributions. up till now several studies correlated global warming to changes in body mass, growth rate or fat content, whereas the effects on immune function and microbiota composition remained almost unexplored. on the contrary, some pioneering studies are showing that increased temperature may influence the insect immune function and the microbiota composition, making global warming in a pivotal position influencing insect survival and adaptation to a warming planet. key words: global warming; immunity; microbiota composition; thermal tolerance; symbionts       58 living in a warming planet temperature is considered one of the most important ecological factors for ectothermic organisms and the ability to tolerate temperature fluctuations is essential for individual survival (overgaard and sørensen, 2008). consequently, global warming may pose a substantial challenge on many natural systems and in particular for tropical ectotherms, living close to their upper critical thermal limits, making them particularly vulnerable to global warming (sala et al., 2000; thomas et al., 2004). insects are among the groups of organisms most likely to be affected by climatic changes because climate has direct influences on their development, reproduction and survival (bale et al., 2002; savage et al., 2004; frazier et al., 2006; menéndez, 2007). nevertheless, insects have short generation times and high reproductive rates, so that they can respond quicker to climate change than long-lived organisms, such as plants and vertebrates (bale et al., 2002; menéndez, 2007). warming can therefore potentially affect several aspects of insect life-cycle and ecology, and potential responses could include changes in phenological patterns and habitat selection and the expansion/contraction of geographic and altitudinal ranges (bale et al., 2002; menéndez, 2007; berg et al., 2010). ___________________________________________________________________________ corresponding author: mauro mandrioli department of biology, university of modena and reggio emilia, via campi 213/d, 41125 modena, italy e-mail: mauro.mandrioli@unimo.it global average surface temperature increased by about 0.6 ºc during the past century, and the third ipcc report predicts that temperatures will continue to rise during the next century, with increases of up to 5.8 ºc by the year 2100 (houghton et al., 2001). the study of how these human-induced changes in climate may affect biodiversity has attracted a vast research effort during the last two decades in order to study the ecological impacts of current warming on a broad range of organisms with diverse life-history traits and geographical distributions (menéndez, 2007). due to the importance of insects for human health and activities (such as agriculture), several studies focused their attention on insects showing that in temperate regions, climate warming is predicted to benefit many insect species since less severe winter months could results in higher overwintering survival and increase in the population sizes (bale et al., 2002; botkin et al., 2007). moreover, climate warming could lengthen the growing season resulting in increased growth, reproduction rate and number of generations per year. the effects could be hampered in cool climate zones, where climate warming could increase insect fitness by bringing them closer to their physiological optima (bale et al., 2002; botkin et al., 2007). is therefore global warming always beneficial for insects? what could happen to tropical insects that already live close to their optimal temperature? can climate warming decrease their fitness by exceeding the physiological optima? aphids, for instance, are not always able to adapt physiologically to high temperatures since they are already living close to their upper temperature limit for survival (neve et al., 2009; hazel et al., 2010). as reported by chiu et al. (2012), global warming seems to affect the fitness of the aphid myzus varians altering several physiological functions, including immunity, development and reproduction. in particular, more than 90% of m. varians nymphs reached adulthood in the temperature regimes with a daily mean temperature of 28.8 and 30.0 °c, whereas no nymphs reached adulthood at 32.5 °c. at the same time, the mean fecundity of aphids reared at 28.8 °c was greater than that for aphids reared at 30.0 °c, showing that even a small increase in mean temperature from 28.8 to 30.0 °c could cause a decline in the fitness of m. varians so that aphid populations could go extinct locally and populations will not rebound even when temperatures become favourable in the fall (chiu et al., 2012).     59 up till now several studies correlated global warming to changes in some aspects, such as body mass, growth rate or fat content as fitness-related parameters (bale et al., 2002; botkin et al., 2007), whereas the effects on immune function and microbiota composition remained almost unexplored. in view of this assumption the present paper has been focussed mainly on the effects of warming on insect immune function and microbiota composition, parameters that could play important roles in insect survival and adaptation to a warming planet. insect immunity and global warming insects present an immune system endowed with only innate immune components consisting of cellular and humoral factors (mandrioli et al., 2003; nappi et al., 2004; malagoli et al., 2007, 2010). cellmediated immunity includes phagocytosis and encapsulation, exerted by specific cell types (ballarin et al., 2008), while humoral mediators comprise several factors among which the antimicrobial peptides (amps) and the components of the pro-phenoloxidase (pro-po) cascade have been the most elucidated (bulet and stöcklin, 2005). it is important to observe that cellular and humoral components have not to be considered as separate elements, because several findings indicated that secreted factors are fundamental for clotting and pathogen recognition and engulfment (bulet and stöcklin, 2005). the maintenance and deployment of an efficient immune response may shift away resources from other functions (such as reproduction), so that immune function results from physiological trade-offs in insects (bonneaud et al., 2003; schmid-hempel, 2003, 2005; rolff et al., 2004). in view of its cost, the immune response is influenced therefore by both biotic and abiotic factors (such as food availability and temperature) (le moullac and haffner, 2000; mydlarz et al., 2006; de block and stoks, 2008; karl et al., 2011). according to these assumptions, the predicted increase in thermal stress due to global warming (diffenbaugh et al., 2005, 2007) is likely to induce cascading effects on other functions such as the fig. 1 global warming can positively influence immunity (by enhancing the activity of some molecules), reproduction (favouring increased egg deposition, faster grow and over-wintering survival) and resource availability (for instance by making faster grow plants). however, global warming also induces heat stress that has costs that could reduce the resources available for reproduction and immune responses. immune response, thus further reducing the individual fitness and favouring the distribution and prevalence of infectious diseases (lafferty, 2009; travers et al., 2009). in order to verify this hypothesis, karl et al. (2011) investigated in the tropical butterfly bicyclus anynana the effects of temperature changes on fitness-related adult traits (such as body mass and fat content) and on phenoloxidase (po) activity and hemocyte numbers that are two key parameters for evaluating the immune function at both cellular and humoral levels. interestingly, results on body mass and fat content suggest that global warming could be beneficial, whereas haemocyte numbers and po activity decreased at increasing temperatures (karl et al., 2011) supporting the hypothesis that immune parameters were negatively affected by global warming. at higher temperatures, insects may therefore increase their rate of growth and reproduction, but may become more susceptible to diseases, leading to reduced lifespan and possibly reduced fitness in the field (figure 1). at the same time, karl et al. (2011) observed that the decrease in po activity with increasing temperature was more evident in food-deprived individuals, in respect to butterflies having access to food showing a trade-off relating energy shortage and immune response. even if the molecular mechanism at the basis of this change in the po activity has not been studied, a direct link between the expression of heat shock proteins and a decrease in immune parameters could be     60 suggested. interestingly, two mediators of the response to stress (norepinephrine and glucocorticoids) are known in mammals to act as immune-suppressors thus decreasing disease resistance (sternberg, 2006). the study of the trade-off between immune response and thermal tolerance is absolutely relevant in medicine and agriculture since if both immune function and reproduction are simultaneously enhanced, then climate change will result in the reproduction of more insects that will be more resistant to diseases. as a consequence, we will have more pest crop insects in our fields with more damages for agriculture, together with larger numbers of insects relevant for the transmission of human diseases. on the contrary, if higher temperatures induce or exacerbate trade-offs between reproduction and immune function, in order to face the global warming insects have to define if put their resources mainly on reproduction or on the immune systems. a possible reply to this question can be obtained from studies performed in the cricket gryllus texensis, where empirical evidence suggested that warmer temperatures lead to a decline in some immune functions (e.g. melanisation) (suwanchaichanda and paskewitz, 1998; adamo and lovett, 2004). in particular, it emerged that g. texensis may become more susceptible to some pathogens at higher temperatures suggesting that reproductive rate and immune function are not simultaneously enhanced at higher temperatures. a further confirmation of the presence of a strict trade-off between immune response, reproduction and temperature has been observed by an interesting set of data published by adamo and lovett in 2004. in particular they reported that elevated temperatures resulted in increased egg laying, faster egg development and greater mass gain in gryllus texensis. in the same experiments a reduction in the resistance to the gram-positive bacterium bacillus cereus was observed both above or below the average field temperature (26°c) suggesting that increased temperatures induce trade-offs between reproduction and disease resistance for some species–pathogen interactions (adamo and lovett, 2004). interestingly, these results also explain the choice of ecological niches by g. texensis that prefers temperatures lower than those corresponding to the optimal reproductive output, but that assure the presence of an efficient immune response. the ecological immunity could suffer global warming bacteria commonly interact with insects in intimate associations known as symbioses, where symbionts increase host fitness (for a review see russell and moran, 2006). several evidences suggested that symbiotic bacteria present in the insect gut resulted to be involved not only in the degradation of specific substances in the food (brummel et al., 2004), but also in other complex interactions protecting the host from invasion by pathogenic microorganisms (a process known as “colonization resistance”) and modulating the insect immune system (dillon and charnley, 1996; ryu et al., 2008). microbiota seems therefore to act in insects (and actually not only in insects) as a sort of ecological immunity or extended immune system being able of affecting the efficiency of the host immune system and limiting the accumulation of pathobionts (ottaviani et al., 2012). according to this proposal, germ-free locusts died prematurely due to infection of pathogens, such as pseudomonas aeruginosa, penicillium spp. and bacillus subtilis (charnley et al., 1995; dillon and charnley, 1996, 2005), suggesting that they are more susceptible to infection than normal insects and that the gut microbiota exerted a protective function out-competing potentially harmful organisms (dillon et al., 2005). the involvement of microbiota in the host immune protection can vary during insect life, since the composition of the bacterial community that populates the insect gut is not stable, but can change during lifespan due to variation in the nutritional composition of the food and the aging process (deveale et al., 2004). in a recent paper, chiu et al. (2012) reported a low survival of nymphs of the aphid m. varians at high temperatures as a consequence of the elimination of endosymbionts, such as buchnera (as previously suggested by ohtaka and ishikawa, 1991). this effect probably results from a temperature-mediated decrease in aphid endosymbionts, which synthesize amino acids essential for their insect hosts (chen et al., 2009). in the last years, different roles have been suggested for symbionts other than the synthesis of amino acids only (russell and moran, 2006). buchnera might, for instance, play a key role in aphid thermal tolerance since endosymbionts code for heat shock proteins, which deter degradation of host protein secondary structure (dunbar et al., 2007). secondary endosymbionts, such as serratia simbiotica, play a similar role in the thermal tolerance of their host strengthening the ability of aphids to evolve further adaptations to overcome the impacts of warming (russell and moran, 2006). buchnera are at least partly able to survive at high temperatures because of constitutive expression of genes that are normally up-regulated in response to heat and aphids could be able to thrive under temperatures as high as 35°c in the laboratory (dunbar et al., 2007). surprisingly, a single nucleotide deletion in the buchnera ibpa gene encoding for a small heat-shock protein virtually eliminates the transcriptional response of ibpa to heat stress and lowers its expression even at cool or moderate temperatures (dunbar et al., 2007). in the presence of this mutant allele, a short heat exposure in juveniles has strong effects on aphids that produce few or no progeny and contain almost no buchnera, in contrast to aphids bearing symbionts without the deletion. the ibpa mutated allele has appreciable frequencies in field populations supporting the view that lowering of ibpa expression improves host fitness under some conditions (dunbar et al., 2007).     61 as previously suggested, the response to stress (including thermal stress) is part of a large trade-off that relates stress response to reproduction and immunity. this mutation by switching off the response to heat stimuli could favor aphid reproduction and immunity. however, the prolonged permanence of aphids at high temperatures (for instance in hot summer with daily mean temperature of 32.5 °c) results in the elimination of buchnera reducing not only the thermal tolerance of aphids, but also their fecundity since the lack of endosymbionts results in a lost synthesis of amino acids essential for the hosts (chiu et al., 2012). according to these results, global warming could be difficultly faced by aphids in tropical regions due to buchnera symbiont depletion. interestingly, in the presence of low density of primary symbionts, secondary symbionts (such as hamiltonella defensa, s. symbiotica, regiella insecticola) could be more present affecting not only the aphid thermal tolerance to high temperatures, but also their symbiont-based immune response (poirié and coustau, 2011). the effects of global warming on the composition of aphid microbiota are of particular interest since, as recently reviewed by poirié and coustau (2011), the immune deficiency (imd) signalling pathway was apparently non functional in aphids and no genes coding for peptidoglycan recognition proteins (pgrps) and several wellconserved antimicrobial peptides, such as defensins and cecropins, have been predicted in the pea aphid acyrthosiphon pisum genome (gerardo et al., 2010), making the microbiota-based immunity essential to protect the host against natural enemies (poirié and coustau, 2011). global warming: threat or opportunity? global warming is a well-studied phenomenon referring to the current temperature of earth’s atmosphere and oceans and it is responsible for climate-driven habitat changes that could influence insect survival and distribution. according to some proposals, the predicted increase in the earth temperature could benefit many insect species since they will face less severe winter months resulting in higher over-wintering survival and increase in the population sizes. moreover, climate warming could lengthen the growing season resulting in increased growth, reproduction rate and number of generations per year. even if these hypotheses are intriguing, the scenario could be more complex in view of the existence of different trade-offs that balance the cost of reproduction and stress and immune responses. as suggested by some pioneering studies, global warming could have detrimental effects not only on insect immune system, but also on the composition of their microbiota making insects more vulnerable to pathogens. the occurrence of increasing temperature could therefore exacerbate the tradeoffs between reproduction and immune function, making few resources available for disease resistance. the study of trade-offs related to global warming has an important value not only from a biological point of view, but also at an ecological level. indeed, in order to balance the use of resource, several insect species that at present live in tropical regions will shift to temperate areas of our planet in order to combine a successful reproductive output to the an efficient immune response suggesting a huge impact of global warming on biodiversity. references adamo sa, lovett mme. some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket gryllus texensis. j. exp. biol. 214: 19972003, 2004. bale js, masters gj, hodkinson id, awmack c, bezemer tm, brown vk, et al. herbivory in global climate change research: direct effect of rising temperature on insect herbivores. global change biol. 8: 1-16, 2002. ballarin l, cammarata m, cima f, grimaldi a, lorenzon s, malagoli d, et al. immuneneuroendocrine biology of invertebrates: a collection of methods. inv. surv. j. 5: 192-215, 2008. berg mp, kiers et, driessen g, van der heijden m, kooi bw et al. adapt or disperse: understanding species persistence in a changing world. global change biol. 16: 587598, 2010. bonneaud c, mazuc j, gonzalez g, haussy c, chastel o, faivre b, et al. assessing the cost of mounting an immune response. am. nat. 161: 367-379, 2003. botkin db, saxe h, araújo mb, betts r, bradshaw rhw, et al. forecasting the effects of global warming on biodiversity. bioscience 57: 227236, 2007. brummel t, ching a, seroude l, simon af, benzer s. drosophila lifespan enhancement by exogenous bacteria. proc. natl. acad. sci. usa 101:12974-12979, 2004. bulet p, stöcklin, r. insect antimicrobial peptides: structures, properties and gene regulation. protein pept. lett. 12: 3-11, 2005. charnley ak, hunt j, dillon ar. the germ-free culture of desert locusts, schistocerca gregaria. j. insect physiol. 31:477-485, 1995. chen b, zhong d, monteiro a. comparative genomics and evolution of the hsp90 family of genes across all kingdoms of organisms. bmc genomics 7: 156, 2009. chiu mc, chen yh, kuo mh. the effect of experimental warming on a low-latitude aphid, myzus varians. entomol. exp. appl. 142: 216222, 2012. de block m, slos s, johansson f, stoks r. integrating life history and physiology to understand latitudinal size variation in a damselfly. ecography 31: 115-123, 2008. deveale b, brummel t, seroude l. immunity and aging: the enemy within? aging cell 3: 195208, 2004.     62 diffenbaugh ns, pal js, trapp rj, giorgi f. finescale processes regulate the response of extreme events to global climate change. proc. nat. acad. sci. usa 102: 15774-15778, 2005. diffenbaugh ns, pal js, giorgi f, gao x. heat stress intensification in the mediterranean climate change hotspot. geophys. res. lett. 34: l11706, 2007. dillon r, charnley ak. colonization of the guts of germ-free desert locusts, schistocerca gregaria, by the bacterium pantoea agglomerans. j. invertebr. pathol. 67: 11-14, 1996. dillon r, charnley k. mutualism between the desert locust schistocerca gregaria and its gut microbiota. res. microbiol. 153: 503-509, 2005. dunbar he, wilson acc, ferguson nr, moran na. aphid thermal tolerance is governed by a point mutation in bacterial symbionts. plos biol. 5: e96, 2007. frazier m, huey r, berrigan d. thermodynamics constrains the evolution of insect population growth rates: “warmer is better”. am. nat. 168: 512–520, 2006. gerardo nm, altincicek b, anselme c, atamian h, barribeau sm, de vos m, et al. immunity and other defenses in pea aphids, acyrthosiphon pisum. genome biol. 11: r21, 2010. guinnee ma, moore j. the effect of parasitism on host fecundity is dependent on temperature in a cockroach-acanthocephalan system. j. parasitol. 90: 673-677, 2004. hazell sp, groutides c, neve bp, blackburn tm, bale js. a comparison of low temperature tolerance traits between closely related aphids from the tropics, temperate zone, and arctic. j. insect physiol. 56: 115-122, 2010. houghton jt, ding y, griggs dj, noguer m, van der linden pj, xiaosu d, et al. climate change 2001: the scientific basis, cambridge university press, cambridge, 2001. karl l, stokcs i, de block m, janowitz sa, fischer k. temperature extremes and butterfly fitness: conflicting evidence from life history and immune function. global change biol. 17: 676687, 2011. lafferty kd. the ecology of climate change and infectious diseases. ecology 90: 888-900, 2009. le moullac g, haffner p. environmental factors affecting immune responses in crustacea. aquaculture 191: 121-131, 2000. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. malagoli d, sacchi s, ottaviani e. lectins and cytokines in celomatic invertebrates: two tales with the same end. inv. surv. j. 7: 1-10, 2010. mandrioli m, bugli s, saltini s, genedani s, ottaviani e. molecular characterization of a defensin in the izd-mb-0503 cell line derived from immunocytes of the insect mamestra brassicae (lepidoptera). biol. cell 95: 53-57, 2003. menéndez r. how are insects responding to global warming? tijdsch. entomol. 150: 355-365, 2007. mydlarz ld, jones le, harvell cd. innate immunity, environmental drivers and disease ecology of marine and freshwater invertebrates. annu. rev. ecol. evol. syst. 37: 251-288, 2006. nappi aj, kohler l, mastore m. signalling pathways implicated in the cellular innate immune responses of drosophila. inv. surv. j. 1: 5-33, 2004. neve bp, hazell sp, groutides c, douglas ae, blackburn tm, bale js. hyperthermic aphids: insights into behaviour and mortality. j. insect physiol. 56, 123-131, 2009. ohtaka c, ishikawa h. effects of heat treatment on the symbiotic system of an aphid mycetocyte. symbiosis 11: 19-30, 1991. ottaviani e, ventura m, mandrioli m, candela m, franchini a, franceschi c. gut microbiota as a candidate for lifespan extension: an ecological/evolutionary perspective targeted on living organisms as metaorganisms. biogerontology 12: 599-609, 2012. overgaard j, sørensen jg. rapid thermal adaptation during field temperature variations in drosophila melanogaster. cryobiology 56: 159-162, 2008. poirié m, coustau c. the evolutionary ecology of aphids' immunity. inv. surv. j. 8: 247-255, 2011. rolff j, van de meutter f, stoks r. time constraints decouple age and size at maturity and physiological traits. am. nat. 64: 559-565, 2004. russell ja, moran na. costs and benefits of symbiont infection in aphids: variation among symbionts and across temperatures. proc. r. soc. b 273: 603-610, 2006. ryu jh, kim sh, lee hy, bai jy, nam yd, bae jw, et al. innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in drosophila. science 319: 777782, 2008. sala oe, chapin fs 3rd, armesto jj, berlow e, bloomfield j, dirzo r, et al. biodiversity: global biodiversity scenarios for the year 2100. science 287: 1770-1774, 2000. savage vm, gillooly jf, brown jh, west gb, charnov el. effects of body size and temperature on population growth. am. nat. 163: 429-441, 2004. schmid-hempel p. variation in immune defence as a question of evolutionary ecology. proc. royal soc. b 270: 357-366, 2003. schmid-hempel p. evolutionary ecology of insect immune defenses. annu. rev. entomol. 50: 529-551, 2005. sternberg em. neural regulation of innate immunity: a coordinated nonspecific response to pathogens. nature rev. immunol. 6: 318-328, 2006. suwanchaichinda c, paskewitz sm. effects of larval nutrition, adult body size, and adult temperature on the ability anopheles gambiae (diptera:     63 culicidae) to melanize sephadex beads. j. med. entomol. 35: 157-161, 1998. thomas cd, cameron a, green re. extinction risk from climate change. nature 427: 145-148, 2004. travers ma, basuyaux o, le goic n. influence of temperature and spawning effort on haliotis tuberculata mortalities caused by vibrio harveyi: an example of emerging vibriosis linked to global warming. global change biol. 15: 1365-1376, 2009. global warming: threat or opportunity? s isj 8: 179-189, 2011 issn 1824-307x review how gene expression profiles disclose vital processes and immune responses in mytilus spp. s domeneghetti1*, c manfrin2*, l varotto1, u rosani1, m gerdol2, g de moro2, a pallavicini2, p venier1 1department of biology, university of padua, padua, italy 2department of life sciences, university of trieste, trieste, italy *equal contribution accepted september 08, 2011 abstract gene expression studies largely support the understanding of gene-environment interactions in humans and other living organisms but the lack of genomic and genetic information often complicates the analysis of functional responses in non-traditional model species. nevertheless, the fast advancement of dna microarray and sequencing technologies now makes global gene expression analysis possible in virtually any species of interest. as regards the mytilus genus, tens of thousands expressed sequence tags (ests) are currently available for m. californianus and m. galloprovincialis, and dna microarrays have been developed. among them, immunochip 1.0 specifically includes 1,820 probes of genes centrally involved or modulated in the innate immune responses of the mediterranean mussel. this review recalls peculiarities and applications of the existing mussel dna microarrays and finally summarizes facts concerning a variety of transcript sequences likely involved in the mussel immunity. beside dna microarrays, next generation sequencing (ngs) technologies now offer new and broader research perspectives, from the whole transcriptome coverage to the mytilus genome sequencing. key words: mytilus; dna microarray; innate immunity; ests; antimicrobial peptides; c1q introduction global gene expression analyses in organisms selected to represent a given ecosystem currently support ecotoxicological investigations and create a conceptual bridge between the early organism responses and late population changes (steinberg et al., 2008). the animal response to a variety of detrimental conditions usually starts with alarm signals followed by adjustment reactions aimed to neutralize the physiological unbalance, and may end up in a general decline of vital processes ultimately marked by disease and death. depending on the stress type and exposure intensity, the expression of definite sets of genes makes available specific proteins and other molecules in cells and tissues. appeared in the 1990s, the dna microarray technology enables the simultaneous expression ___________________________________________________________________________ corresponding author: paola venier department of biology university of padua via u. bassi, 58/b, 35121 padua, italy e-mail: paola.venier@unipd.it measure of thousands of genes represented in the microarray platform by unambiguous polynucleotide probes (schena et al., 1995; lockhart et al., 1996). the gene expression profiles emerging from suitable sampled cells or tissues can provide a dynamic view of biological processes and allow the correct sorting of different functional states. based on the availability of sequence data, dna microarrays can be used to solve a variety of biological questions: from the identification of molecular markers pathognomonic of disease and transcriptional signatures of various stress factors to the understanding of complex phenomena such as the epigenome in normality and disease (martínsubero and esteller, 2011). specific microarray platforms and advanced deep sequencing technologies now support studies on the cellular functions of micrornas and their role in human diseases (thomas et al., 2010). leading research institutions are currently using both the mrna and mirna expression profiling to examine the genomic responses to environmental stresses (nct). central to the toxicogenomics studies is the concept of ‘phenotypic anchoring’ which recalls the 179 fig. 1 number of pubmed publications including the terms "dna-microarray" (blue line) or "mytilus" (purple line) from 1995 to 2010. has the dna microarray revolution reached its peak? importance to correlate the observed gene expression changes to adverse effects defined by conventional parameters of toxicity and pathology. in the controlled vocabulary of the natl. library of medicine, the term ‘dna microarray’ is indexed under the following category which indicates the large application range of such innovative technology (mesh): oligonucleotide array sequence analysisthe hybridization of a nucleic acid sample to a very large set of oligonucleotide probes, which are attached to a solid support, to determine sequence or to detect variations in a gene sequence or expression or for gene mapping. relevant to the gene expression profiling research area is gene expression omnibus, a public repository that archives and freely distributes microarray, next-generation sequencing, and other forms of high-throughput functional genomics data submitted by the scientific community (geo). to fulfil the current standards (minimum information about a microarray experiment) the contents submitted to geo should include the following: raw hybridization data; normalized data from which the main experimental findings can be outlined; description of the tested samples and whole experimental design, with details on the biological and technical replicates; identity and location of all probes and controls of the microarray platform, with external reference in the case of commercial arrays; concise but precise description of laboratory and data processing protocols related to the experiment under submission. according to the aims of the microarray gene expression data society, dating back to the late ‘90s, the compliance to the miame standards should assure the data comparability among different platforms and testing protocols while supporting common work criteria and the reduction of random data variation (rogers and cambrosio, 2007). based on the comparative data analysis, the guidelines for standardization and reporting have been further refined (chen et al., 2007; shi et al., 2008). at present, geo contains as much as 9,000 platform records which can be accessed and browsed in full detail. figure 1 illustrates the annual increase of pubmed records including the term "dnamicroarray" or "mytilus" (subject heading or title/abstract) and suggests that pioneering technologies open the way to new ideas more than an unconventional model organisms. in fact, the gene expression profiling field has substantially diversified: specialized equipments and various related software make today the dna microarrays powerful tools for the study of gene sequence, structure and expression, particularly for the best known model organisms. nonetheless, one must remember that transcription is just one step in gene expression, and post transcriptional events referred to maturation of the primary transcript, rna editing and rna silencing as well as various modifications of the translation products overall influence the final amounts and activity of cellular proteins. mytilus dna microarrays: preparation strategy and applications six geo records refer to mussel dna microarrays at july 2011. mytarray 1.0 (geo platform gpl1799, oct 2006) is composed by 1,712 cdna probes, univocally tagging the 3’-end region of transcripts from the main tissues of adult mussels (mytilus galloprovincialis) and 46 unrelated cdna control probes, all printed in duplicate and twice per slide (1.7 k mussel probes per array, 7.0 k total probes per slide). the probes were designed in the 3’-utr, 180 fig. 2 work diagram referred to the competitive hybridization of two dye-swap-labelled samples on a cdna microarray with two-channel detection of the fluorescence signals (modified from gibson and muse, 2004). one among the least conserved gene regions, so that competition of different mrnas from genes with similar coding sequence and cross-hybridization to the same microarray probe should be minimal. also, the probe size of 400 800 bp is expected to ensure comparable efficiency in the amplification and spotting of the cdna inserts as well as uniform hybridization kinetics (venier et al., 2006). mytarray 1.0 was first used to investigate the specificity of gene transcription in mussel tissues with different functional role and the transcriptional profiles of mussels treated with chemical mixtures or living wild in different sites of the venice lagoon (venier et al., 2006; geo series gse2176, gse2183 and gse2184). sample pairs combined according to dye-swap labelling (reference and test samples labelled with cy3/cy5 cyanine dyes in alternate combinations) were competitively hybridized on the two equal arrays of cdnas spotted on the same slide (fig. 2). gills, digestive 181 gland, tissues involved in contraction/motility (foot, adductor muscles, ligaments) and reproduction (gonads and mantle) displayed specific transcriptional footprints, as expected. the results obtained in mussels treated with mixtures of inorganic metal salts or persistent organic chemicals guided the interpretation of the gene expression profiles of mussels living in the inner industrial canals or at the lagoon border open to the sea (this exercise yielded a provisional list of contamination marker probes). in this study, the evident transcriptional down-regulation detected in the reproductive tissues was consistent with the depleted status of the mussel gonads whereas the greatest variety and abundance of transcripts was found in the digestive gland. additional analysis of these expression data is reported elsewhere (pantzartzi et al., 2010). the same platform was then used to evaluate in a time-course study the gene expression changes in the digestive gland of mussels exposed to okadaic acid (oa) via food contamination for five weeks (manfrin et al., 2010; geo series gse14885). one relevant purpose of the study was the identification of molecular biomarkers which could enable an easy and rapid detection of the diarrhoeic shellfish poisoning biotoxins in marketable mussel stocks, i.e., novel reliable assays complementing the existing diagnostic methods. an unsaturated loop design, combining control and treated samples with different dye-labelling for the competitive hybridization on mytarray 1.0, was adopted to take into account all the time points and the biological replicates, with some combinations only inferred (kerr and churchill, 2001). a considerable number of transcriptional changes was detected in the oa-exposed mussels, with a prevalence of up-regulated probes at 3 days and a subsequent progressive increase of down-regulated probes (from 58 % over-expressed to 76 % underexpressed genes, respectively detected at day 3 and day 35). the biphasic time-related trend of response observed in this study recalls the changes occurring in the mussel digestive gland along different phases of the mussel reaction to the experimental stimulus, from the early acute response to the late overall unbalance of the functional processes. many candidate markers are now under study to evaluate their predictive value in the diagnosis of biotoxin-contaminated mussels. mytarray 1.1 (gpl102699, march 2010) contains the same cdna probes of mytarray 1.0 in a slightly modified platform geometry. it has been used to study the gene expression profiles of m. galloprovincialis with monthly samplings for one year, hence taking into account seasonal differences which are known to influence metabolism rates and gonad development among other vital functions (banni et al., 2011; geo series gse22915, gse23049gse23051). mussels were collected from an anthropized and industrialized lagoon of the southern mediterranean sea (ben said et al., 2009) and competitive hybridizations were performed with dye-swap-labelled samples (dual colour analysis). following a loop design with 3-4 biological replicates and parallel histological evaluation of the gonad status, the authors could analyze the transcriptional profiles of digestive gland tissue of female mussels collected during 12 months, and those of digestive gland and mantle tissues from male and female individuals representing all four gonad maturation stages. in the examined annual period, the transcriptional profiles globally highlighted the higher expression of genes associated to mussel nutrition and digestion in mayaugust compared to the other months, and trends for gonad transcripts consistent with the reproductive mussel status. the same cdna platform contributed to the toxicological evaluation of a neonicotinoid insecticide mixture (dondero et al., 2010), an organophosphate compound (canesi et al., 2011) and to the integrated measure of the functional mussel responses in the estuarine tamar region in uk (shaw et al., 2011). the hofmann_ucsb_mytilus_2.5k_v1.0 record (gpl5795, mar 2008) describes a platform of nearly 2500 spotted cdnas of mytilus californianus consisting of both unsequenced and sequenced clones referring to gill and muscle of environmentally challenged mussels. the related geo series gse8935 include data on latitudinal gene expression changes. five biological replicates from four populations of californian mussels were compared to a common reference sample in dual colour analysis (dye-swap labelling). the hms/somerolab-mytilus-105k array-v1.0 (gpl9676, jun 2010) and hms/somero-mytilus105k agilent-v1.0 salinity stress (gpl11156, jan 2011) are two successive versions of a platform composed by oligomer probes in-situ synthesized by agilent technologies (santa clara, ca, usa). these microarrays include probes of both m. californianus and m. galloprovincialis, and are intended for homologous and heterologous gene expression profiling. the processing and assembling of about 26,000 ests from m. californianus (gracey et al., 2008) and 3,984 ests from m. galloprovincialis (venier et al., 2003) resulted in a total of 12,961 and 1,688 transcript clusters or singletons, respectively. long (60-mer) oligoprobes were designed against the m. californianus series and the resulting 43,969 total unique probes (2.6 probes per transcript sequence) were analyzed through blast searches against the m. galloprovincialis series to support selection and design of related probes (556 probe pairs matching transcripts of both species, with a mean number of 4.6 divergent nucleotide bases per probe). a total of 44,524 unique probes were duplicated or triplicated randomly to fill a microarray of 105,000 elements (105 k probes). these two platforms have been used to investigate the transcriptional responses to thermal and osmotic stresses in m. californianus, m. trossulus and m. galloprovincialis (evans and somero, 2010; lockwood et al., 2010; lockwood and somero, 2011). to control the effects of sequence mismatches in the case of m. galloprovincialis probes included in the gpl9676 platform, only probes experimentally confirmed in the hybridization of 84 samples of both m. galloprovincialis and m. trossulus were used in the related data analysis. following a large set of 182 hybridization experiments and stringent quality control, misleading probes were removed from the dataset and the second platform version (gpl11156/agilent 019153) was generated. in the central and southern coasts of california, m. galloprovincialis has largely displaced the native congener, m. trossulus, and such evidence could be explained by species differences in physiological traits related to the adaptation to warm habitats. to investigate the hypothesis, gene expression profiling was performed on gill rna from mussels subjected to acute heat-stress (geo series gse19031). a total of 1,531 probes, out of 4,488 different genes represented on the microarray and recognizing mrnas of both species, showed temperaturedependent expression changes highly similar in the two congeners whereas 96 probes denoting oxidative stress, proteolysis, energy metabolism, ion transport, cell signalling, and cytoskeleton reorganization outlined species-specific responses to the heat-stress. among them, the one encoding the small heat shock protein 24 was highly induced in the mediterranean mussel and showed only a small change in m. trossulus. six biological replicates per mussel group were included in this study which exemplifies the use of a cross-species microarray as well as heterologous and homologous hybridization. according to the authors and published literature, m. trossulus and m. galloprovincialis are approximately 7.6 million years divergent from m. californianus, and only 3.5 million years divergent from each other: in other words, the heterologous hybridization of target sequences from m. trossulus should occur on microarray probes from m. galloprovincialis without inherent sequence bias and should provide a reliable comparison of their transcriptional responses. though debated, prudent evaluations of the sequence divergence by in silico approaches and phylogenetic data could expand the use of cross-species hybridization as a compromise solution for investigating gene expression in species with unsequenced genomes (costa et al., 2010; nazar et al., 2010; ptitsyn et al., 2010). gene expression profiling was also performed on gill rna from mussels subjected to salinity stress (geo series gse25111). a total of 117 probes, out of 6,777 genes represented on the microarray, showed significant changes similar between m. californianus and m. galloprovincialis whereas 12 probes, denoting mrna splicing, polyamine synthesis, exocytosis, translation, cell adhesion, and cell signaling, outlined species-specific responses. the study was based on alexafluor-labelling (555 and 647 fluorescence dyes) of amplified rna, pooled reference samples, six biological replicates, and competitive hybridization in agreement to the recommended agilent protocols. in addition to the overall stringent processing of the fluorescence signals, the heterologous hybridization design suggested the elimination of data from probes with low signal intensity (signal intensity < 150 % of the local background and hybridized spot diameter < 30 % of the nominal spot diameter). the work performed at the a. gracey’s and g.n. somero’s laboratories (university of southern california -los angeles, ca, u.s.a. and stanford university -palo alto, ca, u.s.a., respectively) on mytilus (geo series gse19031 and gse25111) and other species is facing the fundamental aspects of the organism adaptation to fluctuating environments and global climate changes, and gene expression profiling has been essential to their findings. for instance, the study of gene-expression changes in the californian mussels at different phases in the tidal cycle revealed at least four distinct physiological states, corresponding to metabolism and respiration phase, cell-division phase, and two stress-response signatures linked to moderate and severe heat-stress events. the metabolism and cell-division phases appeared to be functionally linked and anti-correlated in time whereas magnitude and timing of the above states resulted to be influenced by the microhabitat conditions according to the vertical position on the shore (gracey et al., 2008). based on comparative physiology, a recent paper offers an overview on the expected consequences of global climate changes (somero, 2011). finally, the mussel immunochip 1.0 (gpl10758, april 2011) is a spotted oligonucleotide platform consisting of four-replicated 1820 oligomer probes plus unrelated controls prepared at cribi for the purposes of a recent european project (imaquanim). oligomers of 57 bases average length were designed at short distance from the 3’ end of transcript sequences selected previously in mytibase, the interactive knowledgebase of m. galloprovincialis which includes most of the ests publicly available for this species (venier et al., 2009). based on multiple criteria, the subset of transcripts selected from mytibase as putatively immune-related molecules should denote central “players” of the mussel innate immunity or genes whose expression is modulated during the mussel responses to immunostimulation (venier et al., 2011). in the platform description, the probe id is hyperlinked to the relative mytibase record: for instance the probe mgo_07346 relates to mgc07346, a mussel transcript featured by the protein domain ipr000098-interleukin 10 and yet functionally unknown. the performance of immunochip 1.0 was tested with hemolymph samples collected at 3 and 48 h from vibriochallenged mussels (geo series gse23535) according to competitive hybridization of dye-swap labelled amplified rna samples. in agreement with the above descriptions, figure 3 provides an updated summary of the nucleotide and protein sequences publicly available at july 2011 and highlights the importance of est sequencing for the preparation of new dna microarrays. more about the molecular “players” of the innate immunity and the immune responses of m. galloprovincialis is reported in the following paragraph. how much can simple sequences tell us about the mussel immune responses? taking advantage of the continuous increase of the nucleotide and amino acid sequences in the public databases, the current methods of bioinformatics can extract instructive data from 183 fig. 3 number of sequence records available for selected mollusc species at the natl center for biotechnology information at july 2011. dna, rna and protein sequences refer to biomphalaria glabrata (gastropoda) and bivalves belonging to the veneroida, unionoida, mytiloida, ostreoida, pectinoida and pterioida orders. simple sequences: from the analysis of various gene/transcript regions to the evaluation of protein/peptide structure and to the comparative analysis of evolutionary differences across the tree of life. this procedural approach complements and integrates the data derived from long-standing disciplines such as measures of structural changes and protein amounts/activity, among others. the overall analysis of 18,788 high-quality ests rationally organized in 7,112 independent clusters or singletons (mytibase transcript collection) highlighted some particularly abundant transcript groups: namely, transcripts featured by a complement component c1q-like domain, antimicrobial peptide (amp) precursors of all four families known in the mediterranean mussel and many heterogeneous lectins including fibrinogenrelated molecules (venier et al., 2011). to explain the abundance of immune-related molecules in mytibase it is important to remember that such collection has been prepared by 16 primary (5 from hemocytes) and 1 normalized cdna libraries from mussels subjected to various challenges, for instance mussels immune stimulated with preparations of gram positive and gram negative cells and viral-like molecules. searches by protein domain revealed a total of 168 different mytibase transcripts containing the c1q signature ipr001073, almost invariably associated with the overlapping tnf-like ipr008983 motif. curiously, the c1q domaincontaining proteins predicted from the transcript sequences, display a short n-terminal signal peptide and a c-terminal globular domain but no central collagen-like repeats which are instead typical of vertebrate c1q domain-containing proteins. according to the current literature, these mussel proteins could represent secreted globular receptors, components of ancient complement pathways expected to mediate pathogen recognition and lysis (dodds and matsushita, 2007). the modularity and versatility of binding mediated by the globular c1q domain explain the variety of roles currently attributed to this still expanding family of proteins, and also supports their involvement in pathogen pattern recognition (carland and gerwick, 2010). the abundance and variety of mussel c1q domain-containing transcripts are consistent with this view. one among these transcripts, named mgc1q, resulted to be expressed at detectable levels in the main tissues of naïve adult mussels, with the hemocytes showing the highest expression levels, and from 2 h post-fertilization up to 3 months later. the mgc1q expression was significantly modulated after mussel infection with gram positive or gram 184 negative bacteria, data which confirm mgc1q as an immune-related gene. the striking molecular diversity of mgc1q was confirmed at both the dna and cdna levels, hence posing mechanistic questions on the origin of such variation (gestal et al., 2010). experimental findings and sequence analyses support the hypothesis of gene duplication, functional diversification and positive selection of many c1qdc variants in selected taxa, including the mussel lineage (gerdol et al., 2011). defensins, mytilins, myticins and mytimycins are cationic antimicrobial peptides stabilized by 4 intrachain disulphide bonds (6 in mytimycin) in a typical 3-d motif (yeaman and yount, 2007). a remarkable diversity of a new group of myticins, with specific variant profiles detectable in single mussels, was reported in m. galloprovincialis (pallavicini et al., 2008; costa et al., 2009). following the discovery of the myticin-c variants, their molecular diversity and evolution has been further discussed (padhi and verghese, 2008) and the most recent findings indicate myticin c as a chemotactic molecule with antiviral activity and immunoregulatory properties (balseiro et al., 2011). just one singleton and other four similar sequences denote the antifungal amp mytimycin in mytibase (rare transcript). mytimycin is composed by 54 aminoacids (6.2 6.3 kda, 12 cysteines) and two main precursor variants, both featured by a signal peptide and a c-terminal extension, are expressed in mussels from different european regions (sonthi et al., 2011). the presence of a calcium binding (ef hand) motif in the c-terminal extension suggests further characterization of such unusual amp. the "effector" role of the mussel antimicrobial peptides (amps) is confirmed in many experimental studies and a comprehensive review have been recently provided (li et al., 2011). whether these effectors can modulate the mussel immune responses with mechanisms other than membrane disruption, as reported for mammalian amps, it is not clear. based on deep amplicon sequencing, the sequence diversity of mussel amps is now under study in natural mussel populations from different geographical regions and in mussels challenged with bacterial cells. lectins are a rather heterogeneous protein family comprising 8 to 15 subgroups, depending on the scientist’s view (dodd and drickamer, 2001). lectins typically possess carbohydrate binding domains and participate in many cell processes. similarly to the mammalian c1q, the c-terminal fibrinogen-like domain ipr002181 of ficolins forms a tulip-like structure able to bind the carbohydrate residues of foreign and apoptotic cells (with consequent opsonization, phagocytosis and cell clearance) or triggering the proteolytic complement cascade and pathogen lysis. fibrinogen-related lectin proteins (freps) are expressed also in mussels (venier et al., 2011) and are codified by at least 2 (m. edulis) 4 (m. californianus) and 7 genes (m. galloprovincialis) (gorbushin and iakovleva, 2011). these molecules can be regarded as immune pattern-recognition receptors and their involvement in the native immunity is supported by the evidence of species-specific expansion of freps in the snail biomphalaria glabrata and the mosquito anopheles gambiae (waterhouse et al., 2007; zhang et al., 2008). in mussel, freps are significantly up-regulated after bacterial infection or pamp treatment, and display opsonizing activity similar to that of mammalian ficolins; moreover, the different sets of frep sequences detected among and within individuals further emphasize the great complexity of the invertebrate immune systems (romero et al., 2011). other lectin-like sequences expressed in mussels are commented in venier et al. (2011). the cases reported above are a few examples of the many classes of transcripts specifically expressed or modulated during the mussel response to potential pathogens. considering in a dynamic view the behaviour of one cell population only, the versatile mussel hemocytes, one can imagine that almost all cellular processes could be influenced by the contact with pathogen-associated molecular patterns: from the cytoskeleton remodelling supportive of chemotaxis, migration and phagocytosis to the intracellular signalling possibly shaping the inflammatory response and finely tuned expression of many regulatory and effector genes. cross-talking signalling pathways have been traced in mussel and the mytibase collection includes transcripts denoting the regulatory cytokine mif (migration inhibiting factor) and cytokine-related molecules, consistent with the idea of an invertebrate cytokine network (malagoli, 2010). the recent definition of a species-specific immunochip aims to the experimental validation of a selected subset of transcripts: a synopsis of the main gene expression changes detected in mussels at 3 and 48 h after challenge with live bacterial cells is reported in fig. 4. the general amp downregulation observed in this particular laboratory treatment was confirmed by quantitative pcr data and is discussed also in li et al. (2010). concluding remarks est sequencing and dna microarrays have substantially improved the identification of genes expressed in the mytilus species. compared to the first est collection and the related cdna microarray, mytibase includes an interesting variety of immune-related molecules which can be further characterized with traditional and innovative approaches as exemplified by romero et al. (2011). nonetheless, in the mytibase collection about half of the mussel transcripts are still unknown, devoid of functional annotation. hence, much work remains to be done both in silico and in laboratory to provide a comprehensive view of the global gene transcription in mussels, particularly the part of the transcriptome mediating the response to potential invaders (immunome). undoubtedly, the application of the available mussel dna microarray platforms can further reveal expression trends of different gene categories and identify useful markers of functional state, if not global molecular signatures useful to disentangle the complex mussel physiology. depending on the study design and on the type of microarray platform, independent validation of the expression data can be accomplished by quantitative pcr or with other 185 fig. 4 main transcriptional changes detected in mussels at 3 h and 48 h from the injection of live vibrio cells (modified from venier et al., 2011). only relevant molecular "players" represented in the immunochip of m. galloprovincialis are reported (framed). in each frame, the detected expression trends are indicated in red, green and yellow (upand down-regulation and not homogeneous trends, respectively). annotations based only on protein domains are reported in brackets. overall, the figure draws the attention to a number of mussel genes, still not characterized, whose expression is modulated in response to immune stimulation. abbreviation list (fig. 4): aif: allograft inflammatory factor apaf1: apoptotic peptidase activating factor 1 bcl2: baculoviral apoptosis regulator 2 c1-c5: complement component 1-5 calr: calreticulin casp: caspase cd63/limp: tetraspanin-7 (lysosome membrane protein) clr: c-type lectin receptor clr: c-type lectin receptor damps: damage-associated molecular patterns fadd: fas (tnfrsf)-associated via death domain fnbp1: formin-binding protein 1 grp 78/94: glucose-regulated protein 78/94 hsc70: heat shock cognate 70 hsp70/90: heast shock protein 70/90 iap: inhibitor of apoptosis proteins; ikbα: inhibitor of nuclear factor kappa-b kinase alpha ikk: inhibitor of nuclear factor kappa-b kinase complex il: interleukin irak4: interleukin receptor-associated kinase 4 jak: janus kinase klhl: kelch-like protein ldlr: low-density lipoprotein receptor litaf: lps-induced tnfalpha factor lps: lipopolysaccharide mapks: mitogen-activated protein kinases mbl: mannose binding lectin mgd1/2: mytilus galloprovincialis defensin 1 /2 mif: migration inhibitory factor mnk: map kinase-interacting serine/threonine-protein kinase mr1: mannose receptor 1 myd88: myeloid differentiation primary response gene 88 nalps: natch, lrr, and pyr containing proteins nfkb: nuclear factor of kappa light polypeptide gene enhancer in b-cells nlr: nod-like receptor nod: nucleotide binding oligomerization domain p13k: phosphatidylinositol-4,5-bisphosphate 3-kinase pac2: proteasome assembly chaperone 2 pamps: pathogen associated molecular patterns pgrp: peptidoglycan recognition protein pi31: proteasome inhibitor pi31 subunit pim: proto-oncogene serine/threonine-protein kinase pim prr: pathogen recognition receptors rab: ras-related gtp-binding protein rip: receptor-interacting serine-threonine kinase ros: reactive oxygen species sec22: vesicle transport protein sec22 sod: superoxide dismutase stat: signal transducer and activator of transcription protein srcr: scavenger receptor cysteine-rich protein precursor tak: mitogen activated protein kinase kinase timp3: tissue inhibitors of metalloproteinase 3 tnf: tumour necrosis factor tnfr: tumour necrosis factor receptor traf6: tnf receptor-associated factor 6 ub: ubiquitin ubr5: ubiquitin protein ligase e3 (component n-recognin 5) α2: proteasome subunit alpha type 2 β4, β5: proteasome subunit beta type 4/5 186 experimental measures. all the steps of the dna microarray testing could be used to strengthen the final data interpretation, from the microarray preparation strategy to the stringency of the hybridization reaction to the algorithms applied to data processing. the maintenance of the physical collection of the cdnas, i.e., recombinant bacterial clones, is a prerequisite for the use of spotted cdna microarrays (for instance, the current use of mytarray 1.0 slides, printed at the cribi facility depends on long work performed at the department of biology, university of padua). such work is not more affordable as long as the clustered ests increase in number, and external commercial services or deep sequencing become an attractive alternative. as a matter of fact, next-generation sequencing (ngs) technologies are now complementing and challenging the dna microarrays as alternative tools for genome analysis and transcriptome sequencing (hurd and nelson, 2009; morozova et al., 2009). for instance, the so called 454 pyrosequencing has been already applied to the study of tissue-specific expression patterns in m. galloprovincialis (craft et al., 2010) and many laboratories in the world are now investing in this kind of work. acknowledgments this work has been sequentially supported by food-ct-2005-007103 (imaquanim), fp7-kbbe2010-4-266157 (bivalife) and, partially, 20084bej9f (prin08). references balseiro p, falcó a, romero a, dios s, martínezlópez a, figueras a, et al. mytilus galloprovincialis myticin c: a chemotactic molecule with antiviral activity and immunoregulatory properties. plos one. 2011;6(8):e23140. banni m, negri a, mignone f, boussetta h, viarengo a, dondero f. gene expression rhythms in the mussel mytilus galloprovincialis (lam.) across an annual cycle. plos one 6(5): e18904, 2011. ben said o, goñi-urriza m, el bour m, aissa p, duran r. bacterial community structure of sediments of the bizerte lagoon (tunisia), a southern mediterranean coastal anthropized lagoon. microb. ecol. 59: 445-456, 2010. carland tm, gerwick l. the c1q domain containing proteins: where do they come from and what do they do? dev. comp. immunol. 34: 785-790, 2010. chen jj, hsueh hm, delongchamp rr, lin cj, tsai ca. reproducibility of microarray data: a further analysis of microarray quality control (maqc) data. bmc bioinformatics 8: 412, 2007. costa f, alba r, schouten h, soglio v, gianfranceschi l, serra s, et al. use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening. bmc plant biol. 10: 229, 2010. costa mm, dios s, alonso-gutierrez j, romero a, novoa b, figueras a. evidence of high individual diversity on myticin c in mussel (mytilus galloprovincialis). dev. comp. immunol. 33: 162-170, 2009. craft ja, gilbert ja, temperton b, dempsey ke, ashelford k, tiwari b, et al. pyrosequencing of mytilus galloprovincialis cdnas: tissue-specific expression patterns. plos one 5(1): e8875, 2010. dodd rb, drickamer k. lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity. glycobiology. 11(5):71r-79r, 2001.dodds aw, matsushita m: the phylogeny of the complement system and the origins of the classical pathway. immunobiology 212: 233-243, 2007. evans tg, somero gn. phosphorylation events catalyzed by major cell signaling proteins differ in response to thermal and osmotic stress among native (mytilus californianus and mytilus trossulus) and invasive (mytilus galloprovincialis) species of mussels. physiol. biochem. zool. 83: 984-996, 2010. geo. the gene expression omnibus can be accessed at http://www.ncbi.nlm.nih.gov/geo. gerdol m, manfrin c, de moro g, figueras a, novoa b, venier p, et al. the c1q domain containing proteins of the mediterranean mussel mytilus galloprovincialis: a widespread and diverse family of immune-related molecules. dev. comp. immunol. 35: 635-643, 2011. gestal c, pallavicini a, venier p, novoa b, figueras a. mgc1q, a novel c1q-domain-containing protein involved in the immune response of mytilus galloprovincialis. dev. comp. immunol. 34: 926-34, 2010. gibson g, muse sv. a primer of genome science. sinauer associates inc., sunderland, ma usa, 2004. gibson g. the environmental contribution to gene expression profiles. nat. rev. genet. 9: 575581 2008. gorbushin am, iakovleva nv. a new gene family of single fibrinogen domain lectins in mytilus. fish shellfish immunol. 30: 434-438, 2011. gracey ay, chaney ml, boomhower jp, tyburczy wr, connor k, somero gn. rhythms of gene expression in a fluctuating intertidal environment. curr. biol. 18: 1501-1507, 2008. hook se. promise and progress in environmental genomics: a status report on the applications of gene expression-based microarray studies in ecologically relevant fish species. j. fish biol. 77: 1999-2022, 2010. hurd pj, nelson cj. advantages of next-generation sequencing versus the microarray in epigenetic research. brief. funct. genomic. proteomic. 8: 174-183, 2009. imaquanim. facts on this fp6 integrated project are at http://imaquanim.dfvf.dk/info. kerr mk, churchill ga. experimental design for gene expression microarrays. biostatistics 2: 183-201, 2001. larsen pf, schulte pm, nielsen ee. gene expression analysis for the identification of selection and local adaptation in fishes. j. fish biol. 78: 1-22, 2011. 187 http://www.ncbi.nlm.nih.gov/pubmed/21858010 http://www.ncbi.nlm.nih.gov/pubmed/21858010 http://www.ncbi.nlm.nih.gov/pubmed/21858010 http://www.ncbi.nlm.nih.gov/pubmed/21858010 http://www.ncbi.nlm.nih.gov/pubmed?term=%22dodd%20rb%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22drickamer%20k%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed/11425795 http://imaquanim.dfvf.dk/info lenoir t, giannella e. the emergence and diffusion of dna microarray technology. j. biomed. discov. collab. 1: 11, 2006. li h, parisi mg, parrinello n, cammarata m, roch p. molluscan antimicrobial peptides, a review from activity-based evidences to computerassisted sequences. inv. surv. j. 8: 85-97, 2011. li h, venier p, prado-alvárez m, gestal c, toubiana m, quartesan r, et al. expression of mytilus immune genes in response to experimental challenges varied according to the site of collection. fish shellfish immunol. 28: 640-648, 2010. lockhart dj, dong h, byrne mc, follettie mt, gallo mv, chee ms, et al. expression monitoring by hybridization to high-density oligonucleotide arrays. nat. biotechnology 14: 1675-1680, 1996. lockwood bl, sanders jg, somero gn. transcriptomic responses to heat stress in invasive and native blue mussels (genus mytilus): molecular correlates of invasive success. j. exp. biol. 213(pt 20): 3548-3558, 2010. lockwood bl, somero gn. transcriptomic responses to salinity stress in invasive and native blue mussels (genus mytilus). mol. ecol. 20: 517-29, 2011. malagoli d. cytokine network in invertebrates: the very next phase of comparative immunology. inv. sur. j. 7: 146-148, 2010. manfrin c, dreos r, battistella s, beran a, gerdol m, varotto l, et al. mediterranean mussel gene expression profile induced by okadaic acid exposure. environ. sci. technol. 44: 82768283, 2010. martín-subero ji, esteller m. profiling epigenetic alterations in disease. adv. exp. med. biol. 711: 162-177, 2011. mesh. current medical subject headings can be accessed at http://www.ncbi.nlm.nih.gov/mesh. morozova o, hirst m, marra ma. applications of new sequencing technologies for transcriptome analysis. annu. rev. genomics hum. genet. 10: 135-151, 2009. nazar rn, chen p, dean d, robb j. dna chip analysis in diverse organisms with unsequenced genomes. mol. biotechnol. 44: 813, 2010. nct. the national center for toxicogenomics is at the national institute of environmental health sciences (http://www.niehs.nih.gov/research/atniehs/nct.c fm). padhi a, verghese b. molecular diversity and evolution of myticin-c antimicrobial peptide variants in the mediterranean mussel, mytilus galloprovincialis. peptides 229: 1094-10101, 2008. pallavicini a, costa mm, gestal c, dreos r, figueras a, et al. high sequence variability of myticin transcripts in hemocytes of immunestimulated mussels suggests ancient hostpathogen interactions. dev. comp. immunol. 32: 213-226, 2008. pantzartzi c, drosopoulou e, yiangou m, drozdov i, tsoka s, ouzounis ca, et al. promoter complexity and tissue-specific expression of stress response components in mytilus galloprovincialis, a sessile marine invertebrate species. plos comput. biol. 6: e1000847, 2010. ptitsyn a, schlater a, kanatous s. transformation of metabolism with age and lifestyle in antarctic seals: a case study of systems biology approach to cross-species microarray experiment. bmc syst. biol. 4: 133, 2010. rogers s, cambrosio a. making a new technology work: the standardization and regulation of microarrays. yale j. biol. med. 80: 165-178, 2007. romero a, dios s, poisa-beiro l, costa mm, posada d, figueras a, et al. individual sequence variability and functional activities of fibrinogen-related proteins (freps) in the mediterranean mussel (mytilus galloprovincialis) suggest ancient and complex immune recognition models in invertebrates. dev. comp. immunol. 35: 334-344, 2011. romero a, estévez-calvar n, dios s, figueras a, novoa b. new insights into the apoptotic process in mollusks: characterization of caspase genes in mytilus galloprovincialis. plos one 6(2):e17003, 2011. schena m, shalon d, davis rw, brown po. quantitative monitoring of gene expression patterns with a complementary dna microarray. science 270: 467-470, 1995. shaw jp, dondero f, moore mn, negri a, dagnino a, readman jw, et al. integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the tamar estuary, uk mar. environ. res. [in press] 2011. shi l, perkins rg, fang h, tong w. reproducible and reliable microarray results through quality control: good laboratory proficiency and appropriate data analysis practices are essential. curr. opin. biotechnol. 19: 10-18, 2008. somero gn. comparative physiology: a "crystal ball" for predicting consequences of global change. am. j. physiol. regul. integr. comp. physiol. 301: r1-r14, 2011 sonthi m, toubiana m, pallavicini a, venier p, roch p. diversity of coding sequences and gene structures of the antifungal peptide mytimycin (mytm) from the mediterranean mussel, mytilus galloprovincialis. mar. biotechnol. (ny) [in press] 2011. steinberg ce, stürzenbaum sr, menzel r. genes and environment striking the fine balance between sophisticated biomonitoring and true functional environmental genomics. sci. total environ. 400: 142-161, 2008. thomas m, lieberman j, lal a. desperately seeking microrna targets. nat. struct. mol. biol. 17: 1169-1174, 2010. venier p, de pittà c, bernante f, varotto l, de nardi b, bovo g, et al. mytibase: a knowledge base of mussel (m. galloprovincialis) transcribed sequences. bmc genomics 10: 72, 2009. 188 venier p, de pittà c, pallavicini a, marsano f, varotto l, romualdi c, et al. development of mussel mrna profiling: can gene expression trends reveal coastal water pollution? mutat. res. 602: 121-134, 2006. waterhouse rm, kriventseva ev, meister s, xi z, alvarez ks, bartholomay lc, et al. evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. science 316: 1738-1743, 2007. venier p, pallavicini a, de nardi b, lanfranchi g. towards a catalogue of genes transcribed in multiple tissues of mytilus galloprovincialis. gene 314: 29-40, 2003. yeaman mr, yount ny. unifying themes in host defence effector polypeptides. nat. rev. microbiol. 5: 727-740, 2007. zhang sm, zeng y, loker es. expression profiling and binding properties of fibrinogen-related proteins (freps), plasma proteins from the schistosome snail host biomphalaria glabrata. innate immun. 14: 175-189, 2008. venier p, varotto l, rosani u, millino c, celegato b, bernante f, et al. insights into the innate immunity of the mediterranean mussel mytilus galloprovincialis. bmc genomics 12: 69, 2011. 189 minireview isj 9: 134-138, 2012 issn 1824-307x minireview the case of the “serfdom” condition of phagocytic immune cells e ottaviani1, d malagoli1, a grimaldi2, m de eguileor2 1department of life sciences, university of modena and reggio emilia, via campi 213/d,41125 modena, italy 2department of biotechnology and life science, university of insubria, via jh dunant 3, 21100 varese, italy accepted july 24, 2012 abstract in a modern immunological perspective, it may be asserted that the phagocytic cell should not be considered as the "serfdom", but rather the pivot of the immune system. indeed, the invertebrate immunocyte as well as the vertebrate macrophage play a central role in immunity, inflammation and stress response. the evolutionary conserved immune-neuroendocrine effector system have remained of fundamental importance in defense against pathogens, and its efficiency increased through synergy with the new, clonotipical recognition repertoire in vertebrates. key words: invertebrate immunocyte; vertebrate macrophage; immune and neuroendocrine responses; evolution introduction the historical term “serfdom” indicates the low socio-economic status of several peasants during feudalism. as a paradox, it may be stated that a very low level in the functioning of immune system has been attributed for a long time to the vertebrate macrophage and, for extension, also to invertebrate phagocytic immune cells. indeed, expecially among vertebrate immunolgists’ attitude, it appears that these cells do not have the "royal" position occupied in the immune system by the lymphocytes. despite this obsolete point of view, the invertebrate immunocyte/vertebrate macrophage plays a crucial role in immune and neuroendocrine responses in multicellular organisms, and its importance has been neglected for a long time even though it is obviously the pivotal immune cell in forms of life endowed of only innate immunity, as invertebrates. beside this wrong opinion, also the concept that the invertebrate recognition is gross and far less sophisticated than vertebrates was quite diffused before the discovery of molecules such as dscam, 185/333 and frep (watson et al., 2005; bowden et al., 2007; ghosh et al., 2010; romero et al., 2011). before the advances of the last decade in invertebrate immunity, several vertebrate immunologists underestimated the power of invertebrate immune system, since their scientific interest was mainly focused on mammalian models. despite the difficulty of debunking this old concept ___________________________________________________________________________ corresponding author: enzo ottaviani department of life sciences university of modena and reggio emilia via campi 213/d,41125 modena, italy e-mail: enzo.ottaviani@unimore.it among human immunologists, every day new evidence emerge about the role played by phagocytic immune cells. the root of the misconception may be linked to the attitude of the two scientists who formulated the two theories of the immunity, the cellular (elie metchnikoff) and humoral (paul ehrlich). not surprisingly, both of them thought that one component was more significant than the other, but the humoral theory for a long time has played a dominant role as giving responses to unanswered questions on the immunological mechanisms and related diseases. in this minireview we recapitulate the findings that justify why the pahogytic immune cells should be considered the major players of the immune system. phagocyte: morpho-functional characteristics in mammals, macrophage appears to derive from a common progenitor in the bone marrow which gives rise to a separate lineage. all the professional phagocytic cells in humans derive from circulating monocytes and acquire new morphological and physiological characteristics according to the organs and microenvironments in which they settle. this unitarian origin is uncertain for circulating and tissue phagocytes in invertebrates, so we proposed the term “immunocyte” to indicate circulating cells endowed of phagocytic activity, irrespective of their developmental origin (ottaviani, 2011). in our view, the most general characteristic shared by immunocytes and macrophages throughout metazoans is the ability to be activated by not-self material and to react through the release 134 mailto:enzo.ottaviani@unimore.it fig. 1. leech body wall after injection with lps. a) semi-thin section of leech body wall. the adjacent fields of muscle fibers (m) usually in close contact are separated by numerous migrating cells (arrowhead). b) thin section of leech body wall. under the epithelium, among the muscle fibers (m) forming the muscular sac, migrating cells (arrowheads), irregularly shaped (their dimensions vary due to emission of pseudopodia) are recognizable. c, d) cryosections, indirect immunofluorescence staining of migrating cells using anti-d14 (c) and anti-cd68 (d). cd14 and cd68 are typically expressed by macrophages in a wide variety of tissues. e, f) tem. macrophage-like cells moving in the connective tissue show ruffled surface and projections. g) cryosection. cathepsin b (arrowhead), indicated by dark brown signal, is localized in macrophage-like cells migrating among muscle fibers (m). h) sem. area of lesion where macrophage-like cell, showing thin pseudopodia (arrowhead), bridges the epithelial edges. i) semi-thin section of lesioned leech body wall. macrophage-like cells are involved in the obstruction of the wound (arrowheads). migrating cells form a wedge-shaped cluster between muscle fibers (m). bars: a: 25 μm; b: 10 μm; e: 2 μm; f: 5 μm; h: 20 nm. 135 a variety of biologically active molecules, such as cytokines, nitric oxide, reactive oxygen species, hydrolytic enzymes, adrenocorticotropic hormone (acth), β-endorphin and corticotropin-releasing hormone (crh) that affect the immune and the neuroendocrine responses (ottaviani and franceschi, 1997; ottaviani et al., 1998, 2004, 2007). as far as the immune role of the reported molecules is concerned, it has been found that mammalian cytokines, acth peptide fragments and crh are able to influence the chemotactic and phagocytic activity of invertebrate immunocytes and vertebrate macrophages (ottaviani et al., 1990, 1992, 1994, 1997, 2004; genedani et al., 1994). these findings revealed that the pro-chemotactic and prophagocytic effects of the above reported mammalian molecules on invertebrate immunocytes are not straightforward being specie-specific and dose-correlated. the effects on phagocytosis are more uniform than those registered on chemotaxis, making the general assumption that chemotaxis and phagocytosis are strictly correlated, not valid for all invertebrates. the discrepancies observed for the effects of different heterologous peptides on diverse invertebrate immune functions may be attributed to several factors, not excluded the possibility that the immunocyte responds to the same stimulus on the basis of contingency (ottaviani et al., 1997, 2004; malagoli and ottaviani, 2010a). as far as the neuroendocrine responses are concerned, it has been demonstrated that the principle mediators of stress response in invertebrates are fundamentally comparable to those known in vertebrates and have been conserved during evolution (malagoli et al., 2011). we have found that molecules similar to the mediators of mammalian stress, crh, acth, biogenic amines, glucocorticoids and cytokines, are present in and/or released by invertebrate immunocyte (ottaviani and franceschi, 1996; ottaviani et al., 1998; malagoli et al., 2007). it is to underline that even though in invertebrates the molecules are those that participate also in vertebrate stress response, in invertebrates the framework is simpler. in vertebrates, various organs are involved, while all the molecules determining invertebrate stress response are harboured into the immunocyte. in other words, the prototypical response in invertebrates appears to be concentrated into a single, multifunctional cell, representing the best example of an immuneneuroendocrine cell observed so far. during animal diversification, four fundamental evolutionary events that have influenced the formation of the increasingly complex vertebrate immune system, can be recognized. the first evolutionary event is represented by the formation of multicellular organisms, not so much the formation of colonies where the cells are more or less identical to one another, but the association of cells that differ and are able to perform different integrated functions. this series of events is observed for the first time in porifera. the second event is the appearance of a body cavity, i.e., a place in which the circulating cells may be retrieved. the third evolutionary event, is represented by deuterostomia (the blastopore becomes the anus) characteristic of some invertebrate (e.g., echinoderms) and the vertebrate embryos. the fourth event coincides with the transition from the aquatic to the terrestrial area by tetrapode vertebrates. by examining the different species included in the four evolutionary events reported, the key role played by the phagocyte emerges (see turner, 1994). it should be underlined that in animals without a digestive tract, such as porifera and coelenterata, the phagocyte plays a double role i.e., defense and nutrition. hildemann et al. (1979) identified in the immune system of the sponge callyspongia diffusa (porifera), the three functional components as minimal criteria for immunological competence: cytotoxicity, specificity and memory. from invertebrates to vertebrates immune cells are capable of sophisticated performances. as far as immune and neuroendocrine functions are concerned, it is amazing to observe how phagocyting immune cells perform functions ranging from chemotaxis, phagocytosis, encaspsulation, transplantation, wound healing, cytotoxic activity to the stress response (turner, 1994; ottaviani et al., 1997; de eguileor et al., 2000a, b; grimaldi et al., 2004). on the whole, it emerges that phagocytic immune cells are endowed of a vastly dinamic morphology and, beside molluscs (ottaviani, 2011), an interesting example is also represented by hirudo immunocytes (de eguileor et al.,1999, 2000a, b). in leeches, the responses to surgical wounds, grafts or lps injection are similar to those obtained in vertebrates and involve a sequence of events triggered by inflammatory reactions (de eguileor et al., 2003, tettamanti et al., 2003a, b). after immune stimulation a large number of cells migrate through the extracellular matrix from the inner regions of the body close to the gut, towards the superficial body area (figs 1a, b). these migrating cells have been characterized by morphological, histochemical, and immunohistochemical methods. macrophage-like cells, nk-cells and granulocytes are the cell types more represented. in particular several migrating cells involved in leech defense system, display features and behaviour observed also in invertebrate and vertebrate activated macrophages being cd25+, cd14+, cd61+, cd68+, cd11b+ and cd11c+ (de eguileor et al., 2000a). furthermore, immunocytes modify their morphology in relation to the time elapsed after the immune stimulation. during the phase of migration immunocytes change their shape showing ruffled borders (figs 1a-g) and supply enzymes (cathepsin b) to degrade components of extracellular matrix outside the cells, ensuring the movement in the connective tissue (fig. 1g) (grimaldi et al., 2004). in lesioned leeches immunocytes are the first cells that are also involved in closing the wound by using pseudopodia to bridge the epithelial edges (fig. 1h). subsequently additional immunocytes togheter with granulocytes and nk cells complete the obstruction (fig. 1i) (de eguileor et al., 2000a). during wound healing the principal function of immunocytes is phagocitosis. indeed, in their cytoplasm large phagocytic vacuoles containing citoplasmic debris or bacteria or any kind of foreign antigen are always visible (fig. 1e) (de eguileor et al., 2000a). 136 concluding remarks overall, we sustain for the circulating and phagocytic cells the part of principal actor not only during inflammatory response and immunity, as originally suggested by mechnikoff (tauber, 2003), but also during stress response, at least in invertebrates. in this scenario, an unitarian view emerges in which phagocytic immune cells, the most ancestral defense cell of the body, continues to play a fundamental role throughout evolution in the most complex phenomena responsible for body homeostasis, i.e., immunity, inflammation and stress response (ottaviani and franceschi, 1997; ottaviani et al., 2007). however, without minimizing the role played by the cells of acquired immunity, i.e., the vertebrate lymphocytes, they have a more restricted number of functions requiring the involvement of components of innate immunity to be completed. furthermore, the lymphocytes play a key role in immunological memory, an immune function crucial for homeothermic vertebrates, while for the coldblooded ones it seems less relevant (malagoli and ottaviani, 2010b). in this context, the bony fish have their lymphocytes suppressed at low temperatures and, in this situation, found in phagocytic cells the main actors of the immune defense (bly et al., 1990). from these examples it may be argued that in vertebrates, the conserved invertebrate immuneneuroendocrine effector system, centered on the immunocyte, remains of fundamental importance in defense against pathogens, during evolution, and its efficiency increased through synergy with the new, clonotipical recognition repertoire. references bly je, buttke tm, clem lw. differential effects of temperature and exogenous fatty acids on mitogen-induced proliferation in channel catfish t and b lymphocytes. comp. biochem. physiol. 95a: 417-424, 1990. bowden l, dheilly nm, raftos da, nair sv. new immune systems: pathogen-specific host defence, life history strategies and hypervariable immune-response genes of invertebrates. inv. surv. j. 4: 127-136, 2007. de eguileor m, tettamanti g, grimaldi a, boselli a, scarì g, valvassori r et al. histopathological changes after induced injury in leeches. j. invertebr. pathol. 74: 14-28, 1999. de eguileor m, grimaldi a, tettamanti g, valvassori r, cooper el, lanzavecchia g. different types of response against foreign antigens by leech leukocytes. tissue cell 32: 40-48, 2000a. de eguileor m, grimaldi a, tettamanti g, boselli a, valvassori r, cooper el, et al. lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers. tissue cell 32: 437-445, 2000b. de eguileor m, tettamanti g, grimaldi a, congiu t, ferrarese r, perletti g, et al. leeches: immune response, angiogenesis and biomedical applications. curr. pharm. des. 9: 133-149, 2003. genedani s, bernardi m, ottaviani e, franceschi c, leung mk, stefano gb. differential modulation of invertebrate immunocyte motility by crf, acth and its fragments. peptides 15: 203-206, 1994. ghosh j, buckley km, nair sv, raftos da, miller c, majeske aj et al. sp185/333: a novel family of genes and proteins involved in the purple sea urchin immune response. dev. comp. immunol. 34: 235-245, 2010. grimaldi a, tettamanti g, rinaldi l, valvassori r, de eguileor m. role of cathepsin b in leech wound healing. inv. surv. j. 1: 38-46, 2004. hildemann wh, johnson is, jokiel pl. immunocompetence in the lowest metazoan phylum: transplantation immunity in sponges. science 27: 204: 420-422, 1979. malagoli d, accorsi a, ottaviani e. the evolution of pro-opiomelanocortin: looking for the invertebrate fingerprints. peptides 32: 21372140, 2011. malagoli d, di paolo i, ottaviani e. presence of and stress-related changes in urocortin-like molecules in neurons and immune cells from the mussel mytilus galloprovincialis. peptides 28: 1545-1552, 2007. malagoli d, ottaviani e. discrepant effects of mammalian factors on molluscan cell motility, chemotaxis and phagocytosis: divergent evolution or finely tuned contingency? cell biol. int. 34: 1091-1094, 2010a. malagoli d, ottaviani e. life is a huge compromise: is the complexity of the vertebrate immuneneuroendocrine system an advantage or the price to pay? comp. biochem. physiol. 155a: 134-138, 2010b. ottaviani e. immunocyte: the invertebrate counterpart of the vertebrate macrophage. inv. surv. j. 8: 1-4, 2011. ottaviani e, franceschi c. the neuroimmunology of stress from invertebrates to man. prog .neurobiol. 48: 421-440, 1996. ottaviani e, franceschi f. the invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems. immunol. today 18: 169-174, 1997. ottaviani e, franchini a, fontanili p. the effect of corticotropin-releasing factor and proopiomelanocortin-derived peptides on the phagocytosis of molluscan hemocytes. experientia 50: 837-839, 1994. ottaviani e, franchini a, franceschi c. proopiomelanocortin-derived peptides, cytokines and nitric oxide in immune responses and stress: an evolutionary approach. int. rev. cytol. 170: 79-141, 1997. ottaviani e, franchini a, franceschi c. presence of immunoreactive molecules to crh and cortisol in invertebrate haemocytes and lower and higher vertebrate thymus. histochem. j. 30: 6167, 1998. ottaviani e, malagoli d, franceschi c. common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidences to in silico approaches. trends immunol. 28: 497-502, 2007. 137 ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. ancient and complex immune recognition models in invertebrates. dev. comp. immunol. 35: 334-344, 2011. tauber ai. metchnikoff and the phagocytosis theory. nat. rev. mol. cell biol. 4: 897-901, 2003. ottaviani e, petraglia f, genedani s, bernardi m, bertolini a, cossarizza a, et al. phagocytosis and acthand β-endorphin-like molecules in invertebrate (molluscan) and in vertebrate (human) cells: possible significance for the evolution of the immunoneuroendocrine system. ann. ny acad. sci. 594: 454-457, 1990. tettamanti g, grimaldi a, ferrarese r, palazzi m, perletti g, valvassori r, et al. leech responses to tissue transplantation. tissue cell 35: 199212, 2003a. tettamanti g, grimaldi a, valvassori r, rinaldi l, de eguileor m. vascular endothelial growth factor is involved in neoangiogenesis in hirudo medicinalis (annelida, hirudinea). cytokine 22: 168-179, 2003b. ottaviani e, trevisan p, pederzoli a. immunocytochemical evidence for acthand β-endorphin-like molecules in phagocytic blood cells of urodelan amphibians. peptides 13: 227231, 1992. turner rj. immunology. a comparative approach. john wiles & sons, chichester, england, 1994. watson fl, püttmann-holgado r, thomas f, lamar dl, hughes m, kondo m, et al. extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 18741878, 2005 romero a, dios s, poisa-beiro l, costa mm, posada d, figueras a, et al. individual sequence variability and functional activities of fibrinogenrelated proteins (freps) in the mediterranean mussel (mytilus galloprovincialis) suggest 138 fate of pyrene and hydroxypyrene in ilyanassa obsoleta isj 7: 67-78, 2010 issn 1824-307x research report fate and distribution of pyrene in ilyanassa obsoleta exposed through the diet s erskine1, dg beach2,3, c rouleau4, j hellou2,3,5 1department of biology, dalhousie university, halifax, ns, canada 2department of chemistry, dalhousie university, halifax, ns, canada 3bedford institute of oceanography, fisheries and oceans canada, ns, canada 4institut maurice lamontagne, fisheries and oceans canada, québec, canada 5department of oceanography, dalhousie university, ns, canada accepted february 2, 2010 abstract the ability of eastern mud snails to bioaccumulate and biotransform a model polycyclic aromatic hydrocarbon (pah), pyrene (py), was investigated. the contaminant was added to fish at levels ranging from 2 to 5,000 ng/animal fed 20 mg/each and fed to snails. a linear relationship (p=0.03) was observed between the level of py and the sum of metabolites consisting predominantly of 1hydroxypyrene and pyrene-1-sulfate detected in soft tissues of snails. in healthy animals, more py than metabolites was detected, with more biotransformation relative to the parent compound apparent at lower levels of exposure. in ten snails fed together, the body burden of py-related compounds displayed 49% variability as well as a similar mean and median. one snail within that group had five times more metabolites than py and was retracted inside the shell, indicating that the animal was stressed. radio-labelled py was detected in the largest proportion in the kidney of the animals. three snails that died during the exposure had also greater than five times more py metabolites relative to live counterparts. this study is unique in the links that it establishes between stress and the balance of fates of anthropogenic chemicals in biota. key words: body burden; bioaccumulation; biotransformation; pyrene; dose-response; health introduction the bioavailability and bioaccessibility of chemicals labelled as priority pollutants is an area of research that generates continual interest (thorsen et al., 2004; johnsen et al., 2006). such interest results from population expansion that has enhanced the production of these anthropogenic compounds and from a heightened awareness of the associated deleterious effects. an assessment of the presence of contaminants in the abiotic environment provides a means of comparing the actual state of various locations with published guidelines for an acceptable or ideal background state (chapman et al., 1987; ospar, 2009). the latter publication states that “the ultimate aim of achieving concentrations in the marine environment near background values for naturally occurring substances and close to zero for man-made synthetic substances”. questions about the potential impact of exposure on organisms’ health may then be asked. the investigation of the fate of organic ___________________________________________________________________________ corresponding author: jocelyne hellou department of chemistry dalhousie university halifax, ns, canada e-mail: jocelyne.hellou@mar.dfo-mpo.gc.ca compounds in biota reflects the uptake and potential risk caused by exposure to contaminants which bioaccumulate and biotransform. this body burden information can provide the means to link the presence of anthropogenic chemicals to the probability of generating toxic effects (meador et al., 2008). the effects of exposure to contaminants on the health of organisms can cover a broad range of end points. the challenge is to interpret the implications from simple chemical and biochemical levels of organisation, to the more complex of populations, ecosystems and communities (hinton, 1993). contaminants’ mechanism of action differs with dose, where lethality represents an acute endpoint, and does not readily reflect the outcome of exposures. this reality raises the need for additional studies of effects. a balanced assessment based on the “weight of evidence” approach can be achieved (chapman, 2007) by combining knowledge generated in laboratory investigations with the results of field observations. environmental studies aim to discover the state of a site and its inhabitants relative to other or to past situations (myers, et al., 2003; leung et al., 2005). the goal of canada’s oceans strategy is “to ensure healthy and prosperous oceans for the benefit of current and   67 mailto:jocelyne.hellou@mar.dfo-mpo.gc.ca   68 future generations of canadians”. our research has pursued the further development of tools which are the basis of efforts to monitor and assess sediment quality. polycyclic aromatic hydrocarbons (pah) are recognized priority pollutants and pyrene (py) is a commonly abundant pah found in air, freshwater, rain, snow, seawater, sediments and soil (garrigues, narbonne, lafaurie, ribera, lemaire, raoux et al., 1993; lane, leithead, baroi, lee, graham, 2008; lima, farrington, reddy, 2005; williams, meares, brooks, watts, lemieux, 1994). it mainly derives from combustion sources, such as the burning of coal, wood, food and garbage, or the presence of industries involving aluminum smelters, coke ovens, carbon or graphite-electrodes, as well as from natural events such as forest fires and volcanic eruptions. this tetracyclic molecule is always present in complex mixtures with numerous pah and various additional chemicals, especially if sampling is near sewage effluents (soclo et al., 2000; hellou et al., 2002). the physical-chemical properties of py lead to a relatively long residence time in organisms during depuration (hellou and leonard, 2004; hellou, et al., 2009b). in the environment, py has a slower degradation rate than smaller pah, and in sediments, its concentration correlates to that of other large pyrogenic pah associated with mutagenicity and carcinogenicity. these characteristics, along with the availability of a couple of metabolite standards, contribute to choosing py as a model pah for inter-species comparisons of fate (grainger et al., 2005; santella et al., 1994). in the aquatic environment, pah with an octanol water partition coefficient (log kow representing the amount in octanol/amount in water) values of <3.0 reside preferentially in water (mckim, 1994). those with a log kow of 3.0 to 6.0 partition between water and particles, with an increasing tendency to associate with the latter with increasing log kow (mackay et al., 1992). particle-bound pah with log kow >6.0 will be available from lipophilic material such as food. since py has a log kow of 5.2 under steady state conditions, it will be found in small proportion in water and more so in dietary items of the aquatic food web. once taken up by biota, this reactive chemical can undergo a one or two step enzymatic oxidation to 1-hydoxypyrene (pyoh) and it can further conjugate with a biogenic moiety (hellou and leonard 2004; pesch et al., 2007). metabolites represent markers of exposure and effects (perera , et al., 2005). the purpose of biotransformation is to produce more polar and easily eliminated derivatives and represents a biochemical defense mechanism. however intermediate pah products formed during biotransformation such as quinones are toxic (zielinska-park et al., 2004). livingstone (1998) described four types of studies where the role of biotransformation by invertebrates and vertebrates would be of interest, such as in research regarding animal ecology and evolution, in modelling, as biomarkers and in toxicity tests. since less is known about the ability of gastropods to biotransform reactive molecules, our research recently centered on two species of offshore large snails, neptunea lyrata and buccinum undatum, a poisonous and an edible species, respectively (beach et al., 2010, in press; beach and hellou, 2010, in press). an analytical approach was developed for the extraction, separation and identification of bioaccumulation and biotransformation products present in soft tissues of invertebrates exposed to py and pyoh (beach et al., 2009). the accumulation and transformation of these two chemicals yielded up to eight phase i (oxidation) and phase ii (conjugation) metabolites. a smaller readily available related species, the eastern mud snail, ilyanassa obsoleta, is seen at low tide on estuarine beaches where it occupies a variety of habitat surfaces, including fine grain sediments, marsh grasses, pilings or rocks. this mollusc is reported to feed on lettuce, spinach, shrimp or fish when held in tanks, where animals can be easily maintained for experimentation. these snails are found in densities reaching thousands of individuals/m2 (cranford, 1988). they tolerate highly variable weather conditions, surviving temperatures of 40-45 °c for up to 30-90 min and a salinity of 0‰ for more than two days. being surface deposit feeders, as grazers they ingest microalgae and diatoms as well as carrion, where small particles are enriched in lipophilic contaminants. because of their abundance, ilyanassa obsoleta could be used to monitor the availability of contaminants in field, mesocosm or laboratory experiments. the behavioural response of this species towards contaminated sediments and seawater was previously examined in our laboratory (hellou et al., 2009a; marklevitz et al., 2008a, b). in the present study, our goal was to study the fate of py in snails exposed to food containing a range of py levels. the behaviour of the animals was also noted in conjunction to exposure in order to examine potential differences between the behavioural response and fate. the long-term goal of our research aims to support studies evaluating the richness and abundance of fish species from an integrated ecosystem management perspective. such research would also help to link toxicity and contaminants when assessing environmental quality with triad studies (chapman, 1987; 2007) combining toxicity tests, chemical analyses and examining the benthic community. materials and methods care and use of ilyanassa obsoleta snails were collected in summer-early fall from an inter-tidal mud flat in hantsport, nova scotia, canada. sediment and snails were obtained by scraping the upper 5-10 mm surface of the beach and they were transported to the laboratory in halffilled buckets with a couple of cm of additional seawater. upon arrival (1 h drive), animals and sediments were separated. sediments were placed in bags and frozen for future use. our previous work had determined that the grain size of the mud was very fine <50 μm. animals were housed in 1 m2 tanks with flowing seawater maintained at a regulated temperature 15 ± 2 ˚c, with aeration and some sediment (<1cm) placed on the bottom. snails were fed lettuce every other week, sediment was added every month and some was removed to maintain a depth of <1cm in the tank. a smaller 7 l aquaria covered with plastic wrap with pinholes was   69 placed in the holding tank to maintain snails deprived of food for 2.5 days prior to the beginning of experimentation. to select animals for exposures performed in the fall, snails measuring 17-21 mm were removed from the tank, placed in a second one with seawater to determine whether they would take an upright position. they were then distributed randomly among the required 1 l beakers, placing ten per beaker. animals that did not emerge from the shell and begin moving within a few minutes were replaced with active ones. beakers were covered with plastic wrap and punctured with pinholes. behavior of snails all chemical exposures were performed at 1920 °c and snails were counted for their position, on non-immersed glass or in seawater in the 1l beakers containing 800 ml of seawater. experiments were performed over a single season, fall, because of changes previously observed with time. to examine behaviour relative to food preference, exposures were done in the same size beakers with 200 ml of seawater. animals were observed for being upright; in a reversed position lying on their shell with soft tissue extended, “distressed”; or completely within the shell with the operculum covering the soft tissue “retracted” (harry and aldrich, 1963). exposure to spiked food food preference was examined by comparing the consumption of algae, soft tissue of shrimp and filets of whitefish. when fish was offered, the material disappeared more quickly and snails remained on the offered food for a shorter period of time than with the other items. this behavior would indicate less potential loss of contaminant. snails were then fed small pieces of the latter, and it was estimated that after 2.5 days without food, each snail would consume on average 20 mg of fish. therefore, the 200 mg of food required for 10 snails was spiked evenly with <100 μl of an acetone solution while placed in a glass dish, the solvent was evaporated for 10 min and the food was introduced in the beaker and pulled into small pieces with tweezers. in all exposures, animals began to feed readily and consumed most of the food within 15 min, with no traces of fish visible after <60 min. spiking levels were of 100, 1,000, 10,000 and 250,000 ng/g of py in food and represent a dose of 2, 20, 200 and 5,000 ng of py per snail in each beaker. dissection in most cases, because of chemical analyses and the trace levels used in the exposures, animals from one beaker were pooled for analyses, except for any that died during the study, and except at the highest exposure level where each of ten individuals were analyzed separately. for the lipid and chemical extractions, whole animals were frozen until ready for processing. the material was then thawed, the operculum removed and the retractor muscle severed using pointed tweezers. in most cases the snail could then be pulled intact from the shell. when this was not possible, a small screw clamp was used to carefully crack open the shell. prior to weighing, any parts of the shell remaining on the soft tissue were removed. it was important to weigh the snails soon after removing the shells because the moisture evaporated readily and the wet weight became measurably less within minutes of removing the shell. dissection of snails revealed a soft tissue weight of 300-600 mg (wet), with nearly equal weights of muscle and visceral mass. these parts were not divided when processing samples, but only for preliminary physiological determinations. lipid and moisture content lipid content was determined by air-drying tissue in the fume hood overnight with a subsample placed at 70 ˚c in order to confirm the moisture in the air-dried tissue. moisture represents the difference in weight before and after oven drying, expressed as a percentage of wet weight. portions of 200-500 mg of air-dried tissue were weighed into centrifuge tubes and crushed using a manual homogenizing pestle. lipids were extracted using three 10 ml portions of a solution of 1:1 hexane:dichloromethane. this latter solvent was also used to rinse residues off equipment to prevent the loss of material. for each extraction, the tissue and solvent were mixed by vortexing for 30 s and then sonicated for 3 min. after mixing, samples were centrifuged at 2000 rpm for 5-8 min, and the supernatant collected before adding the next aliquot of solvent. combined extract was treated with ovendried sodium sulphate (60-80 °c, continuously), the solution was filtered after 5 min and it was evaporated to dryness. the residue was then transferred to a pre-weighed vial with repetitive rinses of dichloromethane. further evaporation using nitrogen and gravimetric determination of the lipid content was expressed as percent of dry tissue. analysis of soft tissue soft tissue was placed in a pre-weighed aluminum dish using ten animals from each exposure level that were extracted together. the detailed method has been published by beach et al. (2009) and consists of a stepwise methanol extraction repeated four times. ten snails provided 3.9 to 4.7 g of wet tissue. however, smaller samples such as three dead animals of 0.230 g total mass were extracted separately from the remaining ones. the tissue was spiked with 51 ng of 2hydroxyfluorene (floh) using 100 µl of a 0.51 µg/ml solution in acetone and left for 10 min to allow the solvent to evaporate. this phenolic compound represented a surrogate standard to determine the loss of material during processing. tissue was transferred to a centrifuge tube and the petri dish rinsed into a centrifuge tube with 10ml dichloromethane and then 10ml methanol. scoops of dry sodium sulphate were added to ensure that the tissue would deposit in the tube. the mixture was homogenized using a polytron blender, then vortexed for 30 s and sonicated for 3 min. instruments were rinsed with methanol and scraped into the tube to minimize the loss of material. the mixture was centrifuged at 2000 rpm for 5-8 min and the supernatant transferred to a round-bottomed flask. the extraction step was repeated three more times: twice with 10ml methanol and once with 10ml dichloromethane and the extract evaporated using a rotary evaporator at 34-42 ˚c. the flask was carefully rinsed with three 1 ml portions of methanol, blown down under nitrogen,   70 then rinsed again with 2 ml dichloromethane and blown down again. the residue was suspended in 2 ml methanol and filtered through glass wool into another calibrated test tube, rinsing with more methanol. the solvent was evaporated and the residue weighed. a measured amount of methanol was added to give the desired final volume depending on the concentration of py. a subsample of the liquid was transferred to a 2 ml amber hplc vial using a syringe with a filter tip (waters millex hv 4.5 µm) to remove any remaining particles. the extract was analyzed by highperformance liquid chromatography (hplc) with fluorescence detection, and diluted and re-analyzed as necessary to give a concentration within the range of the calibration curves. quality assurance/quality control snail tissue spiked with py, pyoh, pyrene-1sulfate (pyos) and pyrene-1-glucuronide (pyoglca), as well as floh as surrogate standard, were extracted as described above and detailed in beach et al. (2009). a snail matrix spiked with 10100 ng of each of the available standards and processed to examine the efficiency of the approach yielded generally >70% for py and pyoh, while the amount of the more polar compounds was always underestimated. each processed sample was spiked with floh to examine variations in the processing. floh recoveries were above 60% and reaching up to 96%. the concentrations of analytes were therefore adjusted for losses. concentrations are expressed in terms of available standards and there is a need for isotopically labeled standards that could be used to examine the recoveries of the more polar compounds during the work up of each sample. exposure to 14c labeled py and sample preparation a spiking solution was prepared by mixing 22 µl of a 100 µci/ml 14c labeled py in methanol with 35 µl (of the 992 µg/ml of py). the solution was blown dry with a gentle stream of n2 and dissolved in 100 µl of acetone. this amount was applied dropwise to 200 mg of whitefish and allowed to dry for 10 min. the food was given to ten illyanassa held in a 1 l beaker as described above and animals sampled 72 h later. the operculum was removed from live shellfish, they were then cracked with a screw clamp and the soft tissue was removed. each of the animals was slowly lowered into liquid nitrogen with a spoon and held there for 1-2 min. the frozen animal was transferred with forceps to a 50 ml beaker containing 40 ml of carboxymethylcellulose solution (26 g/l). larger forceps were then used to dip the beaker halfway into liquid nitrogen and hold it there until the gel became thoroughly white (5-10 min). as the freezing procedure began, a micro-spatula was used to prevent the tissue from floating to the gel’s surface. finally, the beakers were placed in ziplock bags and stored in the freezer until they were shipped to another location. snails were then processed as described by frouin, et al. (2007) with more than 30 slices generated for each snail, where images were further examined for radiolabeling. results and discussion lipid content the morphometrics and the amount of neutral lipids measured in snails are presented in order to enable comparison by other researchers in future endeavors (table 1). mature animals were chosen according to shell length within a restricted size range having 7% variability (standard deviation/mean). the soft tissue weights of the visceral mass and muscle displayed a wider range of results with 22 and 26% variability, respectively. mean lipid content was 5.8% and varied by 47% over that period. the fish used to feed the snails contained a similar amount of lipid, 5.8%. the mean moisture content of snails and fish was also similar, 83 and 80%, respectively. py food exposure the food preference of the snails was determined by comparing their attraction to, and the time taken to deplete offered items, i.e., algae, shrimp and fish. experiments determined that animals not fed over a period of 2.5 days would be readily attracted to and consume fish more rapidly than other substances. this preliminary conditioning was previously needed in experiments which examined the behavior of snails placed in tanks containing two choices of sediments (marklevitz et al., 2008a, b). this standardization prior to exposure helped to interpret the avoidance/preference response of snails relative to spiked seawater or to single spiked sediment (hellou et al., 2009a). under these holding conditions, an average mature snail readily ingested 20 mg of fish within a few minutes. table 1 morphometric data of some of the snails (n=22) sampled between september and december. shell shell visceral muscle index moisture lipid length width mass mass v/m content content (mm) (mm) (mg) (mg) % % mean 481 19 11 261 1.1 83 5.8 sd 139 1 74 58 0.2 4 2.7 minimum 204 17 9 156 0.6 78 3.4 maximum 861 21 11 431 1.6 87 10.5 fig. 1 body burden of ten snails exposed to the highest level of pyrene (py) spiked in food and representing 5,000 ng per snail for a total of 50,000 ng in 200 mg of fish or 250,000 ng/g fish. behavior towards spiked food behavior was examined because avoidance represents a stress response towards chemical or physical disturbances like spillage of oil or changes in temperature and wind conditions. interest in behavioral research using fish and invertebrates has recently broadened (roudez et al., 2008; robinson, 2009). the stress stages that can develop in snails have been described by harry and aldrich (1963). they consist initially of an attraction-or-avoidance reaction. thereafter animals may be observed to appear “distressed”, soft tissue protruding as they lie on their shells, and eventually to have completely “retracted” soft tissue within their shells. these stages reflect the quality of the snails’ environment and have been detected in exposures to contaminated environments (burris et al., 1990; marklevitz et al., 2008a, b; hellou et al., 2009a). animals were observed during feeding and at intervals during the three-day experiments. none of the offered food was avoided. some variability was detected with time, but without a statistically significant difference in stress (better than 5% level) between exposure levels. in contrast to this behavior towards spiked food, animals exposed to harbor sediments and to sediment spiked with a mixture of seven abundant pah avoided that matrix (marklevitz et al., 2008b). although py has been associated with a range of toxic and therapeutic endpoints (long et al., 1995; law and klungsoyr, 2000; jensen and sverdrup, 2003; clément et al., 2005; culotta et al., 2007) stress was not observed during feeding. variability in contaminants’ uptake: prior to analyzing pools of soft tissue from snails exposed to low levels of py, it was important to determine the distribution in the uptake that would be detected in ten snails exposed together to spiked food. animals exposed to an expected, calculated 5,000 ng of py/animal were therefore analyzed individually. this group of snails was of a restricted mean mass 0.53 g (+0.09 g). assuming that the spiked food was homogenous in py content, since the solvent spike was added uniformly to the food and with great care, then the lowest and highest consumed amount of food interpreted from the body burden consisting of summed py and derivatives was 20 and 170% (fig. 1). the mean and median of the body burden were nearly equal (4,800 + 2,300 and 5,100 ng equivalents of py/animal) and displayed a normal distribution with five animals with concentrations above and below the mean. the inter-individual variability of 49% likely reflects the competition between snails or a snail’s need for food. this investigation provided the standard deviation to be expected with the analysis of pooled tissue of snails exposed to lower levels of py that could not be analyzed individually due to the available instrumentation. concentrations differing by less than 50% between exposure groups would be deemed similar. a high variability has been observed when analyzing animals exposed in the laboratory to spiked food. for example, a wide standard deviation has been reported in studies involving terrestrial and aquatic invertebrates (stroomberg et al., 2004; dam et al., 2006; granberg and forbes, 2006). rates of feeding, excretion and metabolism differ between members of a species even when individuals of a restricted size range are chosen in an experiment in order to minimize the morphometric differences associated with age. fingerprint of uptake the fingerprint of the extracts demonstrated the complexity of the biotransformation process taking place in this species (fig. 2). the proposed structures of the metabolites are based on earlier studies performed in our laboratory and involving larger gastropods, n. lyrata and b. undatum (beach, et al., in press, 2010; beach and hellou, in press, 2010). the presently detected metabolites were identified by comparing the chromatograms of the   71 fig. 2 high performance liquid chromatography (hplc) fluorescence chromatograms displayed on two scales to highlight the fine fingerprint, with pyrene (py) derivatives detected in tissue extracts, with 1-hydroxypyrene (pyoh), fluorenol (floh) representing the surrogate standard, two isomers of pyrene diol monosulfate (pydoms) are followed by two isomers of pyrene diol disulfate (pydods). chromatogram a represents a sample that is more concentrated and injected from a smaller volume of solvent (2 ml), while b is more dilute and injected from a larger volume of solvent (20 ml). larger and smaller species that were injected on the hplc using a common protocol, i.e., same extraction method, guard column and column, solvent gradient, as well as temperature, with injections performed on the same day. the identity of the peaks was previously determined (beach, et al., 2009; beach and hellou, in press, 2010) in part by comparing the retention times and fluorescence spectra of detected peaks to available standards, by performing enzymatic hydrolyses and especially by using a hyphenated chromatography-spectroscopy technique (lc-ms) on an extract fractionated by solid phase extraction. at the highest exposure level, py represented between 61 and 93% of the sum of compounds. the next two more detectable derivatives were pyoh and pyos, followed by two isomers of disulfate derivatives representing between 0.2 and 1.2% of the sum of compounds. two additional diols present as monosulfates were detected at lower levels. pyglca was detected at trace levels. animals with more bioaccumulated py also produced more metabolites, with a significant linear regression between these values (p=0.005, r2=0.6623, with n=9, fig. 3). the exception was for one snail with 1,500 ng of py detected along with 965 ng of metabolites (as py equivalents), where pyoh and pyos were of equivalent proportion. the regression would predict about five times less metabolites or nearly 200 ng. this animal’s behavior differed from that of the others as its body was extended in a manner that is symptomatic of stress. the sum of py and metabolites extracted from this group of snails represented 96% of the amount spiked in food. this result confirmed that the experimental setup was optimal because most of   72 fig. 3 a. linear regression obtained from the analysis of nine healthy individual snails at the higher pyrene exposure, with the star representing the tenth outlier snail. b. linear regression obtained from the analysis of healthy individual or pooled snails (including those in fig. a). the food was consumed. little discharge was apparent in the beakers over the 72 h exposure, reflecting retention of the food or re-ingestion of eliminated material. this has also been observed by beach and hellou (in press, 2010) exposing larger marine gastropods and by dindal and wurzingerref (1971) studying terrestrial snails. it highlights the importance of the recycling of contaminants in the environment with potential trophic transfer by animals consuming detritus. lower levels of exposure three lower exposure levels offering the same amount of white fish with 2, 20 and 200 ng of py per animal were performed. the control and 2 ng group displayed close body burdens (fig. 4). since body burden could be expressed in various units, results obtained for reference animals highlight these differences for an average animal weighing 0.5 g (fig. 4). these body burdens indicate that a similar amount of py was present in the dietary intake of control snails ingesting small particles and animals from the low dietary exposure. in extracts from control snails and the lowest exposure, nearly half, 45-55% of the anthropogenic material, detected in an animal was present as parent py (fig. 4). in extracts from snails exposed to 20 and 200 ng/animal, py represented 65 and 80% of the body burden, respectively, with pyos and pyoh representing the abundant metabolites, and pyos displaying twice the concentration of pyoh. therefore, more biotransformation took place at the lower exposure levels. in healthy animals, tissue extracts contained from 21 to 76% of the py spiked on food. recovery rates of py equivalents were 76 and 96% in the lowest and highest exposures, respectively. therefore, in the middle exposure, either the spike remained in uneaten minute food particles, or py leached out prior to food consumption. the present species handles low levels of xenobiotics through efficient biotransformation better than higher levels (fig. 3). dead animals on a per animal basis, seven times more pyoh and pyos were detected in three dead animals than in surviving animals from the 200ng/snail exposure (fig. 4). these two metabolites were 30% more abundant as py (0.56 vs 0.45 nmol/g), while in healthy animals metabolites represented 20% of the detected py (0.08 vs 0.36 nmol/g). in this mid exposure, metabolites were produced in a somewhat similar opposite ratio as in the distressed animal at the highest food exposure. in the 5,000 ng/animal exposure, healthy animals had metabolites representing 12% of the parent py, while the stressed snail had metabolites representing 60% of the level of py (11 vs 17 nmol/g animal in the one animal compared to a mean of 5.1 vs 42 nmol/g animal in the others). therefore, at least five times more metabolites were produced in animals experiencing toxicity. whether this effect is due to specific derivatives such as quinones (zielinskaet al., 2004) remains to be investigated.   73 fig. 4 a. mean concentrations of pyrene (py) derivatives expressed in different units and detected in reference snails, levels are reported for 1-hydroxypyrene (pyoh), pyrenesulfate (pyos) and pyreneneglucuronide (pyglca). b. concentration of py derivatives detected in animals exposed to various concentrations of pyrene spiked in fish. ref stands for reference, 2, 20 and 200 is the amount of spiked py (ng) expected to be taken up per animal. comparing fate to that in other species the fate of this combustion-derived contaminant has been investigated in the bile of many finfish species from freshwater and marine environments. since the gall bladder bile represents a distinct relatively easy matrix to handle, many studies have examined metabolites in this liquid (solbakken et al., 1982; escartin and porta, 1999). in terrestrial vertebrates, the non-invasive urine analysis has been used most frequently (grainger et al., 2005; walker et al., 2006) with glucuronide and sulfate conjugates detected. interest in the uptake kinetics of pah, in terms of concomitant bioaccumulation and biotransformation, has been relatively restricted. some of the initial studies performed more than two decades ago demonstrated differences in the ability of finfish and shellfish to biotranform vs bioaccumulate pah (varanasi et al., 1985; varanasi et al., 1986; mcelroy, 1990). for example, only one of two species of amphipods was capable of transforming py (reichert et al., 1985). some of the early studies used labeled material to determine the soluble nature of labeled extracts and enzymatic hydrolysis to further pursue the structure of conjugates (collier et al., 1978; solbakken et al., 1979; cravedi and tulliez, 1982; solbakken et al., 1978; mcelroy, 1990). these investigations pursued a balance of fates and the publication by mcelroy (1990) is an excellent example. concern with invertebrates has expanded over the last ten years. a diversity of metabolites has been discovered (eickoff et al., 2003; stromberg, et al., 2004; lee, 2005;) and reviewed in beach et al. (2009). briefly, monohydroxy and dihydroxy pyrene, phase i metabolites, along with glucuronide, sulfate, malonate, glucoside phase ii products, have been identified in tissues of isopod, clam, crab and whelk. the largest number of identified py related compounds was detected in a large gastropod, b. undatum (beach et al., 2010, in press). there are few studies of fates and effects that discuss the presence of metabolites relative to effects (dam et al., 2006). the present observations are unique.   74 fig. 5 example of two images of radio labelled snail tissue with colour associated with the intensity of the labelling and highlighting identified organs. 14[c] labeled pyrene distribution since animals were analyzed whole, an additional exposure to the highest level of py was performed using 14[c] labeled py in food, with identical experimental conditions as used for unlabelled py. the interest was to discover the tissue-specific distribution of py after a three day exposure. as observed with whelks (beach et al., 2010, in press), the most labeling was apparent in the nephridium/kidney (fig. 5). a much lower proportion of labeling was detected in the rest of the digestive tract, particularly in the esophagus, as the images indicate. there was no evidence of labeling in the reproductive system, most likely because of the length of exposure. this species of snails reproduces during may-july under our climate conditions. conclusions and perspectives a linear relationship (p= 0.03) was observed between the level of py accumulated and transformed in soft snail tissues. results demonstrate the complex metabolic capacity of i. obsoleta exposed to realistic levels of food contamination. animals starved for 2.5 days prior to exposure did not avoid py-spiked food. they also did not display significant levels of stress over the following three days, except for one and three snails offered a mean of 5,000 and 200 ng of py per food portion, respectively. these animals displayed more than five times the amount of metabolites detected in soft tissues of healthy members of the respective groups. the difference in the fates of py points to the importance of pursuing the multi-faceted fates of pah to understand the chemical-biological link between fates and effects. with more balanced research, the development of environmental assessment criteria (ospar, 2009) would benefit from the type of research described herein. the eastern mud snails could be used as model organisms for the production and comparison of aromatic metabolites difficult to purchase commercially. the potential presence of a specific py metabolite that would be associated with the   75   76 observed stress needs to be pursued. more investigations should also be performed in order to investigate the link between the fate of other contaminants and stress in various animals. acknowledgements: funding was provided by the national science and engineering research council of canada (jh), by the bedford institute of oceanography (jh) and l’institut maurice lamontagne (cr), of the department of fisheries and oceans. we would like to thank other members of the hellou’s group who helped to collect and maintain snails in our fisheries laboratory and those in the rouleau‘s group who performed the screening of radio-labeled tissue. we also acknowledge the support of dalhousie university to the students (se and dgb) who are co-authors in this publication. references angerer j, mannschreck c, gündel j. biological monitoring and biochemical effect monitoring of exposure to polycyclic aromatic hydrocarbons. int. arch. occup. environ. health 70: 365-377, 1997. beach dg, hellou j. bioaccumulation and biotransformation of 1-hydroxypyrene by the marine whelk neptunea lyrata. int j environ anal chem. [in press]. 2010. beach dg, quilliam m a, hellou j, analysis of pyrene metabolites in marine snails by liquid chromatography using fluorescence and mass spectrometry detection. j. chromatogr. b 877: 2142-2152, 2009. beach dg, quilliam ma, rouleau c, croll rp, hellou j. bioaccumulation and biotransformation of pyrene and 1hydroxypyrene by the marine whelk buccinum undatum. environ. toxicol. chem. [in press]. 2010. burris ja, bamford ms, stewart aj. behavioral responses of marked snails as indicators of water quality. environ. toxicol. chem. 9: 69-76, 1990. chapman pm. sediment quality criteria from the sediment quality triad: an example. environ. toxicol. chem. 5: 957-964, 1986. chapman pm, barrick rc, neff jm, swartz rc. four independent approaches to developing sediment quality criteria yield similar values for model contaminants. environ. toxicol. chem. 6: 723-725, 1987. chapman pm. traditional ecological knowledge (tek) and scientific weight of evidence determinations. mar. poll. bull. 54: 1839-1840, 2007. clément b, cauzzi n, godde m, crozet k, chevron n. pyrene toxicity to aquatic pelagic and benthic organisms in single-species and microcosm tests. polycycl. aromat. comp. 25: 271-298, 2006. collier tk, thomas lc, malins dc. influence of environmental temperature on disposition of dietary naphthalene in coho salmon (oncorhynchus kisutch): isolation and identification of individual metabolites. comp. bioch. phys. 61c: 23-28, 1978. cranford pj. behaviour and ecological importance of a mud snail (ilyanassa obsoleta) population in a temperate macrotidal estuary. can. j. zool. 66: 459-466, 1988. cravedi jp, tulliez j. chronic ingestion of saturated hydrocarbons by rainbow trout: influence of dodecylcyclohexane and pristane on lipid metabolism. arch. environ. contam. toxicol. 11: 719-725, 1982. culotta l, gianguzza a, mannino mr, orecchio s. polycyclic aromatic hydrocarbons (pah) in vulcano island (aeolian archipelago) mud utilized for therapeutic purpose. polycycl. aromat. comp. 27: 281-294, 2007 dam e, styrishave b, rewitz kf, andersen o. intermoult duration affects the succeptibility of shore crabs carcinus maenas (l) to pyrene and their ability to metabolise it. aquat. toxicol. 80: 290-297, 2006. dindal dl, wurzinge.kh. accumulation and excretion of ddt by terrestrial snail, cepaea hortensis. bull. environ. contam. toxicol. 6: 362-371, 1971. eickhoff cv, gobas fapc, law fcp. screening pyrene metabolites in the hemolymph of dungeness crabs (cancer magister) with synchronous fluorescence spectrometry: method development and application. environ. toxicol. chem. 22: 59-66, 2003. escartín e, porte c. assessment of pah pollution in coastal areas from the nw mediterranean through the analysis of fish bile. mar. poll. bull. 38: 1200-1206, 1999. frouin h, pellerin j, fournier m, pelletier e, richard p, pichaud n, et al. physiological effects of polycyclic aromatic hydrocarbons on soft-shell clam mya arenaria. aquat. toxicol. 82: 120134, 2007. garrigues p, narbonne jf, lafaurie m, ribera d, lemaire p, raoux c, et al. banking of environmental samples for short-term biochemical and chemical monitoring of organic contamination in coastal marine environments: the gicbem experience (1986-1990). sci. tot. environ. 139-140: 225-236, 1993. grainger j, huang w, li z, edwards s, walcott c, smith c, et al. polycyclic aromatic hydrocarbon reference range levels in the u.s. population by measurement of urinary mono-hydroxy metabolites polycycl. aromat. comp. 25: 47-65, 2005. granberg me, forbes tl. role of sediment organic matter quality and feeding history in dietary absorption and accumulation of pyrene in the mud snail (hydrobia ulvae). environ. toxicol. chem. 25: 995-1006, 2006. harry hw, aldrich dv. the distress syndrome in taphius glabratus (say) as a reaction to toxic concentration of inorganic ions. malacologia 1: 283-289, 1963. hellou j, steller s, albaiges j. alkanes, terpanes and aromatic hydrocarbons in surficial sediments of halifax harbour. polycycl. aromat. comp. 22: 631-642, 2002. hellou j, leonard j. pah bioaccumulation and biotransformation products in trout exposed through food pellets. int j polyc arom comp 24: 697-712, 2004. hellou j, cook a, lalonde b, walker p, dunphy k, macleod s. escape and survival of corophium volutator and ilyanassa obsoleta exposed to   77 freshwater and chlorotalonyl. j. environ. sci. health. 44:778-790 2009a. hellou j, johnston d, cheeseman k, gronlund a, desnoyers e, leonard j, et al. variables affecting the bioavailability and bioaccumulation of pah in amphipods. int. j. polyc. arom. comp. 29: 12-27, 2009b. hinton de. cells, cellular response, and their markers in chronic toxicity of fishes. in: malins dc, ostrander gk (eds), aquatic toxicology: molecular, biochemical and cellular perspectives. lewis publishers, crc press, pp 207-240, 1993. jensen j, sverdrup le. polycyclic aromatic hydrocarbon ecotoxicity data for developing soil quality criteria. rev. environ. contam. toxicol. 179: 73-97, 2003. johnsen ar, de lipthay jr, reichenberg f, sørensen sj, andersen o, christensen p, et al. biodegradation, bioaccessibility, and genotoxicity of diffuse polycyclic aromatic hydrocarbon (pah) pollution at a motorway site. environ. sci. tech. 40: 3293-3298, 2006. lane da, leithead a, baroi m, lee j, graham la. the detection of polycyclic aromatic compounds in air samples by gcxgc-tofms polycycl. aromat. comp. 28:545-561, 2008. law rj, klungsøyr j. analysis of polycyclic aromatic hydrocarbons in marine samples. int. j. environ. poll. 13: 262-283, 2000. lee rf. fate of petroleum hydrocarbons in marine zooplankton. international oil spill conference, iosc 2005, pp. 2997, 2005. long er, macdonald dd, smith sl, calder fd. incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments. environ. manag. 19: 81-97, 1995. leung kmy, bjørgesæter a, gray js, li, wk, lui gcs, wang y, et al. deriving sediment quality guidelines from field-based species sensitivity distributions. environ. sci .tech. 39: 51485156, 2005. lima alc, farrington jw, reddy cm. combustionderived polycyclic aromatic hydrocarbons in the environment -a review. environ. for. 6: 109131, 2005. livingstone dr. the fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish. comp. biochem. physiol. 120a: 43-49, 1998. mackay d, shiu wy, ma kc. illustrated handbook of physical-chemical properties and environmental fate for organic chemicals, vol ii. lewis publishers, michigan, usa, 1992. marklevitz sac, almeida e, flemming j, hellou j. determining the bioavailability of contaminants and assessing the quality of sediments. part 2: behavioural response of snails, ilyanassa obsoleta, towards contaminated harbour sediments. j. soils sed. 8: 92-97, 2008a. marklevitz sac, almeida e, flemming j, hellou j. determining the bioavailability of contaminants and assessing the quality of sediments. part 1: variables affecting the behavioural response of ilyanassa obsoleta towards contaminated sediments. j. soils sed. 8: 86-91, 2008b. mcelroy ae. polycyclic aromatic hydrocarbon metabolism in the polychaete nereis virens. aq. toxicol. 18: 35-50, 1990. mckim jm. physiological and biochemical mechanisms that regulate the accumulation and toxicity of environmental chemicals in fish. in: hamelink jl, landrum pf, bergman hl, benson wh (eds), bioavailability: physical, chemical and biological interactions. lewis publishers, boca raton, fl, usa, pp 179-201, 1994. meador jp, mccarty ls, escher bi, adams wj. the tissue-residue approach for toxicity assessment: concepts, issues, application, and recommendations. j. environ. mon. 10: 14861498 2008. myers ms, johnson ll, collier tk. establishing the causal relationship between polycyclic aromatic hydrocarbon (pah) exposure and hepatic neoplasms and neoplasia-related liver lesions in english sole (pleuronectes vetulus). hum. ecol. risk. ass. 9: 67-94, 2003. ospar. draft agreement on cemp assessment criteria for the qsr 2010 (asmo 09/8/2-e). ospar meeting of the environmental assessment and monitoring committee (asmo) bonn, germany, 20-24 april 2009. perera fp, rauh v, whyatt rm, tang d, tsai wy, bernert jt, et al. a summary of recent findings on birth outcomes and developmental effects of prenatal ets, pah, and pesticide exposures. neurotoxicology 26: 573-587, 2005. pesch b, kappler m, straif k, marczynski b, preuss r, roßbach b, et al. dose-response modeling of occupational exposure to polycyclic aromatic hydrocarbons with biomarkers of exposure and effect. can. epid. biom. preven. 16: 1863-1873, 2007. reichert wl, le eberhart bt, varanasi u. exposure of two species of deposit-feeding amphipods to sediment-associated [3h]benzo[a]pyrene: uptake, metabolism and covalent binding to tissue macromolecules. aquat. tox. 6: 45-56, 1985. robinson pd. behavioural toxicity of organic contaminants in fish: application to ecological risk assessments (era). can. j. fish. aquat. sci. 66: 1179-1188, 2009. roudez rj, glover t, weis js. learning in an invasive and a native predatory crab. biol. inv. 10: 1191-1196, 2008. santella rm, nunes mg, blaskovic r, perera fp, tang d, beachman a, et al. quantitation of polycyclic aromatic hydrocarbons, 1hydroxypyrene, and mutagenicity in urine of coal tar-treated psoriasis patients and untreated volunteers. can. epid. biom. prev. 3: 137-14, 1994. soclo hh, garrigues p, ewald m. origin of polycyclic aromatic hydrocarbons (pahs) in coastal marine sediments: case studies in cotonou (benin) and aquitaine (france) areas. mar. poll. bull. 40: 387-396, 2000. solbakken je, palmork kh, neppelberg t, scheline rr. distribution of radioactivity in coalfish (pollachius virens) following intragastric administration of [9-14c]phenanthrene. bull. environ. contam. toxicol. 23: 100-103, 1978.   78 solbakken je, knap ah, palmork kh. disposition of (9-14c) phenanthrene in a subtropical marine teleost (haemulon sciurus). bull. environ. contam. toxicol. 28: 285-289, 1982. stroomberg gj, zappey h, steen rjca, van gestel cam, ariese f, velthorst nh, et al. pah biotransformation in terrestrial invertebrates a new phase ii metabolite in isopods and springtail. comp. biochem. physiol. 138c: 129137, 2004. thorsen wa, cope wg, shea d. bioavailability of pahs: effects of soot carbon and pah source. environ. sci. technol. 38: 2029-2032, 2004. varanasi u, reichert wl, stein je, brown dw, sanborn hr. bioavailability and biotransformation of aromatic hydrocarbons in benthic organisms exposed to sediment from an urban estuary. environ. sci. tech. 19: 836841, 1985. varanasi u, nishimoto m, reichert wl, le eberhart b-t. comparative metabolism of benzo(a)pyrene and covalent binding to hepatic dna in english sole, starry flounder, and rat. can. res. 46: 3817-3824, 1986. walker sa, whitten lb, seals gb, lee we, archibong ae, ramesh a. inter-species comparison of liver and small intestinal microsomal metabolism of fluoranthene. food chem. toxicol .44: 380-387, 2006. williams r, meares j, brooks l, watts r, lemieux p. priority pollutant pah analysis of incinerator emission particles using hplc and optimized fluorescence detection. int. j. environ. anal. chem. 54: 299-314, 1994. zielinka-park j, nakamura j, swenberg ja, aiken md. aldehydic dna lesions in calf thymus dna and hela s3 cells produced by bacterial quinone metabolites of fluoranthene and pyrene. carcinogenesis 25: 1727-1733, 2004. care and use of ilyanassa obsoleta isj 8: xx-xx, 2011 isj 8: 247-255, 2011 issn 1824-307x review the evolutionary ecology of aphids' immunity m poirié1,2,3, c coustau1,2,3 1from the evolution and specificity of multitrophic interactions (esim), umr 1301 "biotic interactions and plant health" (ibsv), institut national de la recherche agronomique, inra paca, sophia antipolis, france 2 umr 6243, centre national de la recherche scientifique, cnrs, france 3 université nice sophia antipolis, ufr sciences, france accepted december 19, 2011 abstract aphids comprise 4,400 species that live in close interactions with their host-plants, the parasitoid wasps and fungi they encounter, as well as several bacteria including buchnera aphidicola, an obligatory, nutrient-providing symbiont. aphids also interact with a cohort of facultative secondary symbionts that strongly interfere with their major life history traits such as host-plant specialization, heat tolerance and resistance to natural enemies. here, we present some evolutionary and ecologically-relevant aspects of these interactions, focusing on aphid defenses to parasitism, and considering aphids either as "extended organisms" comprising aphid and symbionts' genomes, or as "single-genome" organisms whose immune components are still poorly known. we highlight the complexity of predicting evolution of aphid immune resistance in the field, due to variable selection pressures, short-term costs, and cross-talk between symbionts. finally, we present perspectives to strongly improve our understanding of the "aphid-symbiont-bacteriophage" meta-organism defenses and to elucidate the interactions between immunity, pathogenicity and symbiosis. key words: aphid; immune defenses; symbionts; parasitoids; extended phenotype, ecological immunity introduction all organisms have to maintain homeostasis and ensure growth and reproduction in changing abiotic and biotic environments. there is no doubt that one of the most challenging environmental condition is the presence of a large diversity of pathogens and parasites, and that the main host defense relies on the immune system. furthermore, the existence of a continuum from pathogenic to beneficial microorganisms is now largely admitted and current research focuses on the immune system as a main factor in the establishment and maintenance of mutualist/symbiotic interactions (slack et al., 2009; login et al., 2011). investigation of the complex interactions between immunity, pathogenicity and symbiosis mostly relies on insect models that are numerous and diversified. indeed, it is roughly estimated that more than 70 % of species host one or more bacterial symbionts (hurst and darby, 2009). ___________________________________________________________________________ corresponding author: marylène poirié evolution and specificity of multitrophic interactions (esim) umr 1301 "biotic interactions and plant health" (ibsv) institut national de la recherche agronomique inra paca, 400 route des chappes, sophia antipolis, 06903, france e-mail:marylene.poirie@sophia.inra.fr the complex pathways of innate immunity have been at least partly deciphered in model species, allowing comparative analyses to be performed. of course, as immunity is a major fitness-related trait, interest is also given to variation of some immune components in relation with biological characteristics such as the developmental stage, the morph, the sex, or the occurrence of a previous immune challenge, as well as with physical environmental characteristics, such as the temperature. evolutionary important features as the existence of trade-offs between constitutive or induced immunity and, for instance, survival or reproduction, are also considered, and the evolution of the level of specificity of the immune response is largely discussed (sadd and schmidt-hempel, 2009; schulenburg et al., 2009). however, data on all these aspects are scarce in homopteran insects and notably in one of the most representative groups, the aphids. aphids are remarkable organisms at the evolutionary and ecological level that have adapted to drastic nutritional and ecological constraints thanks to specific life-history traits and complex polymorphisms. firstly, their life-cycle is characterized by a succession of sexual and asexual morphs, dependent on the environmental 247 mailto:francedominique.colinet@sophia.inra.fr conditions. their impressive ability to proliferate thus largely relies on clonal multiplication while the resulting lack of genetic diversity is mainly compensated by a high phenotypic plasticity. dissemination and colonization of new host plants is ensured by the production of winged individuals when resources become scarce (le ralec et al., 2010). secondly, the biology of aphids is characterized by multiple inter-specific interactions. they are sapfeeding insects that establish a durable interaction with their host plant, managing to avoid or control the plant defenses, and manipulating the plant physiology to ensure the compatibility of the interaction (giordanengo et al., 2010). adaptation to the restricted phloem sieve diet is ensured by an obligate (primary) symbiosis with the bacteria buchnera aphidicola which is essential in providing the missing nutrients (amino acids) (brinza et al., 2009). aphids can also carry secondary, facultative symbionts that are not required for survival but can be mutualistic in affecting positively various life history traits such as suitability to the plant host, heat tolerance, or protection against natural enemies (montlor et al., 2002; oliver et al., 2010). finally, like all living organisms, aphids are attacked by natural enemies such as pathogenic fungi and parasitoid wasps. the immunity of aphids is of particularly interest as it likely affects the network of inter-specific interactions, therefore playing a central role in their ecology and evolution (fig. 1). understanding the functioning and evolutionary ecology of aphid immune defenses is therefore of central importance for future development of control strategies involving pathogenic agents or targeting the aphid-symbionts equilibrium. however, data in this area remain scarce. here, we present a brief overview of our current knowledge, mainly focusing on the pea aphid a. pisum whose genome has been sequenced, and which represents a good example of aphids' functioning as a meta-organism. we then discuss the evolutionary and ecologically-relevant traits of aphid biology that should be explored in the next future in relation to immune findings. the aphid complex biology aphid diversity and plasticity aphids belong to the aphidoidea and the phylloxeroidea super families of hemiptera and they comprises about 4,400 species (blackman and eastop, 1994). among these, about 250 species are agricultural pests, mainly because they vehicle and transmit plant viruses. most aphid species are found in temperate regions but some have adapted to tropical environments (dixon et al., 1986) or even extreme climates such as sub-antarctic (hullé et al., 2003), resulting in a world-wide distribution. aphids can feed on virtually all plant families, the majority of species being specialized to a single host plant, while some have a broad host-plant range (pecoud et al., 2010). aphid speciation and diversification is thought to be widely driven by their specific adaptation to host plants (pecoud et al., 2010). aphid life cycles involve a succession of sexual and asexual morphs. in the simplest cycles, such as that of the pea aphid acyrthosiphon pisum, a single sexual generation occurring in autumn alternates with several parthenogenetic generations where each female produces hundreds of viviparous offspring (helle, 1987). changes in sexual fate and reproductive mode are condition-dependent and they illustrate the aphid extraordinary developmental plasticity in response to environmental cues. altogether, whether variability of a given trait of aphids results from an existing genetic diversity among clones, as evidenced for their adaptation to the host plant, or from high phenotypic plasticity, is sometimes difficult to establish. aphid multiple inter-specific interactions one of the major characteristics of nearly all aphids is their adaptation to plant feeding through association with the obligatory nutrient-providing bacterial symbiont buchnera aphidicola. this gramnegative proteobacterium has co-evolved with aphids for 160-280 millions years (moran and baumann, 1994; wilson et al., 2010). bacteria are located only in specialized cells, the bacteriocytes, and they are transmitted vertically to the embryos. buchnera has a dramatically reduced genome (<1mb), typical of well-integrated obligatory intracellular endosymbionts, where genetic and metabolic redundancy has been minimized (gil et al., 2002; toft and andersson, 2010). it has been estimated that nearly 10 % of the coding capacity is devoted to biosynthesis of 10 essential amino acids that are lacking in the aphid phloem sap diet (wilson et al., 2010). while metabolic interactions between aphids and buchnera have been extensively studied (hansen and moran, 2011), the role of the aphid immune system in the establishment and maintenance of this mutualistic interaction remains to be examined. aphids are attacked by various enemies, notably parasitoid wasps. primary parasitoids of aphids belong to two specialized taxa, the subfamily aphidiinae (hymenoptera: braconidae) and the genus aphelinus (hymenoptera: aphelinidae). female wasps lay eggs in different developmental stages of aphids, from larvae to adults. by the time the parasitoid larvae is fully developed, the aphid dies and its cuticle hardens to form a so-called "mummy" from which an adult wasp will emerge (le ralec et al., 2010). aphids are also infected by various fungi, which generally induce death within a few days (butt et al., 1990). differences in aphid susceptibility/resistance to parasitoids or pathogens have been reported in the field (henter and via, 1995) but the underlying mechanisms are still largely unknown. finally, aphids also interact with bacterial secondary endosymbionts (oliver et al., 2010). they are facultative and found free in the hemolymph as well as within various cell types including bacteriocytes (oliver et al., 2010). interestingly, secondary symbionts can impact important fitnessrelated traits, further complicating the evolutionary ecology of aphids. for instance, serratia symbiotica has a beneficial effect on a. pisum reproduction and viability under heat stress (montllor et al., 2002), thus providing a functional explanation to the previous observations that its frequency reached 80 248 fig. 1 aphid’s immunity is likely involved in interactions with the host plant, the primary and secondary symbionts, as well as the pathogens or parasitoids. % in hot places (oliver et al., 2010). another reported symbiont effect is the change in aphid color. a recent study indeed evidenced that the presence of rickettsiella induces a body color change from red to green (tsuchida et al., 2010). such a modification is likely to affect prey-predatorparasite interactions since ladybird beetles preferentially consume red aphids while parasitoids are more attracted by green ones. finally, a largely affected fitness-related trait is adaptation to the host plant. in particular, results from several independent studies revealed a complex association between infection by regiella insecticola, the aphid genotype, and the host plant use (ferrari et al., 2007). host-plant specialization of aphids can also be directly affected, as infection by r. insecticola would improve aphids' fitness specifically on clover (tsuchida et al., 2004). most fitness-related traits and interactions of aphids with other species can therefore be diversely affected by the presence of symbionts, whether alone or in combination (oliver et al., 2006). symbiont transmission in contrast to b. aphidicola, secondary symbionts are generally transmitted vertically. however, occasional horizontal transmission has been reported. for instance, one secondary symbiont was shown to be possibly transmitted through artificial diet, and its presence was reported in aphid honeydew as well as siphuncular fluid samples (darby and douglas, 2003). the lateral transfer of symbionts may not only generate exchanges between otherwise independent clonal lines but also allow a much quicker spread of symbionts among populations. most interestingly, symbiont transmission was also reported to differ between the parthenogenetic and sexual reproduction stages. first, the maternal transfer of symbionts appeared to be far more imperfect during sexual reproduction than during parthenogenesis, which might be a source of uninfected aphids (moran and dunbar, 2006). besides, paternal transfer of symbionts could lead either to infection of previously non-infected aphids, to double infections, or to replacement of the maternal symbiont (moran and dunbar, 2006). the occurrence of paternal transfer of symbionts likely impacts their population dynamics, notably because of the possible establishment of double infections. new combinations of symbionts might indeed confer new characteristics to the host, as well as generate synergistic or antagonistic interactions. in addition, recombination events and phage gene exchanges might occur, representing a source for rapid evolutionary changes. the evolutionary ecology of aphids immune interactions one major difficulty in understanding how ecological factors, either biotic or abiotic, shape the evolution of the immune system is probably that this question that defines ecological immunology (schulenburg, 2009) is at the interface between different scientific areas. as secondary symbionts are main components of the biotic environment of aphids and strongly influence their resistance to pathogens, the study of aphid defenses belongs naturally to ecological immunity. the use of classical tools to estimate overall defenses (survival to pathogens, encapsulation ability, hemocyte numbers, phagocytic activity, phenol oxidase activity, antimicrobial activity, quantitative pcr on 249 immune-relevant genes, etc.) under various biotic and abiotic conditions is thus a major approach to explain and predict the complex interactions between symbiosis and immunity in the aphid model. an important feature at this time is also the definition of the "organism" to be considered. aphids can indeed be perceived either as species whose immune phenotype is mainly determined by their own genome, or as "extended organisms"1 (in the sense of dawkins' extended phenotype) or metaorganisms, whose defenses may result from the intricate effect of different genomes, including that of symbionts. aphids as "extended organisms" though the aphid meta-organism was reported to interact with host-plants, parasitoids and pathogenic fungi, studies have mainly focused on the "resistance to parasitoids" phenotype, and more specifically on the resistance associated with secondary symbionts. clonal resistance to braconid parasitoids has been described in populations of a. pisum (hufbauer and via, 1999; ferrari et al., 2001), myzus persicae (von burg et al., 2008) and aphis fabae (vorburger et al., 2009), although it is quite rare. in a resistant aphid, failure of the parasitoid can occur either at an early stage when the egg fails to develop or at the larval stage (li et al., 2002), and it is the "larval stage" resistance that is largely influenced by secondary symbionts. in order to understand how symbionts increase aphids' resistance, several studies have experimentally manipulated the symbiotic associations, either by suppressing symbionts using antibiotics treatment, or by introducing a new symbiont thanks to microinjection. for instance, aphidius ervi parasitism success on a. pisum was shown to be reduced by 42 % and 23 % in aphid lines harboring h. defensa and s. symbiotica respectively (oliver et al., 2003). when aphids were experimentally super-infected with both symbionts, the reduction in parasitism success reached 60 %, a benefit that correlated with a marked decrease in fecundity (oliver et al., 2006). h. defensa was also reported to provide resistance to a. ervi against aphidius eadyi (ferrari et al., 2004; oliver et al., 2009), and more recently to aphis fabae against lysiphlebus fabarum (vorburger et al., 2009). it is noteworthy that different strains of h. defensa confer various levels of protection against a. ervi (oliver et al., 2005). strikingly, however, a toxin-encoding bacteriophage, apse (a. pisum secondary endosymbiont), was demonstrated to be required, and likely responsible, for the protective phenotype (oliver et al., 2009). more recently, an independent study evidenced that a. pisum clones infected with both h. defensa and the newly discovered symbiont paxs displayed a high resistance to a. ervi (guay et al., 2009). another symbiont, regiella insecticola, was previously known to confer resistance to a fungal pathogen (scarborough et al., 2005) but not to parasitoids. however, unlike other strains of this bacterium, a specific isolate from myzus persicae provides a protection against the wasp aphidius colemani (vorburger et al., 2010). altogether, these data suggests that the ability to protect the host against natural enemies may evolve readily in different endosymbiotic bacteria, maybe in relation with occurrence of genetic exchanges and gene transfer among symbionts or phages in double-infected hosts (see above). to date, the only described mechanism explaining the symbiont-associated protection is a direct effect, involving the use of bacteriophage' toxins. however, bacterial toxins might be used as well, since most symbionts, including hamiltonella have retained virulence-associated genes in their genomes (degnan et al., 2009). alternatively, symbionts may act indirectly, through an existing host system, and, for instance, positively affect the host immunity. the evolution of symbiont-associated resistance in populations depends on the selection pressures induced by parasitism rates, on the costs associated with the presence of a given symbiont and of the cross-talk between the symbiotic companions in case of multi-infection (oliver et al., 2006). interestingly, the symbiont-associated cost may itself vary. most facultative symbionts have detrimental effects on their host fitness under sexinducing conditions (simon et al., 2011), and the estimated cost on aphis fabae longevity associated with hamiltonella depends on genotype×genotype interactions between the host and the symbiont (vorburger and gouskov, 2011). the selective advantage provided by symbionts can also vary. for instance, the resistance associated with hamiltonella's bacteriophage evolves quickly due to repeated losses of the phage under laboratory conditions, probably because of an imperfect vertical transmission (oliver et al., 2009). resistance to a. ervi in the presence of h. defensa can also change from complete protection to high susceptibility depending on the temperature (bensadia et al., 2006). finally, parasitoids exposed to h. defensa-harboring resistant clones rapidly gain virulence over time (dion et al., 2011), so that resistance is overcome, but they also experience a reduction in fitness. the case of hamiltonella well illustrates the prediction that symbiont-associated resistance may be less stable than genetic resistance (hurst and darby, 2009). indeed, the rate of vertical transmission is not always 100 %, so that bacteria can be lost, and the presence of symbionts is possibly costly, at least energetically. the complex pattern of selective advantages and disadvantages may then explain the large fluctuations of hamiltonella frequency and aphid resistance to parasitoids reported in the field. also, it may facilitate the acquisition/evolution of new resistanceassociated secondary symbionts (maybe explaining the observed resistance conferred by paxs in association with hamiltonella, guay et al., 2009). 1 the term "extended-species" we initially choose do not strictly apply to the aphid model since the host and the symbionts, including buchnera, are considered as different taxonomic units. however, we have clear examples from the long-term evolution that an intricate symbiosis can ultimately lead to the formation of a unique taxonomic entity. 250 http://www.ncbi.nlm.nih.gov/pubmed?term=%22vorburger%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22gouskov%20a%22%5bauthor%5d aphids as a "single genome" species vorburger et al. (2008) has suggested that aphid parasitoids may be confronted with two lines of defense: the "innate defences” and the “acquired defences” provided by secondary endosymbionts, which likely differ in their effectiveness and specificity. significant clonal variation in resistance was indeed observed in several studies, which suggest the existence of an aphid innate resistance. for example, susceptibility of the pea aphid to a. ervi was shown to vary among clones of a single population (henter and via, 1995). although occurrence of such a "genetic" variation suggested a potential for resistance to evolve in response to selection, the average resistance remained unchanged between aphids from this population collected early or late in the summer and exposed meanwhile to a high parasitism rate (henter and via, 1995). the authors hypothesized that the lack of response to selection was due to trade-offs between resistance and other fitness-related traits. significant clonal variation and co-variation in resistance of a. pisum to two parasitoid wasps and to a pathogenic fungi was also reported, without evidence of a trade-off between resistance and fecundity (ferrari et al., 2001). on the contrary, myzus persicae effectiveness to survive a. colemani attacks was correlated with a loss of fecundity in individuals surviving the attack (vorburger et al., 2008). in other words, clones that were more resistant to the parasitoid experienced a higher loss in fecundity when attacked. such a trade-off may impair selection for resistance in natural populations and participate to the maintenance of genetic variation for resistance (vorburger et al., 2008). finally, recent work from dion et al. (2011) also demonstrated a large clonal variation in resistance to a. ervi in the absence of secondary symbionts. importantly, caution must be taken in concluding on the genetic basis of a variation in resistance since the absence of secondary symbionts has not always been assessed or accurately demonstrated. when tested, the presence of symbionts was assessed through pcr analysis based on known sequences, while novel aphid secondary symbionts are regularly described (guay et al., 2009; tsushida et al., 2010). although these studies nevertheless highlight natural variation in aphid ability to fight pathogens, the mechanisms underlying this variation are totally unknown and the involvement of the immune system has not been investigated. understanding resistance to parasitoids in aphids and their evolution thus requires a thorough study of aphids' own immune defences, as well as of the parasitoid strategies selected to avoid or circumvent all aphid defense categories. the aphid immune system: what do we know? intriguingly, neither the physiology nor the molecular biology of the immune defenses of aphids have ever attracted attention. possible reasons for that are the small size of most aphid species. also, insect immunity was primary studied on diptera and lepidoptera that are submitted to frequent bacterial challenges, while aphids belong to the homoptera and were mainly described as interacting with parasitoid wasps. ecological immunologists often estimate the immune response level by counting the number of immune cells, and measuring the phenol oxidase (po) activity. however, there are very few available descriptions of immune cells in aphids, the most detailed being by far the one of boiteau and perron (1976), which described six hemocyte categories in macrosiphon euphorbiae: prohemocytes, oenocytoids, plasmatocytes, granulocytes, spherulocytes and wax cells. surprisingly, the first data on a. pisum (laughton et al., 2011), reported only three morphologically distinct types of hemocytes: prohemocytes, granulocytes that may phagocyte bacteria, and oenocytoids. more accurate, thorough analyses, including ultra-structural description of the cells, and description of their adhesion profiles, are strongly needed to perform functional analyses. comparison of aphid hemocyte numbers from different morphs and under different environmental conditions nevertheless remains a complicated task, due to the low cell number and the quantity of debris and symbionts in the hemolymph. regarding the phenoloxidase pathway, detailed annotation work of a. pisum genome suggests that it exists in the pea aphid (gerardo et al., 2010), and a constitutive phenoloxidase activity can be measured (m poirié, personal data). whether or not it differs between morphs and can be activated by a pathogen challenge remains to be established. the question of the immune molecular processes underlying the biotic interactions of aphids is then far from being elucidated. most information comes from the recent sequencing of the first aphid genome (a. pisum) by the international aphid genomics consortium (iagc) (iagc, 2010) that has raised novel evolutionarily and functionally-relevant questions. for instance, a total of approximately 34,000 genes were predicted, which is nearly twice as described for other insect sequenced genomes belonging to diptera, coleoptera and hymenoptera (iagc, 2010; tagu et al., 2010). this is at least partly explained by the existence of many gene duplications (tagu et al., 2010). in a first search for immune-related genes in a. pisum genome, gerardo and collaborators (2010) identified key elements of the toll and janus kinase/signal transducer (jak/stat) pathways, as well as corresponding recognition and effector genes. surprisingly, however, the immune deficiency (imd) signaling pathway was apparently non functional, with some of the genes missing, and no peptidoglycan recognition proteins (pgrps) were found. in addition, well-conserved antimicrobial peptides such as defensins and cecropins could not be predicted (gerardo et al., 2010). experimental analyses were designed to characterize immune response through the isolation of rna transcripts from immune-challenged pea aphids but they uncovered few immune-related products. these data and the low expression levels of some characterized aphid immune genes suggested a low overall antibacterial immune response (altincicek, 2008; gerardo, 2010) in agreement with aphid susceptibility to experimental bacterial infection (grenier, 2006; altincicek, 2011). 251 fig. 2 studies on aphid immune-related traits should be performed in a sequential manner aimed at understanding the respective influence of aphid genotype (level 1), of the presence of secondary symbionts (level 2), and possibly the presence of phages in secondary symbionts (level 3). the parasitoid effect should be tested in combination with all these levels and the temperature effect will likely have to be considered as well. note that secondary symbionts may belong to different species or may represent different strains of a given species, therefore complexifying the approach. different evolutionary hypotheses have been proposed to explain this surprising result. for instance, aphid increased investment in reproduction following infection, or symbiontmediated host protection might "compensate" for the "deficient" immunity. this latter hypothesis implies of course that symbionts do not act indirectly through manipulation of host immunity. also, the reduced antibacterial defense was suggested to be an adaptation for the symbiosis with the bacterium buchnera aphidicola, that is known to elicit an immune response in drosophila s2 cells (douglas et al., 2011). this selection to "accommodate" the bacterial partner could have also ended in a reduced antibacterial defense specific to the bacteriome as reported in a weewil species (anselme et al., 2008) given that buchnera cells are rarely encountered outside the bacteriome. a main concern in answering the question of a “deficient” or a “different” immune system in aphids is the lack of information both on genes potentially involved in the anti-parasitoid response, and on occurrence of resistance to bacterial pathogens. besides, it is possible that a substantial part of the aphid immune genes escaped annotation due either to assembly problems or to biases, since gene prediction and identification strictly rely on similarities with genes previously described in other models. the "deficient immunity" hypothesis thus remains first to be tested accurately, taking into account other elements of the immune response such as the signaling pathways involved in cellular responses (including mapk pathways), the receptors involved in phagocytosis ability, or the reactive oxygen species-mediated defenses. future directions aphid’s life history traits, including immune performances, must be viewed as extended phenotypes (dawkins, 1989) resulting not only from the expression of the aphid genome itself, but also from the expression of genes from their bacterial symbionts and eventually from the bacterial phages. in many insects, including drosophila, bacterial symbionts can indeed positively or negatively impact host defenses against pathogens and even participate in the formation of the immune system (xie et al., 2010; weiss et al., 2011). characterization of immune traits thus have to be performed in aphids naturally or artificially deprived of secondary symbionts. this will allow subsequent comparison of different genetic backgrounds and different morphs, under different conditions (fig. 2). in a second step, it will be possible to compare immune components between genetically identical lines without secondary symbionts or with a single secondary symbiont, or different strains of this symbiont, with or without associated phages (fig. 2). this will provide essential information on how the aphid immune system and the symbionts interfere with each other, depending of the symbiont strain or species. strikingly, understanding the immune ecology of aphids as meta-organisms will also require addressing the important question of the multiple-infections and of the impact of abiotic conditions. in the field, future studies aimed at characterizing immune processes or at examining an immune-related trait, such as the ability to fight infection by a particular pathogen or parasitoid, should be carefully designed to control or characterize the extended genotype (fig. 2). the diagnostic of the presence of microorganisms by observatory methods (such as microscopy, immunelabeling or pcr) is restricted to the known symbiont species. however, the rapid progression of genomesequencing methods and facilities should allow characterization of the metagenome of aphid clones in a close future, opening the way to comparative 252 genomics of clones presenting different immunerelated traits (i.e., resistance/susceptibility to a pathogen). applied to human gut, microbial metagenomics recently revealed more than 1,000 prevalent bacterial species in a single cohort of 124 individuals, each individual hosting at least 160 of such species (qin et al., 2010), whether commensal or potentially pathogenic. this commensal microbiota is now known to shape the host immune system. aphids host a comparatively much smaller number of bacteria but they have highly intricate relationships with most of them, then appearing as good models for deciphering the interactions between immunity, pathogenicity, and symbiosis. they are also important models to address the central question of how to use our increasing knowledge on the symbiont-mediated modification of essential life-history traits for improving human and plant health. acknowledgments we are thankful to jc simon, jl gatti and a schmitz for fruitful discussions as well as to an anonymous reviewer for comments and suggestions to improve the manuscript. references altincicek b, gross j, vilcinskas a. woundingmediated gene expression and accelerated viviparous reproduction of the pea aphid acyrthosiphon pisum. insect mol. biol. 17: 711716, 2008. altincicek b, ter braak b, laughton am, udekwu ki, gerardo nm. escherichia coli k-12 pathogenicity in the pea aphid, acyrthosiphon pisum, reveals reduced antibacterial defense in aphids. dev. comp. immunol. 35: 1091-1097, 2011. anselme c, pérez-brocal v, vallier a, vincentmonegat c, charif d, latorre a, moya a, heddi a. identification of the weevil immune genes and their expression in the bacteriome tissue. bmc biol. 6:43, 2008. bensadia f, boudreault s, guay jf, michaud d, cloutier c. aphid clonal resistance to a parasitoid fails under heat stress. j. insect physiol. 52: 146-157, 2006. blackman rl, eastop vf. aphids on the world’s trees. cab international, wallingford, united kingdom, 2004. boiteau g, perron j.-m. etude des hémocytes de macrosiphum euphorbiae (thomas) (homoptera: aphididae). can. j. zool. 54: 228234, 1976. brinza l, viñuelas j, cottret l, calevro f, rahbé y, febvay g, et al. systemic analysis of the symbiotic function of buchnera aphidicola, the primary endosymbiont of the pea aphid acyrthosiphon pisum. cr biol. 332: 1034-1049, 2009. butt tm, becket a, wilding n. a histological study of the invasive and developmental processes of the aphid pathogen erynia neoaphidis (zygomycotina: entomophthorales) in the pea aphid acyrthosiphon pisum. can. j. bot. 68: 2153-2163, 1990. darby ac, douglas ae. elucidation of the transmission patterns of an insect-borne bacterium. appl. envir. microbiol. 69: 44034407, 2003. dawkins r. the extended phenotype. oxford university press, oxford, 1989. degnan ph, yu y, sisneros n, wing ra, moran na. hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors. proc. natl. acad. sci. usa 106: 9063-9068, 2009. dion e, zélé f, simon jc, outreman y. rapid evolution of parasitoids when faced with the symbiont-mediated resistance of their hosts. j. evol. biol. 24: 741-750, 2011. dixon afg, kindlmann p, leps j, holman j. why there are so few species of aphids, especially in the tropics. am. nat. 129: 580-592, 1986. douglas ae, bouvaine s, russell rr. how the insect immune system interacts with an obligate symbiotic bacterium. proc. biol. sci. 278: 333338, 2011. ferrari j, muller cb, kraaijveld ar, godfray hcj. clonal variation and covariation in aphid resistance to parasitoids and pathogen. evolution 9: 1805-1814, 2001. ferrari j, darby ac, daniell h, godfray hcj, douglas a. linking the bacterial community in pea aphids with host-plant use and natural enemy resistance. ecol. entomol. 29: 60-65, 2004. ferrari j, scarborough cl, godfray hcj. genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids. oecologia 153: 323-329, 2007. gerardo nm, altincicek b, anselme c, atamian h, barribeau sm, de vos m, et al. immunity and other defenses in pea aphids, acyrthosiphon pisum. genome biol. 11: r21, 2010. giordanengo p, brunissen l, rusterucci c, vincent c, van bel aje, dinant s, et al. compatible plant-aphid interactions: how do aphids manipulate plant responses. cr biol. 333: 516523, 2010. gil r, sabater-munoz b, latorre a, silva fj, moya a. extreme genome reduction in buchnera spp.: toward the minimal genome needed for symbiotic life. proc. natl. acad. sci. usa 99: 4454-4458, 2002. grenier am, duport g, pagès s, condemine g, rahbé y. the phytopathogen dickeya dadantii (erwinia chrysanthemi 3937) is a pathogen of the pea aphid. appl. environ. microbiol. 72: 1956-1965, 2006. guay jf, boudreault s, michaud d, cloutier c. impact of environmental stress on aphid clonal resistance to parasitoids: role of hamiltonella defensa bacterial symbiosis in association with a new facultative symbiont of the pea aphid. j. insect physiol. 55: 919-926, 2009. hansen ak, moran na. aphid genome expression reveals host-symbiont cooperation in the production of amino-acids. proc. natl. acad. sci. usa 108: 2849-2854, 2011. helle w. aphids, their biology, natural enemies and control, vol. a, b, c. ak minks and p harrewijn eds, elsevier science publishers, 1987. 253 http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2583.2008.00835.x/abstract http://www.ncbi.nlm.nih.gov/pubmed/21527277 http://www.ncbi.nlm.nih.gov/pubmed/21527277 http://www.ncbi.nlm.nih.gov/pubmed/21527277 http://www.ncbi.nlm.nih.gov/pubmed/21527277 http://www.ncbi.nlm.nih.gov/pubmed?term=%22anselme%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22p%c3%a9rez-brocal%20v%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22vallier%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22vincent-monegat%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22vincent-monegat%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22charif%20d%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22latorre%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22moya%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22heddi%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22heddi%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed/18925938 http://www.ncbi.nlm.nih.gov/pubmed/16307754 http://www.ncbi.nlm.nih.gov/pubmed/16307754 http://www.ncbi.nlm.nih.gov/pubmed/21261770 http://www.ncbi.nlm.nih.gov/pubmed/21261770 http://www.ncbi.nlm.nih.gov/pubmed/21261770 http://www.ncbi.nlm.nih.gov/pubmed/20719775 http://www.ncbi.nlm.nih.gov/pubmed/20719775 http://www.ncbi.nlm.nih.gov/pubmed/20719775 http://www.ncbi.nlm.nih.gov/pubmed/20178569 http://www.ncbi.nlm.nih.gov/pubmed/20178569 http://www.ncbi.nlm.nih.gov/pubmed/20178569 http://www.ncbi.nlm.nih.gov/pubmed?term=%22grenier%20am%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22duport%20g%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22pag%c3%a8s%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22condemine%20g%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22rahb%c3%a9%20y%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=grenier%202006%20and%20aphid henter hj, via s. the potential for coevolution in a host-parasitoid system. i. genetic variation within an aphid population in susceptibility to a parasitic wasp. evolution 49: 427-438, 1995. hufbauer ra, via s. evolution of an aphidparasitoid interaction: variation in resistance to parasitism among aphid populations specialized on different plants. evolution 53: 1435-1445, 1999. hullé m, pannetier d, simon jc, vernon p, frenot y. aphids of sub-antarctic isles crozet and kerguelen: species diversity, host range and spatial distribution. antarctic sci. 15: 203-209, 2003. hurst gdd, darby ac. the inherited microbiota of arthropods and their importance in understanding resistance and immunity. in: rolff j, st reynolds st (eds), insect infection and immunity, oxford university press, 2009. international aphid genomics consortium. genome sequence of the pea aphid acyrthosiphon pisum. plos biol,8(2): e1000313, 2010. laughton am, garcia jr, altincicek b, strand mr, gerardo nm. characterisation of immune responses in the pea aphid, acyrthosiphon pisum. j. insect physiol. 57: 830-839, 2011. le ralec a, anselme c, outreman y, poirié m, van baaren j, le lann c, et al. evolutionary ecology of the interactions between aphids and their parasitoids. cr biol. 333: 554-565, 2010. li s, falabella p, giannantonio s, fanti p, battaglia d, digilio mc, völkl w, et al. pea aphid clonal resistance to the endophagous parasitoid aphidius ervi. j. insect physiol. 48: 971-980, 2002. login fh, balmand s, vallier a, vincent-monégat c, vigneron a, weiss-gayet m, et al. antimicrobial peptides keep insect endosymbionts under control. science 334: 362-365, 2011. milner rj. distribution in time and space of resistance to the pathogenic fungus erynia neoaphidis in the pea aphid acyrthosiphon pisum. entomol. exp. appl. 37: 235-240, 1985. montllor cb, maxmen a, purcell ah. facultative bacterial endosymbionts benefit pea aphids acyrthosiphon pisum under heat stress. ecol. entomol. 27: 189-195, 2002. moran n, baumann p. phylogenetics of cytoplasmically inherited microorganisms of arthropods. trends ecol. evol. 9: 15-20, 1994. moran n, dunbar he. sexual acquisition of beneficial symbionts in aphids. proc. natl. acad. sci. usa 103 : 12803-12806, 2006. oliver km, russell ja, moran na, hunter ms. facultative bacterial symbionts in aphids confer resistance to parasitic wasps. proc. natl. acad. sci. usa 100: 1803-1807, 2003. oliver km, moran na, hunter ms. variation in resistance to parasitism in aphids is due to symbionts not host genotype. proc. natl. acad. sci. usa 102: 12795-12800, 2005. oliver km, moran na, hunter ms. costs and benefits of a superinfection of facultative symbionts in aphids. proc. r. soc. b 273: 12731280, 2006. oliver km, degnan ph, hunter ms, moran na. bacteriophages encode factors required for protection in a symbiotic mutualism. science 325: 992-994, 2009. oliver km, degnan ph, burke gr, moran na. facultative symbionts of aphids and the horizontal transfer of ecologically important traits. ann. rev. entomol. 55: 247-266, 2010. peccoud j, simon jc, von dohlen c, cœur d’acier a, plantegenest m, vanlerberghe-masuti f, et al. evolutionary history of aphid-plant associations and their role in aphid diversification. cr biol. 333: 474-487, 2010. qin j, li r, raes j, arumugam m, burgdorf ks, manichanh c, et al. a human gut microbial gene catalogue established by metagenomic sequencing. nature 464: 59-65, 2010. sadd bm, schmidt-hempel p. ecological and evolutionary implications of specific immune responses. in: rolff j, st reynolds st (eds), insect infection and immunity, oxford university press, 2009. scarborough cl, ferrari j, godfray hc. aphid protected from pathogen by endosymbiont. science 310: 1781, 2005. schulenburg h, kurtz j, moret yt, siva-jothy m. introduction. ecological immunology. philos. trans. r soc. lond. b biol. sci. 364: 3-14, 2009. simon jc, boutin s, tsuchida t, koga r, le gallic jf, frantz a, et al. facultative symbiont infections affect aphid reproduction. plos one, 6(7): e21831, 2011. slack e, hapfelmeier s, stecher b, velykoredko y, stoel m, lawson ma, et al. innate and adaptive immunity cooperate flexibly to maintain hostmicrobiota mutualism. science 325: 617-620, 2009. schulenburg h, kurtz j, moret y, siva-jothy mt. introduction: ecological immunology. philos. trans. r. soc. lond. b biol. sci. 364: 3-14, 2009. tagu d, dugravot s, outreman y, rispe c, simon jc, colella s. the anatomy of an aphid genome: from sequence to biology. cr biol. 333: 464-473, 2010. toft c, andersson sg. evolutionary microbial genomics: insights into bacterial host adaptation. nat. rev. genet. 11: 465-475, 2010. tsuchida t, koga r, fukatsu f. host plant specialization governed by facultative symbiont. science 303: 1989, 2004. tsuchida t, koga r, horikawa m, tsunoda t, maoka t. matsumoto s, et al. symbiotic bacterium modifies aphid body color. science 330: 1102-1104, 2010. von burg s, ferrari j, müller cb, vorburger c. genetic variation and covariation of susceptibility to parasitoids in the aphid myzus persicae: no evidence for trade-offs. proc. biol. sci. 275: 1089-1094, 2008. vorburger c, gouskov a, von burg s. genetic covariation between effectiveness and cost of defence in aphids. biol. lett. 4: 674-676, 2008. vorburger c, sandrock c, gouskov a, castañeda le, ferrari j. genotypic variation and the role of defensive endosymbionts in an allparthenogenetic host-parasitoid interaction. evolution 63:1439-1450, 2009. 254 http://www.ncbi.nlm.nih.gov/pubmed/20186266 http://www.ncbi.nlm.nih.gov/pubmed/20186266 http://www.ncbi.nlm.nih.gov/pubmed/20186266 http://www.ncbi.nlm.nih.gov/pubmed/21439291 http://www.ncbi.nlm.nih.gov/pubmed/21439291 http://www.ncbi.nlm.nih.gov/pubmed/21439291 http://www.ncbi.nlm.nih.gov/pubmed/20541166 http://www.ncbi.nlm.nih.gov/pubmed/20541166 http://www.ncbi.nlm.nih.gov/pubmed/20541166 http://www.ncbi.nlm.nih.gov/pubmed/12770044 http://www.ncbi.nlm.nih.gov/pubmed/12770044 http://www.ncbi.nlm.nih.gov/pubmed/12770044 http://www.ncbi.nlm.nih.gov/pubmed/22021855 http://www.ncbi.nlm.nih.gov/pubmed/22021855 http://www.ncbi.nlm.nih.gov/pubmed/22021855 http://www.ncbi.nlm.nih.gov/pubmed/21236755 http://www.ncbi.nlm.nih.gov/pubmed/21236755 http://www.ncbi.nlm.nih.gov/pubmed/21236755 http://www.ncbi.nlm.nih.gov/pubmed?term=%22scarborough%20cl%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ferrari%20j%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22godfray%20hc%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=scarborough%202005%20and%20aphid http://www.ncbi.nlm.nih.gov/pubmed/21818272 http://www.ncbi.nlm.nih.gov/pubmed/21818272 http://www.ncbi.nlm.nih.gov/pubmed/19644121 http://www.ncbi.nlm.nih.gov/pubmed/19644121 http://www.ncbi.nlm.nih.gov/pubmed/19644121 http://www.ncbi.nlm.nih.gov/pubmed/18926970 http://www.ncbi.nlm.nih.gov/pubmed/20541158 http://www.ncbi.nlm.nih.gov/pubmed/20541158 http://www.ncbi.nlm.nih.gov/pubmed/20517341 http://www.ncbi.nlm.nih.gov/pubmed/20517341 http://www.ncbi.nlm.nih.gov/pubmed/18270153 http://www.ncbi.nlm.nih.gov/pubmed/18270153 http://www.ncbi.nlm.nih.gov/pubmed/18270153 http://www.ncbi.nlm.nih.gov/pubmed?term=%22vorburger%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22sandrock%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22gouskov%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22casta%c3%b1eda%20le%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22casta%c3%b1eda%20le%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ferrari%20j%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed/19228189 vorburger c, gehrer l, rodriguez p. a strain of the bacterial symbiont regiella insecticola protects aphids against parasitoids. biol. lett. 6: 109111, 2010. vorburger c, gouskov a. only helpful when required: a longevity cost of harboring defensive symbionts. j. evol. biol. 24: 1611-1617, 2011. wilson acc, ashton pd, calevro f, charles h, colella s, febvay g, et al. genomic insight into the amino acid relations of the pea aphid, acyrthosiphon pisum, with its symbiotic bacterium buchnera aphidicola. insect mol. biol. 19: 249-258, 2010. weiss bl, wang j, aksoy s. tsetse immune system maturation requires the presence of obligate symbionts in larvae. plos biol, 9(5): e1000619, 2011. xie j, vilchez i, mateos m. spiroplasma bacteria enhance survival of drosophila hydei attacked by the parasitic wasp leptopilina heterotoma. plos one, 5(8): e12149, 2010. 255 http://www.ncbi.nlm.nih.gov/pubmed/19776066 http://www.ncbi.nlm.nih.gov/pubmed/19776066 http://www.ncbi.nlm.nih.gov/pubmed/19776066 http://www.ncbi.nlm.nih.gov/pubmed?term=%22vorburger%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22gouskov%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=vorburger%20and%20gouskov%2c%202011 http://www.ncbi.nlm.nih.gov/pubmed?term=%22weiss%20bl%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22wang%20j%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22aksoy%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=weiss%202011%20and%20wigglesworthia http://www.ncbi.nlm.nih.gov/pubmed/20730104 http://www.ncbi.nlm.nih.gov/pubmed/20730104 http://www.ncbi.nlm.nih.gov/pubmed/20730104 the interaction insect-symbiont, rather than insect-pathogen, may open new perspectives in the understanding of the host choice in bacteria isj 6: 98-101, 2009 issn 1824-307x visions and perspectives insect-symbiont: the key relationship to get in-depth insight on the host choice of bacteria m mandrioli department of animal biology, university of modena and reggio emilia, modena, italy accepted june 26, 2009 abstract insects are extremely successful animals in view of their great adaptability to a wide range of terrestrial niches. symbiotic bacteria gave a precious contribution to such a success playing crucial roles in different contexts such as nutrition, development, reproduction, immunity, defense against natural enemies and speciation. recently, the study of symbiosis furnished precious data not only on insect evolution, but also on the mechanisms involved in the bacterial host choice giving us new perspectives to study this process that was poorly understood up to date through the study of pathogenic interactions. key words: symbiosis; host choice; symbiont genome degeneration; insect-bacteria interaction introduction insects are undoubtedly one of the most successful animal group in nature in view of the high number of species and the high number of individuals observed in insect population. the success of insects is due to their remarkable adaptability to a vast array of terrestrial habitats, including those that are strongly limited or imbalanced in nutrients and to their ability to face pathogens (such as bacteria). nevertheless, insect success is also due to the collaboration with bacteria in term of symbiosis since bacteria play crucial roles in the biology and life cycle of most insects species, affecting nutrition, development, reproduction, immunity, defense against natural enemies and speciation (buchner, 1965; moran and baumann, 2000; moran, 2001; moran, 2006).  up to date several papers faced the relationship between bacteria and insects in term of insect defense so that most of the attention has been put on insect pathogens and antimicrobial peptides synthesized by insects or onto other strategies that they set up to avoid bacterial infection (mandrioli et al., 2003; brivio et al., 2005; schmidt et al., 2005; brown and hancock, 2006; lemaitre and hoffmann, 2007; lazzaro, 2008; müller et al., 2008). ___________________________________________________________________________ corresponding author: mauro mandrioli department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: mauro.mandrioli@unimo.it however, in the last years the interaction between insects and bacteria has been studied with particular attention to symbiosis and, interestingly, the study of mutualism and symbiosis is furnishing several intriguing evidences about the host choice giving us new data about this process that has been poorly understood up to date through the study of pathogenic interactions (mandel et al., 2009). symbioses are categorized according to the extent of dependence between the host and the symbionts, which generally depends on evolutionary antiquity of the symbiosis (moran and baumann, 2000; moran, 2001). while obligate primary symbionts are essential for the host survival and/or reproduction, secondary are facultative and thought to be of more recent acquisition, even though they can contribute to the fitness of the host, e.g., conferring resistance to parasites (moran and baumann, 2000; moran, 2001). most primary symbionts are vertically transmitted to the progeny with a process starting at early stages of oogenesis or embryogenesis. vertical transmission is common also in secondary symbionts, but they can also colonize novel hosts through horizontal transmission among host individuals belonging to the same or different species (dale and moran, 2006). sequencing of bacterial genomes is facilitating our understanding of the relationships between insects and their symbionts bringing to a better comprehension of the genome interdependence that occurs between host and bacteria (zientz et al.,     98 fig. 1 whole mount in situ hybridization: analysis of the distribution of fitc-labeled (green) asaia bacteria in anopheles gambiae salivary glands stained with propidium iodide (red) observed by confocal microscopy. bar = 100 μm (a). magnification of a portion of the left salivary gland showing the presence of asaia in the gland duct. bar = 100 μm (b). 2001; feldhaar and gross, 2009). in particular, symbiosis results in a genome reduction in endosymbiotic bacterial lineages that loose preferentially genes involved in catabolic pathways, since these functions may be played by the insect metabolism (zientz et al., 2001; feldhaar and gross, 2009). genome reduction may also affect the anabolic pathways if symbionts succeed in recruiting metabolic precursors from the host cell metabolisms bringing to a further rationalization of the symbiont’s genome (andersson and kurland, 1998; andresson and andersson, 1999; goebel and gross, 2001; moran and mira, 2001). interestingly, genome degeneration could be a key aspect not only in the study of symbiont genome evolution, but in the understanding of the host choice, since genome degeneration can not affect genes that are essential for the interaction with the host, neither genes that serve to avoid the exposition of bacteria to the host’s immune system. therefore, the occurrence of smaller genomes can make symbionts perfect experimental models to test the role of different genes in the host-bacteria interaction. examples include three genomes of buchnera aphidicola strains from different aphid hosts, two of candidatus blochmannia species from ants, one of wigglesworthia glossinidia from tsetse flies, and one each of candidatus baumannia cicadellinicola and candidatus sulcia muelleri from leafhoppers (dale and moran, 2006; mccutcheon and moran, 2007). their genomes are below one megabase in size and are known to encode as few as 500 genes. an extreme case is that of carsonella ruddii, a primary symbiont of psillids, that has a genome of 160 kb, the smallest bacterial genome described so far (nakabachi et al., 2006). in contrast, escherichia coli and other free-living relatives in this group have genomes of about four to five megabases encoding some 5000 genes. despite these advances, however, the mechanisms by which host-symbiont specificity develops in animal-bacterial interactions are not clear. many animals, including humans, are born devoid of symbionts and must recruit their microbiota from the environment and the process by which hosts and symbionts find each other to initiate a mutualism must be sensitive enough to identify the correct partner even when the symbiont is a minority constituent of the microbial community, and specific enough to exclude interlopers from gaining access to the host (mandel et al., 2009). the species specificity is also poorly understood for pathogenic interactions and at present is very difficult to explain why similar congeneric bacteria have distinct host ranges as reported, for example, in salmonella and brucella species (edwards et al., 2002; rajashekara et al., 2004). attempts to understand the molecular basis of host specificity have been unsuccessful in many pathogen-host animal interactions, including humans. salmonella enterica serovar typhi, for instance, can infect humans only, whereas serovar typhimurium has a broad range of hosts that includes mice, although the genomes of these two strains are over 97 % identical (edwards et al., 2002). similarly, different brucella species share over 98 % identity across 90 % of their genes, but exhibit strict host specificity (rajashekara et al., 2004). in contrast, the study of mutualism is providing insights into how specificity develops. for     99     100 instance, works from many laboratories has established nitrogen-fixing, nodulating rhizobia as the best-understood system for the development and evolution of host specificity in plant-associated bacteria (long, 2001). a strong confirmation of the hypothesis that symbionts may favour our understanding of the mechanisms involved in host choice better than pathogens has been recently published by mandel and colleagues (2009) showing that a single regulatory gene is sufficient to alter host range in an animal-bacterial mutualism, suggesting that the same could be true in the host-pathogen interaction. despite the relevance of this paper, however, the fundamental biological questions on how animalbacterial partnerships are established is still difficult to access and it is still impossible to define when bacteria passed the thin line that separates patogenicity and mutualism/symbiosis (gilmore and ferretti, 2003). in insects, some good candidates for taking a glance into the mechanisms involved in host choice are already present and in particular bacteria of the genus asaia could be perfect experimental models since they are cultivable in vitro (that is not a common feature for symbionts), can be manipulated at a chromosomal level in order to obtain stable transgenic strains and can be used for study of colonization of the insect body (favia et al., 2007). asaia belong to the group of the acetic acid bacteria that can be identified in virtue of their ability to oxidize ethanol into acetic acid even if asaia differentiates because it does not (or weakly) oxidize ethanol to acetic acid. besides tropical plants, where it was originally isolated (malimas et al., 2008), asaia has thus far been found associated to the insects scaphoideus titanus, the leafhopper vector of the phytoplasma causing flavescence dorée, a severe disease of grapevine (marzorati et al., 2006), and three mosquito vectors of malaria, anopheles stephensi, anopheles maculipennis and anopheles gambiae. in particular asaia has been found stably associated with larvae and adults of a. stephensi, dominating the microbiota of the mosquito (favia et al., 2007). the distribution of asaia in the body of a. stephensi has been investigated by the use of a strain, previously isolated from the mosquito, after genetic modification to express a green fluorescent protein (gfp). the gfp-tagged strain efficiently colonized the gut, salivary glands, and male and female reproductive organs. it is noteworthy that asaia, after assumption with a sugar-based diet by females, was detected in the gut and then in the salivary glands of the insect (fig. 1), crucial organs for the development of the cycle of the malaria parasites plasmodium spp. (favia et al., 2007). by using fluorescent strains it was shown that in a. stephensi, asaia is vertically transmitted from the mother to offspring (favia et al., 2007), but also undergoes paternal transmission to the progeny, by the way of venereal transfer from male to female during mating (damiani et al., 2008). the efficient capacity of asaia of colonizing adults and larvae of a. stephensi and the discovery of this bacterium in other insect vectors (i.e., other anopheles species and scaphoideus titanus) rise the question of whether this bacterium can crosscolonize different insect hosts. the reply to this question could be very useful not only in order to better understand asaia biology, but also to verify which asaia genes may be involved in the host choice that is necessary for establishing a symbiotic interaction. finally, it is important to underline that the investigation of the basis of host-symbiont interaction could be very useful also from an applicative point of view since asaia represents a promising bacterial species for the development of asaia-based symbiotic control approaches to block parasite transmission by insect vectors (favia et al., 2008). the symbiotic control approach would utilize bacteria capable of colonizing the insect body to produce effector molecules (natural or transgenic in the paratransgenic models) that kill or inhibit the causative agent of the disease or interfere with the survival of parasitic insects (beard et al., 2001). considering the localization in the insect body, the capability of colonizing very different hosts the culturability and the genetic transformability, asaia may be also accounted as potential interesting agents for natural or paratransgenic symbiotic control opening new perspectives also from an applicative point of view. acknowledgements this work is supported by the grant "far" from the university of modena and reggio emilia (mm). references andersson jo, andersson sg. insights into the evolutionary process of genome degradation. curr. opin. genet. dev. 9: 664-671, 1999. andersson sg, kurland cg. reductive evolution of resident genomes. trends microbiol. 6: 263268, 1998. beard cb, dotson em, pennington pm, eichler s, cordon-rosales sc, durvasula sr. bacterial symbiosis and paratransgenic control of vectorborne chagas disease. int. j. parasitol. 31: 621-627, 2001. brivio mf, mastore, m, pagani m. parasite-host relationship: a lesson from a professional killer. inv. surv. j. 2: 41-53, 2005. brown kl, hancock re. cationic host defense (antimicrobial) peptides. curr. opin. immunol. 18: 24-30, 2006. buchner p. endosymbiosis of animals with plant microorganisms. wiley interscience, new york, 1965. dale c, moran, na. molecular interaction between bacterial symbionts and their hosts. cell 126: 453-465, 2006. damiani c, ricci i, crotti e, rossi p, rizzi a, scuppa p, et al. paternal transmission of symbiotic bacteria in malaria vectors. curr. biol. 18: r1087-r1088, 2008. edwards ra, olsen gj, maloy sr. comparative genomics of closely related salmonellae. trends microbiol. 10: 94-99, 2002. favia g, ricci i, damiani c, raddadi n, crotti e, marzorati m, et al. bacteria of the genus asaia     101 stably associate with anopheles stephensi, an asian malarial mosquito vector. proc. natl. acad. sci. usa 104: 9047-9051, 2007. favia g, ricci i, marzorati m, negri i, alma a, sacchi l, et al. bacteria of the genus asaia: a potential paratransgenic weapon against malaria. adv. exp. med. biol. 627: 49-59, 2008. gilmore ms, ferretti jj. the thin line between gut commensal and pathogen. science 299: 19992001, 2003. goebel w, gross r. intracellular survival strategies of mutualistic and parasitic prokaryotes. trends microbiol. 9: 267-273, 2001. lazzaro bp. natural selection on the drosophila antimicrobial immune system. curr. opin. microbiol. 11: 284-289, 2008. lemaitre b, hoffmann j. the host defense of drosophila melanogaster. ann. rev. immunol. 25: 697-743, 2007. long sr. genes and signals in the rhizobiumlegume symbiosis. plant physiol. 125: 69-72, 2001. malimas t, yukphan p, takahashi m, kaneyasu m, potacharoen w, tanasupawat sy, et al. asaia lannaensis sp. nov., a new acetic acid bacterium in the alphaproteobacteria. biosci. biotechnol. biochem. 72:666-667, 2008. mandel mj, wollenberg ms, stabb ev, visick kl, ruby eg. a single regulatory gene is sufficient to alter bacterial host range. nature 458: 215218, 2009. mandrioli m, bugli s, saltini s, genedani s, ottaviani e. molecular characterization of a defensin in the izd-mb-0503 cell line derived from immunocytes of the insect mamestra brassicae (lepidoptera). biol. cell. 95: 53-57, 2003. marzorati m, alma a, sacchi l, pajoro m, palermo s, brusetti l, et al. a novel bacteroidetes symbiont is localized in scaphoideus titanus, the insect vector of flavescence dorée in vitis vinifera. appl. environ. microbiol. 72: 14671475, 2006. mccutcheon jp, moran na. parallel genomic evolution and metabolic interdependence in an ancient symbiosis. proc. natl. acad. sci. usa 104: 19392-19397, 2007. moran na. bacterial menagerie inside insects. proc. natl. acad. sci. usa 98: 1338-1340, 2001. moran na. symbiosis. curr. biol. 16: r866-r871, 2006. moran na, baumann p. bacterial endosymbionts in animals. curr. opin. microbiol. 3: 270-275, 2000. moran na, mira a. the process of genome shrinkage in the obligate symbiont buchnera aphidicola. genome biol. 2:research0054, 2001. müller u, vogel p, alber g, schaub ga. the innate immune system of mammals and insects. contrib. microbiol. 15: 21-44, 2008. nakabachi a, yamashita a, toh h, ishikawa h, dunbar h, moran na, et al. the 160-kilobase genome of the bacterial endosymbiont carsonella. science 314: 267, 2006. rajashekara g, glasner jd, glover da, splitter ga. comparative whole-genome hybridization reveals genomic islands in brucella species. j. bacteriol. 186: 5040-5051, 2004. schmidt o, rahman mm, ma g, theopold u, sun y, sarjan m, et al. mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides (hypothesis). inv. surv. j. 2: 8290, 2005. mauro mandrioli acknowledgements isj 9: yyy-xxx, 2012 isj 9: 163-168, 2012 issn 1824-307x minireview utilization of a silkworm model for understanding host-pathogen interactions c kaito, h yoshikai, k sekimizu graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan accepted september 27, 2012 abstract studies of the interactions between humans and pathogenic microorganisms require adequate representative animal infection models. further, the availability of invertebrate models overcomes the ethical and financial issues of studying vertebrate materials. insects have an innate immune system that is conserved in mammals. the recent utilization of silkworms as an animal infection model led to the identification of novel virulence genes of human pathogenic microorganisms and novel innate immune factors in the silkworm. the silkworm infection model is effective for identifying and evaluating novel factors involved in host-pathogen interactions. key words: insect model; innate immune factor; bacteria; fungi; virulence factor advantages of the silkworm as an animal infection model invertebrate animals possess an innate immune system, but lack an acquired immune system. many aspects of the innate immune system of invertebrate animals are conserved in mammals. for example, cationic antimicrobial peptides and toll receptors recognizing pathogens are found in both invertebrate animals and mammals (okada and natori, 1983; hoffmann, 1995; natori, 2010). therefore, studies using invertebrate animals can be performed to develop a better understanding of the host-pathogen interactions in mammals without the ethical and financial issues (seabra and bhogal, 2009). silkworms are larvae of the moth bombyx mori, a lepidopteran species (fig. 1). silkworms form cocoons where they develop into pupae. humans have used these cocoons as raw materials for silk for over 5000 years (goldsmith et al., 2005). bombyx mori is the only domesticated insect species, and the silkworm cannot survive in the natural world, probably due to their ineffective locomotion. in contrast to wild insects, silkworms can barely bite human fingers or escape from a breeding cage. silkworms typically consume mulberry leaves, but an artificial diet for silkworms has also been established and is commercially available. thus, rearing silkworms in the laboratory is easy. studies of host-pathogen interactions require quantitative evaluation of the virulence properties of ___________________________________________________________________________ corresponding author: chikara kaito graduate school of pharmaceutical sciences the university of tokyo 7-3-1, hongo, bunkyo-ku, tokyo, 113-0033, japan e-mail: kaito@mol.f.u-tokyo.ac.jp pathogenic microorganisms. to evaluate pathogenic virulence quantitatively, injection of a precise amount of the pathogen solution into model animals is essential. the large body size of the fifth instar silkworm (~ 5 cm) allows for the injection of a very precise amount of the pathogen solution into the silkworm hemolymph using a tuberculin syringe equipped with a 27-gauge needle (kaito and sekimizu, 2007), whereas injection of a precise sample amount is more difficult in small body-sized invertebrates such as drosophila melanogaster and caenorhabditis elegans. injection of human pathogenic bacteria such as staphylococcus aureus and pseudomonas aeruginosa into the silkworm hemolymph kills the silkworm (kaito et al., 2002). s. aureus injected into silkworms proliferates in the hemolymph. the lethal effects of s. aureus injection in silkworms are blocked by the injection of antibiotics. these observations suggest that the lethal effects of s. aureus in silkworms require bacterial proliferation (kaito et al., 2002). the silkworm-s. aureus infection model allows for the identification of biologic molecules involved in the ability of s. aureus to escape various innate immune factors of the silkworm and to proliferate in the silkworm hemolymph. importantly, infection experiments using silkworms can be performed at 37 ˚c, the temperature at which most human pathogenic microorganisms exhibit high virulence properties (kaito et al., 2011). genetic and biochemical analyses of silkworms are essential for identifying biologic molecules of silkworms that are involved in host-pathogen interactions. the bombyx mori genome project was recently completed and genome data are now 163 fig. 1 5th instar larvae of bombyx mori. tuberculin syringe equipped with a 27-gauge needle is shown above the silkworm. available on line (shimomura et al., 2009). in addition, construction of transgenic silkworms is established (tomita, et al., 2003). for biochemical analysis, biologic molecules from crude silkworm biomaterials must first be purified and identified. a fifth instar silkworm weighs around 2 grams, and thus an adequate amount of silkworm biomaterial can easily be prepared for purifying biologic molecules. identification of bacterial and fungal virulence factors using silkworms s. aureus is a pathogenic gram-positive bacterium present in the noses of 30 % of healthy individuals. to identify novel virulence factors of s. aureus, 100 hypothetical genes that are conserved among bacteria were disrupted and examined for lethal activity against silkworms. gene-disrupted mutants of three novel genes, named cvfa, cvfb, and cvfc (conserved virulence factor a, b, and c), exhibited attenuated lethality in silkworms (kaito et al., 2005) (table 1). these gene-disrupted mutants also showed attenuated virulence in mice, indicating that these genes contribute to the virulence of s. aureus not only in insects but also in mammals (kaito et al., 2005; matsumoto et al., 2007; marincola et al., 2012). streptococcus pyogenes is a human pathogenic gram-positive bacterium that causes various diseases, including adenoiditis and necrotizing fasciitis. the cvfa gene is also required for the lethality of s. pyogenes in silkworms and mice, and it is involved in the expression of various genes in s. pyogenes (kaito et al., 2005; kang et al., 2010; kang et al., 2012) (table 1). the cvfa gene is required for hemolysin production in both s. aureus and s. pyogenes. cvfa protein is a cyclic phosphodiesterase that cleaves a 2’,3’-cyclic phosphodiester linkage at the 3’-terminal nucleotide of rna (kaito et al., 2005; nagata et al., 2008). the cvfb gene contributes to s. aureus hemolysin production via a virulence regulatory gene, agr (matsumoto et al., 2007). crystal structure analysis revealed that cvfb has a novel l-shaped structure comprising three s1 rna binding domains and a winged-helix domain (matsumoto et al., 2010). the cvfc gene contributes to s. aureus resistance to detergents via the expression of thymidylate synthetase (ikuo et al., 2010). these novel virulence factors are conserved in many human pathogenic bacteria and their molecular functions are different from those of other well-known virulence factors. to determine whether s. aureus virulence factors against mammals contribute to s. aureus lethality in silkworms, s. aureus gene-disrupted mutants of hemolysins, cell wall proteins, and virulence regulators were examined for their attenuated lethality against silkworms (miyazaki et al., 2012) (table 1). the results demonstrated that s. aureus hemolysins are not required for virulence in silkworms. in contrast, several cell wall proteins and virulence regulators are required for s. aureus lethality in silkworms. thus, although not all s. aureus virulence factors against mammals can be evaluated in silkworms, silkworms are useful for evaluating the effects of s. aureus cell wall proteins and virulence regulators. that is, interactions between the host animal and s. aureus cell wall proteins or between the host animal and s. aureus virulence regulators are conserved among invertebrates and vertebrates. the silkworm model is also applicable for evaluating virulence factors of gram-negative human pathogenic bacteria. enterohemorrhagic escherichia coli (ehec) is a human pathogen that causes encephalopathy and nephropathy. ehec o157:h7 produces shiga toxins that are toxic to mammalian cells. the ehec gene-deleted mutant of shiga toxin exhibits attenuated virulence in a mouse infection model (eaton et al., 2008), but not in a silkworm model (miyashita et al., 2012). in contrast, the ehec gene-deleted mutant of lipopolysaccharide (lps) o-antigen synthase showed attenuated lethality in both silkworms and mice (miyashita et al., 2012) (table 1). the lps o-antigen mutant of ehec is sensitive to both silkworm and porcine antimicrobial factors (miyashita et al., 2012). therefore, lps o-antigen is required for the lethal effects of ehec in silkworms and mice via conferring resistance against innate immune factors of insects and mammals. a transposon mutant library of serratia marcescens, a 164 table 1 summary of biologic molecules identified in the silkworm infection model pathogenic microorganism gene category function references gram-positive bacteria staphylococcus aureus cvfa regulator 2', 3'-cyclic phosphodiesterase (kaito et al., 2005) cvfb regulator rna binding protein (matsumoto, et al., 2010) cvfc regulator conributing to detergent resistance (ikuo et al., 2010) sarz regulator transcription factor (kaito et al., 2006) agr regulator transcription factor and regulatory rna (miyazaki et al., 2012) saers regulator a two-component regulatory system (miyazaki et al., 2012) arlrs regulator a two-component regulatory system (miyazaki et al., 2012) srta cell wall protein anchoring proteins to cell wall (miyazaki et al., 2012) clfb cell wall protein binding mammalian cytokeratins (miyazaki et al., 2012) fnbb cell wall protein binding mammalian fibronectin (miyazaki et al., 2012) sdrc cell wall protein adherence to mammalian epithelial cells (miyazaki et al., 2012) streptococcus pyogenes cvfa regulator 2', 3'-cyclic phosphodiesterase (kaito et al., 2005) gram-negative bacteria enterohemorrhagic escherichia coli rfbe lipopolysaccharide lipopolysaccharide o-antigen synthesis (miyashita et al., 2012) waal lipopolysaccharide lipopolysaccharide o-antigen ligation (miyashita et al., 2012) serratia marcescens weca lipopolysaccharide lipopolysaccharide o-antigen synthesis (ishii et al., 2012) flhd flagella flagella synthesis (ishii et al., 2012) flir flagella flagella synthesis (ishii et al., 2012) pseudomonas aeruginosa toxa toxin exotoxin a (chieda et al., 2011) exos toxin type iii effector protein (okuda et al., 2010) sodm stress response manganese-superoxide dismutase (iiyama et al., 2007) sodb stress response iron-superoxide dismutase (iiyama et al., 2007) fungi cryptococcus neoformans gpa1 regulator g-protein alpha subunit (matsumoto et al., 2012) pka1 regulator catalytic subunit of protein kinase a (matsumoto et al., 2012) cna1 regulator catalytic subunit of calcineurin (matsumoto et al., 2012) candida albicans cmp1 regulator protein phosphatase (hanaoka et al., 2008) yvh1 regulator protein phosphatase (hanaoka et al., 2008) sit4 regulator protein phosphatase (hanaoka et al., 2008) ptc1 regulator protein phosphatase (hanaoka et al., 2008) candida glabrata cyb2p metabolism lactate dehydrogenase (ueno et al., 2011) host animal silkworms apolp-ii/i virulence inhibitor suppressing s. aureus hemolysin production (hanada et al., 2011) pp cytokine inducing innate immune responses (ishii, et al., 2010) silkworm hybrid (kinshu × showa) was used in studies of p. aeruginosa (iiyama et al., 2007; chieda et al., 2011). silkworm hybrid (hu • yo × tukuba • ne) was used in other studies. 165 human pathogenic gram-negative bacterium, was screened for its attenuated lethality in silkworms, leading to the identification of lps o-antigen synthase as the factor required for silkworm lethality (ishii et al., 2012). exotoxin a, a type iii effector protein exos, and superoxide dismutase of p. aeruginosa, which are virulence factors in mammals, are also required for killing silkworms (iiyama et al., 2007; okuda et al., 2010; chieda et al., 2011) (table 1). in contrast, p. aeruginosa pyocyanin, which is a virulence factor in mammals, is not required for killing silkworms (chieda et al., 2008). many factors in gram-negative bacteria are required for virulence in both silkworms and mammals, although some factors are specifically required for virulence in mammals. several virulence factors of human pathogenic fungi, including cryptococcus neoformans, candida glabrata, and candida albicans, were identified by infecting silkworms with gene-deletion mutants (hanaoka et al., 2008; ueno et al., 2011; matsumoto et al., 2012). gene-deletion mutants of the virulence factors of c. neoformans and c. albicans in mammals showed attenuated virulence in silkworms (hanaoka et al., 2008; matsumoto et al., 2012) (table 1). cyb2p of c. glabrata and ptc2 of c. albicans have been identified as virulence factors in silkworms and these genes are also required for virulence in mice (hanaoka et al., 2008; ueno et al., 2011) (table 1). these results suggest that human pathogen virulence factors of gram-positive bacteria, gram-negative bacteria, and fungi can be identified and evaluated in a silkworm model by infecting silkworms with gene-disrupted mutants. identification of innate immune factors in silkworms injection of s. aureus hemolysins into silkworms kills silkworms (hossain et al., 2006). in contrast, s. aureus hemolysin gene-deleted mutants did not exhibit attenuated killing ability against silkworms (miyazaki et al., 2012). these findings suggest that silkworm hemolymph contains a factor that inhibits s. aureus hemolysin production. a lipid carrier protein, apolipophorin (apolp), purified from silkworm hemolymph shows inhibitory activity against s. aureus hemolysin production (hanada et al., 2011) (table 1). the addition of apolp to s. aureus culture decreases the expression of saers, which is a positive regulator of s. aureus hemolysin genes. injection of anti-apolp antibodies into silkworms sensitizes silkworms against s. aureus. these findings suggest that apolp inactivates s. aureus saers and decreases hemolysin expression, leading to silkworm resistance against s. aureus. mammalian mucin also inhibits s. aureus hemolysin production, indicating that resistance to infection by the inhibition of hemolysin production is conserved among insects and mammals. most innate immune factors contribute to infection resistance by killing pathogenic microorganisms. novel innate immune factors that do not inhibit bacterial proliferation and inhibit bacterial virulence are not well understood. in addition to apolp, apolipoprotein b in mammalian blood and hydrogen peroxide produced by macrophages inhibit s. aureus virulence (rothfork et al., 2004; peterson et al., 2008). apolp is the first invertebrate biologic molecule found to inhibit bacterial virulence. silkworm hemolymph contains a cytokine-like peptide named paralytic peptide (pp) (ishii et al., 2008) (table 1). pp is synthesized as an inactive precursor and constitutively exists in silkworm hemolymph. bacterial peptidoglycans and fungal glucans induce reactive oxygen species (ros) from silkworm hemocytes and ros activate serine protease. the activated serine protease digests the pp precursor to produce matured pp. the matured pp activates humoral and cellular immune responses, including phagocytosis by silkworm hemocytes, phosphorylation of p38 mitogen-activated protein kinase, and production of antimicrobial peptides (ishii et al., 2010). because injection of the anti-pp antibody into silkworms sensitizes silkworms against s. aureus (ishii et al., 2008), pp contributes to silkworm resistance against s. aureus. pp was originally identified as a biologic molecule that induces muscle contraction in silkworms (ha et al., 1999). the biologic significance of the muscle-contracting activity of pp in the innate immune system is unknown. concluding remarks this minireview describes biologic molecules identified in the silkworm infection model. in most cases, the biologic molecules identified in the silkworm infection model are involved in mammalian host-pathogen interactions. utilization of a multitude of silkworms allows for quantitative evaluation of the virulence of many gene-disrupted mutants of pathogenic microorganisms. the silkworm infection model will be a powerful tool to further our understanding of host-pathogen interactions. references chieda y, iiyama k, lee jm, kusakabe t, yasunaga-aoki c, shimizu s. inactivation of pyocyanin synthesis genes has no effect on the virulence of pseudomonas aeruginosa pao1 toward the silkworm, bombyx mori. fems microbiol. lett. 278: 101-107, 2008. chieda y, iiyama k, lee jm, kusakabe t, yasunaga-aoki c, shimizu s. virulence of an exotoxin a-deficient strain of pseudomonas aeruginosa toward the silkworm, bombyx mori. microb. pathog. 51: 407-414, 2011. eaton ka, friedman di, francis gj, tyler js, young vb, haeger j, et al. pathogenesis of renal disease due to enterohemorrhagic escherichia coli in germ-free mice. infect. immun. 76: 3054-3063, 2008. goldsmith mr, shimada t, abe h. the genetics and genomics of the silkworm, bombyx mori. annu. rev. entomol. 50: 71-100, 2005. ha sd, nagata s, suzuki a, kataoka h. isolation and structure determination of a paralytic peptide from the hemolymph of the silkworm, bombyx mori. peptides 20: 561-568, 1999. hanada y, sekimizu k, kaito c. silkworm apolipophorin protein inhibits staphylococcus aureus virulence. j. biol. chem. 286: 166 39360-39369, 2011. hanaoka n, takano y, shibuya k, fugo h, uehara y, niimi m. identification of the putative protein phosphatase gene ptc1 as a virulence-related gene using a silkworm model of candida albicans infection. eukaryot. cell 7: 1640-1648, 2008. hoffmann ja. innate immunity of insects. curr. opin. immunol. 7: 4-10, 1995. hossain ms, hamamoto h, matsumoto y, razanajatovo im, larranaga j, kaito c, et al. use of silkworm larvae to study pathogenic bacterial toxins. j. biochem. 140: 439-444, 2006. iiyama k, chieda y, lee jm, kusakabe t, yasunaga-aoki c, shimizu s. effect of superoxide dismutase gene inactivation on virulence of pseudomonas aeruginosa pao1 toward the silkworm, bombyx mori. appl. environ. microbiol. 73: 1569-1575, 2007. ikuo m, kaito c, sekimizu k. the cvfc operon of staphylococcus aureus contributes to virulence via expression of the thya gene. microb. pathog. 49: 1-7, 2010. ishii k, hamamoto h, kamimura m, sekimizu k. activation of the silkworm cytokine by bacterial and fungal cell wall components via a reactive oxygen species-triggered mechanism. j. biol. chem. 283: 2185-2191, 2008. ishii k, hamamoto h, kamimura m, nakamura y, noda h, imamura k, et al. insect cytokine paralytic peptide (pp) induces cellular and humoral immune responses in the silkworm bombyx mori. j. biol. chem. 285: 28635-28642, 2010. ishii k, adachi t, imamura k, takano s, usui k, suzuki k, et al. serratia marcescens induces apoptotic cell death in host immune cells via a lipopolysaccharideand flagella-dependent mechanism. j. biol. chem. 2012 [in press]. kaito c, sekimizu k. a silkworm model of pathogenic bacterial infection. drug discov. ther. 1: 89-93, 2007. kaito c, akimitsu n, watanabe h, sekimizu k. silkworm larvae as an animal model of bacterial infection pathogenic to humans. microb. pathog. 32: 183-190, 2002. kaito c, usui k, kyuma t, sekimizu k. isolation of mammalian pathogenic bacteria using silkworms. drug discov. ther. 5: 66-70, 2011. kaito c, morishita d, matsumoto y, kurokawa k, sekimizu k. novel dna binding protein sarz contributes to virulence in staphylococcus aureus. mol. microbiol. 62: 1601-1617, 2006. kaito c, kurokawa k, matsumoto y, terao y, kawabata s, hamada s, et al. silkworm pathogenic bacteria infection model for identification of novel virulence genes. mol. microbiol. 56: 934-944, 2005. kang so, caparon mg, cho kh. virulence gene regulation by cvfa, a putative rnase: the cvfa-enolase complex in streptococcus pyogenes links nutritional stress, growth-phase control, and virulence gene expression. infect. immun. 78: 2754-2767, 2010. kang so, wright jo, tesorero ra, lee h, beall b, cho kh. thermoregulation of capsule production by streptococcus pyogenes. plos one 7: e37367, 2012. marincola g, schäfer t, behler j, bernhardt j, ohlsen k, goerke c, et al. rnase y of staphylococcus aureus and its role in the activation of virulence genes. mol. microbiol. 85: 817-832, 2012. matsumoto y, kaito c, morishita d, kurokawa k, sekimizu k. regulation of exoprotein gene expression by the staphylococcus aureus cvfb gene. infect. immun. 75: 1964-1972, 2007. matsumoto y, miyazaki s, fukunaga dh, shimizu k, kawamoto s, sekimizu k. quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with cryptococcus neoformans. j. appl. microbiol. 112: 138-146, 2012. matsumoto y, xu q, miyazaki s, kaito c, farr cl, axelrod hl, et al. structure of a virulence regulatory factor cvfb reveals a novel winged helix rna binding module. structure 18: 537-547, 2010. miyashita a, iyoda s, ishii k, hamamoto h, sekimizu k, kaito c. lipopolysaccharide o-antigen of enterohemorrhagic escherichia coli o157:h7 is required for killing both insects and mammals. fems microbiol. lett. 333: 59-68, 2012. miyazaki s, matsumoto y, sekimizu k, kaito c. evaluation of staphylococcus aureus virulence factors using a silkworm model. fems microbiol. lett. 326: 116-124, 2012. nagata m, kaito c, sekimizu k. phosphodiesterase activity of cvfa is required for virulence in staphylococcus aureus. j. biol. chem. 283: 2176-2184, 2008. natori s. molecules participating in insect immunity of sarcophaga peregrina. proc. jpn. acad. ser. b phys. biol. sci. 86: 927-938, 2010. okada m, natori s. purification and characterization of an antibacterial protein from haemolymph of sarcophaga peregrina (flesh-fly) larvae. biochem. j. 211: 727-734, 1983. okuda j, hayashi n, okamoto m, sawada s, minagawa s, yano y, et al. translocation of pseudomonas aeruginosa from the intestinal tract is mediated by the binding of exos to an na,k-atpase regulator, fxyd3. infect. immun. 78: 4511-4522, 2010. peterson mm, mack jl, hall pr, alsup aa, alexander sm, sully ek, et al. apolipoprotein b is an innate barrier against invasive staphylococcus aureus infection. cell host microbe 4: 555-566, 2008. rothfork jm, timmins gs, harris mn, chen x, lusis aj, otto m, et al. inactivation of a bacterial virulence pheromone by phagocyte-derived oxidants: new role for the nadph oxidase in host defense. proc. natl. acad. sci. usa 101: 13867-13872, 2004. seabra r, bhogal n. hospital infections, animal models and alternatives. eur. j. clin. microbiol. infect. dis. 28: 561-568, 2009. shimomura m, minami h, suetsugu y, ohyanagi h, satoh c, antonio b, et al. kaikobase: an integrated silkworm genome database and data mining tool. bmc genomics 10: 486, 2009. tomita m, munetsuna h, sato t, adachi t, hino r, 167 hayashi m, et al. transgenic silkworms produce recombinant human type iii procollagen in cocoons. nat. biotechnol. 21: 52-56, 2003. ueno k, matsumoto y, uno j, sasamoto k, sekimizu k, kinjo y, et al. intestinal resident yeast candida glabrata requires cyb2p-mediated lactate assimilation to adapt in mouse intestine. plos one 6: e24759, 2011. 168 isj 5: 110-xxx, 2008 isj 5: 110-123, 2008 issn 1824-307x review oxidative stress and bivalves: a proteomic approach d sheehan1, b mcdonagh2 1proteomics research group, dept. biochemistry, university college cork, lee maltings, prospect row, mardyke, cork, ireland 2departamento de bioquímica y biología molecular, university of cordoba, campus de rabanales, córdoba 14071, spain accepted september 3, 2008 abstract bivalves are of major importance in aquatic ecology, aquaculture, are widely used as sentinel species in environmental toxicology and show remarkable plasticity to molecular oxygen. excess reactive oxygen species (ros) arising from molecular oxygen can cause oxidative stress and this is also a consequence of exposure to many common environmental pollutants. indices of oxidative stress have therefore found favor as biomarkers of exposure and effect in environmental toxicology. however, there is a growing body of literature on the use of discovery-led proteomics methods to detect oxidative stress in bivalves. this is because proteins absorb up to 70 % of ros leading to complication of the proteome. this article explores the background to these developments and assesses the practice and future potential of proteomics in the study of oxidative stress in bivalves. key words: bivalve; oxidative stress; mussel; clam; ecotoxicology introduction molecular oxygen (o2) first accumulated on earth ~ 2.3 billion years ago due to the appearance of photosynthesis. the redox characteristics of the earth’s atmosphere fundamentally altered from reducing to strongly oxidizing and living cells, with reducing internal environments, for the first time needed to expend considerable energy to survive the surrounding oxidizing environment. oxygen is paradoxical in that it is on the one hand essential for the most efficient form of energy metabolism; aerobic metabolism. on the other hand, it is a potential chemical threat because it can lead to formation of reactive oxygen species (ros) (halliwell and gutteridge, 2007; winterbourn, 2008). these include species such as h2o2, the hydroxyl and superoxide radicals (fig. 1) which are naturally formed in living cells especially in subcellular organelles such as the mitochondrion and endoplasmic reticulum. cells developed elaborate ___________________________________________________________________________ corresponding author: david sheehan proteomics research group, dept. biochemistry, university college cork, lee maltings, prospect row, mardyke, cork, ireland e-mail: d.sheehan@ucc.ie strategies to cope with ros as part of adaptation to their changed redox situation including antioxidant enzyme activities (e.g., catalases), small antioxidant molecules (e.g., glutathione, gsh; vitamin e) and redoxins (e.g., thioredoxins). under normal circumstances, these defenses cope well with the levels of ros produced and cells exist in a state of redox homeostasis. however, in certain circumstances, the balancing act between ros production and antioxidant defences tilts in favor of build-up of ros in the cell leading to a state of oxidative stress (halliwell and gutteridge, 2007). bivalves (informally including mussels, clams, oysters and scallops) comprise animals of the class bivalvia in the phylum mollusca which first appeared late in the cambrian explosion (~ 530 million years ago) and eventually came to dominate over brachiopods in the palaezoic era (gould and calloway, 1980). they are characterized by a shell which is divided from front to back into left and right valves, have adapted their gills as filter-feeding organs and often have a well-developed byssus apparatus allowing them to attach to rocky substrates (bayne, 1976). the precise reason for the evolutionary success of bivalves is a matter for conjecture (gould and calloway, 1980; miller, 1998), but it is evident that they have shown considerable resilience and now occupy niches in a 110 mailto:d.sheehan@ucc.ie fig. 1 oxidative stress arises when there is an imbalance between production and neutralisation of ros. these can arise from endogenous enzyme mechanisms or from exogenous sources such as metals and pahs. oxidative stress can result in modification of biomolecules, especially proteins and lead to toxicity. wide range of aquatic environments. they are often the major macrofauna on rocky substrates of littoral, shallow sub-littoral and deep-sea vents (bayne, 1976; lutz and kennish, 1993). their filter-feeding habit adds greatly to their ecological significance in that bivalves are important calcium and carbon accumulators, they link primary producers (bacteria and phytoplankton) with higher organisms in aquatic food-chains and are responsible in tidal zones for filtration of the water body (newell, 2004). since most human interaction with the aquatic environment is concentrated along rivers, coastlines and estuaries (halpern et al., 2008), bivalves have importance in addition to their evolutionary and ecological significance. they are an important foodsource for human populations and can be cultured for food and other reasons (naylor et al., 2000; newell, 2004). bivalves have genomes generally comparable in size to the human genome yet they are poorly represented in dna sequence databases. for this reason, there is growing interest in bivalve genomics as a means of elucidating evolutionary, genetic and toxicological relationships (mckillen et al., 2005; tanguy et al., 2008). because of their sedentary lifestyle, filterfeeding habit, abundance, ease of identification, tolerance to pollution and wide geographical distribution bivalves have found particular applications as sentinel species in environmental toxicology (ecotoxicology) (bayne, 1979; goldberg and bertine, 2000). this has led to a considerable body of research focusing on using levels of particular biomolecules (biomarkers) to reveal effects of environmental pollutants and give insights to the pollution status of specific sampling sites (cajaraville et al., 2000; handy et al., 2003; depledge and galloway, 2005; galloway, 2006). since many environmental pollutants such as heavy metals, polyaromatic hydrocarbons (pahs), polychlorinated biphenyls (pcbs) and nanomaterials are known to be strongly pro-oxidant, much of this research has focused on biomarkers reflecting oxidative stress (viarengo et al., 1991; lópez-barea and pueyo, 1998; lesser, 2006; valavanidis et al., 2006). it has increasingly become clear that such studies also need to allow for effects due to non-pollution variables such as seasonality, nutritional status and the normal redox variations inherent in the bivalve lifestyle (power and sheehan, 1996; manduzio et al., 2004). more recently, interest has grown in extending this essentially hypothesis-driven biomarker approach to a discovery-driven approach in which highthroughput proteomics techniques are applied to bivalves to identify novel biomarkers for exposure to environmental pollution. this review explores how bivalves cope with ros under normal circumstances, describes the biomarker approach to study of oxidative stress and reviews the technology and opportunities offered by proteomics in this important area of research. bottlenecks to exploitation of this experimental approach are briefly described. reactive oxygen species and oxidative stress in aerobic metabolism, molecular oxygen is eventually reduced to water. in this process, a 111 variety of oxygen derivatives, collectively called ros, are naturally produced. these include the superoxide anion radical (o2• -), hydrogen peroxide (h2o2), peroxyl radicals (roo•), nitrogen oxide (no•) and hydroxyl radical (ho•) (lesser, 2006; winterbourne, 2008). some ros contain unpaired electrons (i.e. they are free radicals) whilst others are non-radical species (e.g., h2o2) (fig.1). ros can also be formed in response to a wide range of exogenous agents including radiation (x-ray, gamma, uv, or visible light in the presence of a sensitizer), metal ions, solvents, particulate matter, nitrogen oxides, ozone etc. (davies, 2005). the ho• is the most important ros in biology with an oxidation rate constant for protein components comparable to the rate of diffusion ~ 108-10 m-1 s-1 (davies, 2005; winterbourn, 2008). due to its high reactivity ho• is quite non-specific in its targets for oxidation, whereas ros with lower rate constants react more specifically (davies, 2005; winterbourn, 2008). ros are generally removed rapidly by antioxidant mechanisms as they can affect major cellular components including lipids, proteins, carbohydrates and dna and can ultimately lead to cell death (fig. 1). ros can cause serious toxicity because they are capable of interacting rapidly and efficiently with important biomolecular targets (davies, 2005; valavanidis et al., 2006; winterbourne, 2008). the most toxic ros is ho• which can attack biological membranes in a diffusion-controlled fashion, initiating free radicalmediated chain reactions (davies, 2005; lesser, 2006; winterbourn, 2008). ros capable of diffusion across biological membranes (e.g., h2o2) can enter into numerous other reactions and so cause effects at a distance from their site of formation. the extent of damage which ros can generate is dependent on a number of factors including: the concentration of target, the rate constant for reaction of oxidant with the target, the location of the target when compared to the site of oxidant formation, the occurrence of secondary events, the occurrence of oxidant-scavenging reactions and repair reactions (davies, 2005; winterbourn, 2008). oxidative stress in bivalves: what is “normal”? oxidative stress refers to a state where there is an imbalance between the generation and neutralisation of ros by antioxidants, caused by excessive production of ros, loss of antioxidant defenses or both (halliwell and gutteridge, 2007). several aspects of bivalve biology make oxidative stress of especial significance. bivalves can be exposed to relatively high levels of pro-oxidants as a consequence of their filter-feeding habit. common environmental pollutants known to be pro-oxidants such as pcbs, pahs, heavy metals and organochlorines are bioconcentrated within bivalves leading to oxidative stress (viarengo et al., 1991; rodríguez-ariza et al., 1992, 1993, 2003; cheung et al., 2001; downs et al., 2002; rodríguez-ortega et al., 2002; valavanidis et al., 2006). moreover, intertidal animals exposed to air during the tidal cycle face particular challenges. these only survive dehydration because of the integrity of the seal between the two halves of their shell. however, this seal also cuts off access to oxygen from the animals’ surroundings and oxygen inside the shell is rapidly depleted (stachowitz et al., 2007). depending on local conditions, individual animals may be exposed to air for a comparatively short time-period (at the lower end of the intertidal zone) or for up to 7 h in every 12 (at the highest end of the intertidal zone). when exposed to air, animals soon experience hypoxia which can turn to anoxia in the more extreme circumstances of the highest part of the intertidal. this is a significant cause of mortality and individual animals can physiologically adapt to this challenge (widdows et al., 1979; altieri, 2006; stachowitz et al., 2007). normal aerobic metabolism is very quickly depressed with glycolytic fermentation progressively increasing (widdows et al., 1979; widdows and shick, 1985). on reimmersion, the bivalve opens its shell and experiences a rapid increase in oxygen level. simultaneously, oxidative metabolism resumes (widdows and shick, 1985) resulting in a burst of ros similar to the reperfusion-ischemia injury of mammals (grace, 1994). thus, as a routine part of their life-cycle, bivalves must have sufficient plasticity to cope with relatively large fluctuations in oxygen levels which can be further modulated by factors such as seasonality, pollution exposure and nutritional status (viarengo et al., 1991; rodríguezariza et al., 1992, 1993, 2003; power and sheehan, 1996; manduzio et al., 2004; lesser, 2006). moreover, recent research (primarily in mammalian systems) suggests that, even in sub-stress scenarios, redox modifications may be an important aspect of normal cell signalling capable of triggering apoptosis (biswas et al., 2006; ying et al., 2007; oktyabrsky and smirnova, 2007; poole and nelson, 2008). while outside the scope of this review, bivalve species will no doubt be a fruitful area for future research into this important role of ros. oxidative stress in bivalves: the biomarker approach bivalves have found especial favor as “sentinels” of environmental pollution and have come to be widely used in pollution surveillance programmes (bayne, 1979; goldberg and bertine, 2000). biomarkers are indices of the presence of chemical pollutants in particular environmental contexts (cajaraville et al., 2000; handy et al., 2003; depledge and galloway, 2005; galloway, 2006). as has been pointed out (power and sheehan, 1996; manduzio et al., 2004) effects of environmental pollutants are to some extent dependent on biological (seasonal, nutritional and reproductive status) and environmental variables (e.g., temperature, exposure to sunlight). bivalves have evolved robust biochemical and physiological defenses against chemical pollution and the varying levels of oxygen exposure to which they are subject (widdows et al., 1979; widdows and shick, 1985). they have an extensive battery of antioxidant defenses (winston, 1991; valavanidis et al., 2006) and variations in these were found to be useful in environmental monitoring (rodríguez